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0121 micromax ditel

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Valid till 2017/5/25



Micromax Canvas Mega 2 Q Price in India (, March 29th): Rs. 6, Check Micromax Canvas Mega 2 Q Specs And Reviews. Compare Micromax Canvas Mega 2 Q Prices From Various Stores/5(9). Mar 26, · Checkout the best price to buy Micromax Canvas 2 in India. Know full specification of Micromax Canvas 2 Mobile Phone along with its features. Buy Micromax Q Plus at All mobile phones are % Original and carry full Manufacturers Warranty since we procure directly from.
Normalizing chromosomes for the determination and verification of common and rare chromosomal aneuploidies. The washing step can be omitted between Ditel rounds of annealing, Micromax extension and strand separation, if it is desired to maintain the same templates in the vicinity of immobilised primers. Another concern is the lack of new antibiotics to treat infections Ditel resistant pathogens. Methods and compositions for generation a variety of copies of nucleic acid sequences and methods 0121 detection thereof. In another preferred embodiment, the enzyme is selected from Micromax group consisting of esterases, cerebrosidases, and carbohydrases that 0121 a linkage that effects release of a cell-surface protein or carbohydrate.
Micromax Canvas Mega 2 Q Price in India (, March 29th): Rs. 6, Check Micromax Canvas Mega 2 Q Specs And Reviews. Compare Micromax Canvas Mega 2 Q Prices From Various Stores/5(9). Mar 26, · Checkout the best price to buy Micromax Canvas 2 in India. Know full specification of Micromax Canvas 2 Mobile Phone along with its features. Buy Micromax Q Plus at All mobile phones are % Original and carry full Manufacturers Warranty since we procure directly from.

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Two flasks were used to seed a L fermentor filled with L of the same steam sterilized medium. The protein concentration was measured at approximately This was accompanied by an increased use of therapeutic antibiotics that resulted in an overall increase in the use of antibiotics as well as increased meat animal production costs. Mice were euthanized by injecting each with 0. Feed was fed in mash form on an ad libitum basis throughout the entire trial test period day 0 to day

See Friedman and Pastan, Proc. See Jacobs et al. There are several examples of host cell GPI-anchored proteins that are the binding sites for viruses. See Clarkson et al.

See Barbis and Parrish, Brazilian J. These include structures such as cholesterol esters Rostand and Esko, J. To this end, a key aspect of the present invention is the demonstration that an enzyme, which not only is active in cleaving cell surface components, can be administered orally as an anti-infection agent and be effective in vivo.

Notably, while a representative suitable enzyme, PI-PLC, has been available since the s, this approach has not been suggested heretofore. Feed preparations that contain an endo-1,4-D-mannanase are known, and some reports have proposed an antifuigal activity for mannanase.

In these instances, mannanase is combined with a recognized antibiotic, although the prospect of enzyme use in antibiotic-free feed has been discussed generally. Adams, Feed Mix Special, at pages Further, the present invention contemplates a composition that contains such an enzyme, including a mannanase, but that contains no other anti-infection agent.

This result is unexpected because B. For example, see Stein and Logan, J. It is surprising, therefore, that an extracellular enzyme from a pathogen which causes gastroenteritis would have a curative effect, pursuant to the present invention, in relation to a disease caused by a bacterial infection.

In the aforementioned enzyme preparation, an extracellular phosphatidylinositol-specific phospholipase C [E. Enzyme treatment of the present invention worked effectively as a coccidiostat and antibiotic.

Therefore, it is an effective and commercially viable approach for the treatment of digestive tract infections particularly as currently used substances are banned.

Surprisingly, addition of the PI-PLC to the animal feed without coating results in effective treatment of pathogenic infections. Dried, shelf-stable formulations of enzymes that are suitable, pursuant to the present invention, for incorporation in tablets or capsules, for example, can be prepared by freeze-drying, spray drying in fluidized bed dryer with inert or carbohydrate carrier, or by using evaporative techniques in conjunction with glass-forming stabilizers.

See Franks et al. Another approach involves salt precipitation, for example, ammonium sulfate precipitate or solvent precipitate, as with acetone for powder formation, followed by drying and blending with a carrier.

Exemplary carbohydrates for use as carriers are xylose, fructose, glucose, sorbitol and maltotriose, among others, as described by Franks, supra. Choice of a carbohydrate carrier is based on compatibility with the enzyme, low hydroscopic tendency, and a favorable glass transition curve.

The stabilizer trehalose is particularly suitable for producing ambient temperature-stable biologics. Such solutions typically are sterile-filtered, prior to pharmaceutical use.

Feeds are composed mainly of grain material, a protein source, vitamins, amino acids, and minerals. The grain material typically includes corn, sorghum, wheat, barley, or oats. The source of proteins can be beans or peas, for example.

Exemplary minerals, amino acids and vitamins include B 12 , A, pantothenic acid, niacin, riboflavin, K, DL-methionine, L-lysine, choline chloride, folic acid, dicalcium phosphate, magnesium sulfonate, potassium sulfate, calcium carbonate, sodium chloride, sodium selenite, manganous oxide, calcium iodate, copper oxide, zinc oxide, and D-activated animal sterol.

Other preservatives for feed such as sodium benzoate, propylparaben, sodium or potassium sorbate, and ascorbyl palmitate are examples of approved chemical preservatives that can alsobe used to prevent potential spoilage by microbial growth in the product.

These preservatives can be applied to feeds by post-pelleting with a large dilution by automated spraying technology. See Fodge et al. Such liquid preparations may contain stabilizing carbohydrates such as sorbitol or glycerol, if compatible.

Materials that are desired components of feed, such as other enzymes or vitamins that are heat-labile, may be included for increased efficiency. Such dry enzyme concentrates are prepared by first concentrating the liquid enzyme preparation, using a 10 Kd NMWC or other suitable ultra-filter, to achieve a high percentage of enzyme content, and then by blending with a very dry carrier, such as corn grits, soy grits or even an inert material or insoluble salt that is approved for use in feeds.

It is well known that modifying protein structure, primarily through changing the encoding DNA sequence or, secondarily, through chemical modification can render enzymes more stable against inactivation.

One illustration in this regard is the use of chemical cross-linking of enzyme crystals. See Collins et al. Another approach to increasing the stability of enzyme for the present invention entails changing amino acids by mutagenesis of the gene that codes for the enzyme of interest, or obtaining genes or parts of genes for shuffling.

See Crameri et al. For example, see Giver et al. Thus, known protocols could be employed in this regard to make modified enzyme, for testing, according to the examples, to gauge suitability in the inventive treatment methodology.

The expression system Rygus and Hillen, Appl. Strains of this type, with amplified expression, provide a feasible means for producing commercially useful amounts of PI-PLC, to incorporate into animal feed in accordance with the present invention.

See Kamogashira et al. In the examples detailed below, the possibility of involving these two antibiotics was eliminated by excluding possible low molecular-weight antibiotics from the enzyme preparation.

The concentrate, containing protein larger than 10 Kd, was used for further processing. The small molecular-weight antibiotics, such as those described by Handelsman, supra, would pass through this filter.

Moreover, ammonium sulfate precipitation was employed to precipitate high molecular weight proteins, leaving low molecular weight materials in solution.

After the ammonium sulfate precipitate was re-dissolved, the resultant enzyme solution was dialyzed against buffer, yet another treatment that removes low molecular-weight antibiotics. Finally, the protein was precipitated a second time, again with ammonium sulfate, to relegate any remaining, low molecular-weight compounds to the solution.

The fermentation broth also was tested, with an E. When the culture density reached OD reading of 1. The initial batch volume was 9. The initial pH was adjusted to 7.

The OD of the initial culture was 0. At six hours, the fermentation was stopped with final OD of The fermentations were run without pH control and the final pH was 8.

In this case the seed cultures were used at six hours with OD of 2. The main fermentations were run for 6. Final pH was 8. Chilled broth was pumped through the filters with a peristaltic pump at about 2 liters per minute with recycling back to the holding reservoir.

The permeate containing the enzyme was collected in a reservoir chilled on ice. The initial cell-containing broth volume of about 9 liters was concentrated down to about 2 liters at which point diafiltation was started with 10 mMTris-HCl, pH 8.

After a total volume of about 14 liters of permeate was collected, the cell washing was terminated. The same pumping method with recycle of concentrate was used except the permeate was discarded.

Final concentrate with a volume of about mL from each fermentor was saved for the next processing step. The supernatant was discarded, and the precipitate was dissolved in a minimal volume of 10 mMTris-HCl, 0.

Table 1 below summarizes the assay data and predicts the approximate purity and total enzyme on a pure basis obtained for each of the four preparations.

Ammonium sulfate g was added slowly and the solution was mixed on ice for several minutes. The solution was centrifuged to collect the precipitate. The pellet fraction was dissolved in a minimal amount of 20 mM phosphate buffer pH 7.

The protein concentration was measured at approximately See Kuppe et al. The shake flask fermentation was used to evaluate the phosphatidylinositol specific phospholipase C production in Bacillus megaterium.

At OD of about 0. After three hours, supernatant was harvested by centrifugation. The phosphatidylinositol-specific phospholipase C activity was measured by a fluorescent substrate method Hendrickson, et al.

The pH was adjusted to 7. After growth to about 1. Two flasks were used to seed a L fermentor filled with L of the same steam sterilized medium. Tetracycline was sterile filtered 0.

The operating conditions for the first fermentor seed stage were as follows: The pH was controlled at 6. D reached, the contents were used to seed a L fermentor containing L of the same medium.

The second rate was held until all The fermentation was terminated at 20 hours. A test for the presence of antibiotic was conducted with E. The test was conducted as a. See Brantner, Pharmazie 52 1: No clearing zone indicating antibiotic activity was observed around the cylinders containing the enzyme samples.

The substrate 4-methylumbelliferyl-myo-inositolphosphate, N-methyl-morpholine salt was obtained from Biosynth Naperville, Ill. Enzyme dilutions if needed are made into 0. The reaction at pH 8. Also, at pH 9.

For the purpose of efficacy testing in animal feeding experiments, the units measured using this assay were converted to the equivalent Hendrickson Unit to facilitate comparison with the first tests before this assay was used.

The enzyme was extracted from feed test materials by weighing 4 g feed and adding it to 20 mL of 10 mM Tris-Cl pH 7. Appropriate dilutions of the extracts were made in 0. Sample reactions should be foil-covered to protect the substrate from light.

Finally, samples were centrifuged at 12, RPM in a microcentrifuge for 5 minutes. Background control levels were subtracted. A rate of fluorescence units production per minute was calculated.

Fluorescense units were converted to micromoles of reaction product and enzyme units extracted per original pound of feed was calculated. Broiler Chicken Feeding Trial I. Water and feed were provided ad libitum throughout the 21 day test period.

A randomized block design was used to allocate chicks to cages and cages to treatment groups. All cages, feeders and waterers were sanitized prior to the beginning of the test.

Lighting was continuous 24 hour per day with incandescent lamps. Body weights were determined at day one and day. Feed consumption was measured at day On day 7, all birds were further infected with, Clostridium perfringens through the water supply.

In the negative control T1 there was no treatment for infection. In the positive control T2 a coccidiostat and antibiotic were added to the feed. The study was done in randomized battery cages, on a blinded basis, to test the effect of PI-PLC made from the natural source, Bacillus cereus wild type PI-PLC, or a recombinant Bacillus megaterium on male broiler performance reared to 21 days of age.

The natural source also contains other extracellular enzymes but the PI-PLC prepared from Bacillus megaterium is highly purified and was further purified by ultrafiltration using a Kd NMWC membrane.

Birds were challenged at 8 days of age with Avian coccidia, E. Each of the nine treatments Table 6 had 10 replications or cages. Dead birds, if present, were not replaced after the 8 th day. Feed was fed in mash form on an ad libitum basis throughout the entire trial test period day 0 to day Thereafler, nine treated diets, in mash form, were fed from days of age.

Birds were challenged at 8 days of age with avian coccidia 75, E acervulina oocysts and 1, E. Dead birds were removed from the cages when they were detected and were not replaced. Feed was fed in MASH form on an ad libitum basis throughout the entire trial test period day 0 to day In the case of E.

The results are shown in Table 9. Birds were challenged at 8 days of age with coccidia 75, E acervulina oocysts and 1, E. Each of the treatments had 8 replications or cages.

Birds were not replaced. Feed and water was fed ad libitutim throughout the entire trial test period Days 0 to Day Diets were fed in MASH from days of age. Importantly, this study shows that either the PI-PLC or mannanase when combined with salinomycin, but without the antibiotic BMD Tests 6 and 5 restores the performance to basically the level of the uninfected tests No.

This provides evidence for antibacterial function. For cell pretreatment, cell cultures were overlaid With dilutions of enzymes as described in Table 11, and examined for morphological changes From 5 to 45 min post overlay.

After 45 minutes, the monolayer cultures were washed twice, then inoculated with untreated E. For application during infection, sporozoites were suspended in the appropriate dilution of the enzyme and inoculated immediately into the cell cultures.

After 45 minutes incubation, cultures were fixed, stained, and the invasion was quantified. At the enzyme levels used in these experiments, no morphological change was noted. The sporozoite invasion of the cultured cells was measured after the two methods of enzyme treatment, as well as without enzyme treatment, by histological staining and microscopy procedures.

Even at dose approximately one-half of the B. Thus, pretreatment of the cells with the enzyme and washing away of the enzyme was as effective as adding the enzyme concurrently during the infection.

However, based on the experience with extracting enzyme from feed, the two wash steps likely do not remove all of the enzyme. These positive results included one experiment with each pathogen type.

Thus, where heating is used to separate a newly synthesised nucleic acid strand from its template, the nucleic acid polymerase is preferably heat stable at the temperature used.

Such heat stable polymerases are known to those skilled in the art. They are obtainable from thermophilic micro-organisms. The nucleic acid polymerase need not however be DNA dependent.

It may be RNA dependent. Thus it may be a reverse transcriptase—i. Such a temperature range will normally be maintained during primer extension. Once sufficient time has elapsed to allow annealing and also to allow a desired degree of primer extension to occur, the temperature can be increased, if desired, to allow strand separation.

They can be used to control the timing of colony initiation, e. Following strand separation e. The washing step can be omitted between initial rounds of annealing, primer extension and strand separation, if it is desired to maintain the same templates in the vicinity of immobilised primers.

This allows templates to be used several times to initiate colony formation. It is preferable to provide a high concentration of template molecules initially so that many colonies are initiated at one stage.

The size of colonies can be controlled, e. Other factors which affect the size of colonies can also be controlled. Once colonies have been formed they can be used for any desired purpose.

A surface comprising immobilised nucleic acid strands in the form of colonies of single stranded nucleic acid molecules is also within the scope of the present invention. Normally each immobilised nucleic acid strand within a colony will be located on the surface so that an immobilised and complementary nucleic acid strand thereto is located on the surface within a distance of the length of said immobilised nucleic acid strand i.

This allows very high densities of nucleic acid strands and their complements to be provided in immobilised form. Preferably there will be substantially equal proportions of a given nucleic acid strand and its complement within a colony.

A nucleic acid strand and its complement will preferably be substantially homogeneously distributed within the colony. It is also possible to provide a surface comprising single stranded nucleic acid strands in the form of colonies, where in each colony, the sense and anti-sense single strands are provided in a form such that the two strands are no longer at all complementary, or simply partially complementary.

Such surfaces are also within the scope of the present invention. Normally, such surfaces are obtained after treating primary colonies, e. Once single stranded colonies have been provided they can be used to provide double stranded molecules.

Thus surfaces comprising colonies of non-bridged double stranded nucleic acid molecules are also within the scope of the present invention. Using the present invention, small colonies can be provided that contain large numbers of nucleic acid molecules whether single or double stranded.

Many colonies can therefore be located on a surface having a small area. Colony densities that can be obtained may therefore be very high, as discussed supra. Different colonies will generally be comprised of different amplified nucleic acid strands and amplified complementary strands thereto.

Thus the present invention allows many different populations of amplified nucleic acid molecules and their complements to be located on a single surface having a relatively small surface area.

The surface will usually be planar, although this is not essential. The present invention also provides an apparatus for providing a surface comprising colonies of the immobilised nucleic acid molecules discussed supra.

Such an apparatus can include one or more of the following:. Other apparatuses are within the scope of the present invention. These allow immobilised nucleic acids produced via the method of the present invention to be analysed.

They can include a source of reactants and detecting means for detecting a signal that may be generated once one or more reactants have been applied to the immobilised nucleic acid molecules.

They may also be provided with a surface comprising immobilised nucleic acid molecules in the form of colonies, as described supra. Desirably the means for detecting a signal has sufficient resolution to enable it to distinguish between signals generated from different colonies.

Apparatuses of the present invention of whatever nature are preferably provided in automated form so that once they are activated, individual process steps can be repeated automatically.

The present invention will now be described without limitation thereof in sections A to I below with reference to the accompanying drawings. It should be appreciated that procedures using DNA molecules referred to in these sections are applicable mutatis mutandis to RNA molecules, unless the context indicates otherwise.

It also illustrates the annealing, elongation and denaturing steps that are used to provide such colonies. Referring now to FIG. Each primer 1 is attached to the surface by a linkage indicated by a dark block.

This may be a covalent or a non-covalent linkage but should be sufficiently strong to keep a primer in place on the surface. In practice however longer sequences would generally be provided. Between the two ends any sequence to be amplified or the complement of any sequence to be amplified can be provided.

When primer extension is complete, as shown in FIG. The target molecule can then be separated from the extended immobilised strand e. This separation step frees the extended, immobilised strand so that it can then be used to initiate a subsequent round of primer extension, as shown in FIGS.

Primer extension is shown occurring in FIG. Each of these strands can then themselves be used as templates in further rounds of primer extension initiated from new primers 3 and 4 , as shown in FIGS.

Four single stranded, immobilised strands can be provided after two rounds of amplification followed by a strand separation step e. Two of these have sequences corresponding to the sequence of the target molecule originally used as a template.

The other two have sequences complementary to the sequence of the target molecule originally used as a template. In practice a given immobilised strand and its immobilised complement may anneal once.

It will therefore be appreciated that a given sequence and its complement can be provided in equal numbers in immobilised form and can be substantially homogeneously distributed within a colony.

Further rounds of amplification beyond those shown in FIG. Only a single template need be used to initiate each colony, although, if desired, a template can be reused to initiate several colonies. It will be appreciated that the present invention allows very high densities of immobilised extended nucleic acid molecules to be provided.

Within a colony each extended immobilised molecule will be located at a surface within one molecule length of another extended immobilised molecule. Thus position 3 shown in FIG.

A flat plate is shown schematically in plan view having primers immobilised thereon in a square grid pattern the primers are indicated by small dots. A regular grid is used solely for simplicity: At the position indicated by arrow X a template molecule has annealed to a primer and an initial bout of primer extension has occurred to provide an immobilised, extended nucleic acid strand.

Following strand separation, an end of that strand becomes free to anneal to further primers so that additional immobilised, extended nucleic acid strands can be produced. This is shown having occurred sequentially at positions indicated by the letter Y.

For simplicity, the primer chosen for annealing is positioned next to the primer carrying the nucleic acid strand: However, this primer will obviously be within a distance equal to the length of the nucleic acid strand.

It will be appreciated that annealing at only one rather than at all of these positions is required for colony cell growth to occur. After immobilised, extended, single-stranded nucleic acid molecules have been provided at the positions indicated by letter Y, the resultant molecules can themselves anneal to other primers and the process can be continued to provide a colony comprising a large number of immobilised nucleic acid molecules in a relatively small area.

It also depicts the typical observations that can be made, as can be seen on the examples shown in FIGS. The simultaneous amplification and immobilisation of nucleic acids using solid phase primers has been successfully achieved using the procedure described in Examples 1, 2 and 3 below:.

Microtitre wells with p57 or p58 were prepared as follows. The biotinylated probe was diluted in to a concentration of 2. The control reaction shows very few fluorescent spots, since the sequence of the flanking regions on the template do not correspond to the primer sequences grafted onto the well.

Referring now to FIGS. Here two different immobilised primers are used to provide primer extension. The embodiment shown in FIGS. The possibility of annealing occurring between both ends of an immobilised complement to a target molecule can also be avoided.

Several different concentrations of PCR template have been tested approximately 1, 0. These objects have an irregular shape, are 20 to micro-meters in size and have a thickness much larger than the field depth of the observation.

They present a circular shape, they are 1 to 5 micro meters in size and do not span the field of view. The number of spots depends on the concentration of the template used for initiating colony formation.

From the observed size of the colonies, one can estimate that more than 10, distinct colonies can be arrayed within 1 mm 2 of support. S1 and 52 are base pair and bp fragments, respectively, which have been cloned into pBlueScript Skminus plasmids and subsequently amplified through a PCR using P1 and P2 as primers.

A base pair fragment corresponding to the central sequence of the S1 fragment, but not including the Pl or P2 sequence was amplified by PCR as previously described. The biotinylated probes were hybridized to the samples in EasyHyb buffer Boehringer-Mannheim, Germany, using the following temperature scheme in the PTC thermocycler: The processing consisted in inversion and linear contrast enhancement, in order to provide a picture suitable for black and white print-out on a laser printer.

It can be seen from FIG. Amplified, single stranded nucleic acid molecules present in colonies provided by the present invention can themselves be used as templates to synthesise additional nucleic acid strands.

Colonies will usually comprise both a given nucleic acid strand and its complement in immobilised form FIG. Thus they can be used to provide additional copies not only of a given nucleic acid strand but also of its complement.

One way of doing this is to provide one or more primers primers TTA and TGG in solution that anneal to amplified, immobilised nucleic acid strands present in colonies FIG. These primers may be the same as primers initially used to provide the immobilised colonies, apart from being provided in free rather than immobilised form.

Primer extension, using AmpliTaq DNA polymerase and the four deoxyribonucleoside triphosphates labeled or unlabeled can then be used to synthesise complementary strands to immobilised nucleic acid strands or at least to parts thereof step iii.

Once newly formed strands FIG. The process can then be repeated if desired using the PCR reaction, to provide large number of such strands in solution FIG. Strands synthesised in this manner, after separation from the immobilised strands, can, if desired, be annealed to one another i.

Alternatively they can be separated from one another to provide homogenous populations of single-stranded nucleic acid molecules in solution. It should also be noted that once single-stranded molecules are provided in solution they can be used as templates for PCR or reverse PCR.

Therefore it is not essential to continue to use the immobilised nucleic acid strands to obtain further amplification of given strands or complementary strands thereto. It should be noted that where a plurality of colonies are provided and nucleic acid strands in different colonies have different sequences, it is possible to select only certain colonies for use as templates in the synthesis of additional nucleic acid molecules.

This can be done by using primers for primer extension that are specific for molecules present in selected colonies. Alternatively primers can be provided to allow several or all of the colonies to be used as templates.

Such primers may be a mixture of many different primers e. Sl and S2 are base pair and b. The temperature control was performed in a PTC thermo-cycler. Reactions were stopped by rinsing the wells with TNT buffer.

PCR 25 cycles, 30 sec. P70 and P71 are suited for the amplification of both S1 and S2, since primer P70 contains the sequence of primer P1 and p7l contains P2.

The pictures of the gels are presented in FIGS. It is also possible to modify initially formed colonies to provide different colonies i. As a starting point, the primary colony FIG. A single-strand specific DNA exonuclease, might be used to remove all primers which have not been elongated.

Secondly and independently, the DNA molecules forming the colonies can be cleaved by using endonucleases. If desired, the enzymatically cleaved colony FIG. In any case, the secondary primers are available after denaturation e.

An important case is when the exonuclease digests only a few bases of the DNA molecule before being released in solution, and when digestion can proceed when another enzyme binds to the DNA molecule FIG.

In this case the exonuclease digestion will proceed until there remain only single stranded molecules which, on average, are half the length of the starting material, and are without any complementary parts which could form partial duplexes remaining in the single stranded molecules in a colony FIG.

In all cases, these treatments result in single-stranded fragments grafted onto a support which correspond to the sequence of the original template and that can be used for new DNA colony growing if an appropriate new template is provided for colony initiation FIG.

Templates useful for secondary colony growing may include molecules having known sequences or complements of such sequences. Alternatively templates may be derived from unsequenced molecules e.

In either event the templates should be provided with one or more regions for annealing with nucleic acid strands present in the primary colonies. Following a denaturing step ii, reannealing step iii and DNA polymerase step iv cycle, a replica of the original primary colony will be formed FIG.

The maximum size of a secondary colony provided by this embodiment of the present invention is restricted by the size of the primary colony onto which it grows. Several secondary growing processes can be used sequentially to create colonies for specific applications i.

The same procedure could be applied to a support covered with colonies or secondary primers as described in section E. Different immobilised primers are shown present in different regions of the support represented by squares.

Apparatuses of the present invention can be used for various procedures some of which will be described later on. Various preparation procedures are described below:. Here is described a method to prepare DNA originating from one biological sample or from a plurality of samples for amplification in the case where it is not necessary to keep track of the origin of the DNA when it is incorporated within a colony.

This can be done e. In order to standardise experimental conditions, the extracted and cut DNA fragments can be size-fractionated, e. Fragments obtained within a single fraction can be used in providing templates in order to reduce the variability in size of the templates.

Alternatively, the template DNA fragments can be inserted into a biological vector at a site that is flanked by the sequence of the primers that are grafted on the support.

This cloned DNA can be amplified within a biological host and extracted. Obviously, if one is working with a single primer grafted to the solid support for DNA colony formation, purifying fragments containing both PI and P2 primers does not pose a problem.

Hereafter, the DNA fragments obtained after such a suitable process are designated by the expression: Here it is described how to prepare DNA originating from a plurality of biological samples in the case where it is necessary to keep track of the origin of the DNA when it is incorporated within a colony.

The procedure is the same as that described in the previous section except that in this case, the oligonucleotide linkers used to tail the randomly cut genomic DNA fragments are now made of two parts; the sequence of the primers grafted onto the surface P1 and P2, FIG.

Note that for each sample, the tag may not be unique, but a plurality of tags could be used. This tagging procedure can be used for providing colonies carrying a means of identification which is independent from the sequence carried by the template itself.

This could also be useful when some colonies are to be recovered specifically using the procedure given in section D. This could also be useful in the case the recovered colonies are further processed, e.

The DNA of interest can first be extracted from a biological sample by any means known by those skilled in the art as mentioned supra. Hereafter, we will designate the DNA fragments obtained after such a suitable process by the expression: Note that for each sample, a plurality of tags might be used, as in ii supra.

Potential uses of tags are the same as in ii, supra. The procedure is similar to the procedures described for preparing DNA fragments in the previous sections except that the starting point is to extract mRNA by any means known to those skilled in the art e.

Certainly, the tags and primers described supra can be used in conjunction with the process of double-stranded cDNA synthesis to allow their incorporation into the templates.

Hereafter, we will designate the mRNA fragments obtained after such suitable processes by the expressions: In assay procedures of the present invention labels may be used to provide detectable signals.

Labels for use in the present invention are preferably attached. Staining agents can be used in the present invention. With certain staining agents the result can be observed with a suitable fluorescence imaging apparatus.

DNA colonies are first prepared for hybridisation. Then they are hybridised with a probe labelled or unlabelled. If required, the hybridised probed is assayed, and the result is observed.

This can be done with an apparatus of the present invention e. In a preferred embodiment of the present invention colonies are treated with a DNA restriction endonuclease which is specific either for a sequence provided by a double stranded form of one of the primers originally grafted onto the surface where colonies are formed or for another sequence present in a template DNA molecule see e.

After restriction enzyme digestion, the colonies can be heated to a temperature high enough for double stranded DNA molecules to be separated. After this thermal denaturing step, the colonies can be washed to remove the non-hybridised, detached single-stranded DNA strands, leaving a remaining attached single-strand DNA.

A further alternative is simply to heat denature DNA in the colonies. Single-stranded nucleic acid probes labelled or unlabelled can be hybridised to single-stranded DNA in colonies at the appropriate temperature and buffer conditions which depends on the sequence of each probe, and can be determined using protocols known to those skilled in the art.

A hybridised probe provided initially in unlabelled form can be used as a primer for the incorporation of the different or a subset of the different labelled or a mix of labelled and unlabelled deoxyribonucleoside triphosphates with a DNA polymerase.

The incorporated labelled nucleotides can then be detected as described supra. Firstly, the DNA colonies can be prepared for hybridisation by the methods described supra. Then they can be hybridised with a probe labelled or initially unlabelled.

If required, hybridised labelled probes are assayed and the result is observed with an apparatus as described previously. The probe may then be removed by heat denaturing and a probe specific for a second DNA sequence may be hybridised and detected.

These steps maybe repeated with new probes as many times as desired. Secondly, the probes can be assayed as described supra for unlabelled probes, except that only a subset preferably 1 only of the different labelled or unlabelled nucleotides are used at each cycle.

The colonies can then be assayed for monitoring the incorporation of the nucleotides. This second process can be repeated until a sequence of a desired length has been determined. DNA colonies can be generated from templates and primers, such that a RNA polymerase promoter sequence is positioned at one end of the double-stranded DNA in the colony.

The detection can be done non-specifically e. In another embodiment of the present invention, colonies can be analysed in order to determine sequences of nucleic acid molecules which form the colonies.

Since very large numbers of the same nucleic acid molecules can be provided within each colony the reliability of the sequencing data obtained is likely to be very high. The sequences determined may be full or partial.

Sequences can be determined for nucleic acids present in one or more colonies. A plurality of sequences may be determined at the same time. In some embodiments the sequence of a complementary strand to a nucleic acid strand to be sequenced or of a part thereof may be obtained initially.

However this sequence can be converted using base-pairing rules to provide the desired sequence or a part thereof. This conversion can be done via a computer or via a person. It can be done after each step of primer extension or can be done at a later stage.

Sequencing can be done by various methods. For example methods relying on sequential restriction endonuclease digestion and linker ligation can be used. This method comprises the steps of: However in a preferred method of the present invention, amplified nucleic acid molecules preferably in the form of colonies, as disclosed herein are sequenced by allowing primers to hybridise with the nucleic acid molecules, extending the primers and detecting the nucleotides used in primer extension.

Preferably, after extending a primer by a single nucleotide, the nucleotide is detected before a further nucleotide is used in primer extension step-by-step sequencing. One or more of the nucleotides used in primer extension may be labelled.

The use of labelled nucleotides during primer extension facilitates detection. Preferably the label is not present in naturally-occurring nucleotides. Ideally, labels are non-radioactive, such as fluorophores.

However radioactive labels can be used. Where nucleotides are provided in labelled form the labels may be the same for different nucleotides. If the same label is used each nucleotide incorporation can be used to provide a cumulative increase of the same signal e.

Alternatively different labels may be used for each type of nucleotide which may be detected at different wavelengths. In some embodiments of the present invention a mixture of labelled and unlabelled nucleotides may be provided, as will be described in greater detail later on.

In a preferred embodiment of the present invention the sequencing of nucleic acid molecules present in at least 2 different colonies is performed simultaneously. More preferably, sequencing of nucleic acid molecules present in over 10, over, over or even over 1,, different colonies is performed simultaneously.

Thus if colonies having different nucleic acids molecules are provided, many different sequences full or partial can be determined simultaneously—i. If desired, controls may be provided, whereby a plurality of colonies comprising the same nucleic acid molecules are provided.

By 50 determining whether or not the same sequences are obtained for nucleic acid molecules in these colonies it can be ascertained whether or not the sequencing procedure is reliable.

One sequencing method of the present invention is illustrated in FIG. In the example outlined in FIG. After several repetitions of the addition of single deoxyribonucleoside triphosphates, it will be possible to determine any sequence.

For example sequences of at least 10, at least 20, at least 50 or at least bases may be determined. If colonies are provided initially in a form comprising doublestranded molecules the colonies can be processed to provide single-stranded molecules for use in sequencing as described above.

It should however be noted that double stranded molecules can be used for sequencing without such processing. For example a double stranded DNA molecule can be provided with a promoter sequence and step-bystep sequencing can then be performed using an RNA polymerase and labeled ribonucleotides cf FIG.

Another alternative is for a nick to be introduced in a double stranded DNA molecule so that nick translation can be performed using labeled deoxyribonucleotides.

One way of processing double-stranded molecules present in colonies to provide single-stranded colonies as described later with reference to FIG. Here double-stranded immobilised molecules present in a colony which may be in the form of bridge-like structures are cleaved and this is followed by a denaturing step.

Alternatively a denaturing step could be used initially and could be followed by a cleavage step. Preferably cleavage is carried out enzymatically. However other means of cleavage are possible, such as chemical cleavage.

An appropriate cleavage site can be provided in said molecule. Denaturing can be performed by any suitable means. Once single-stranded molecules to be sequenced are provided, suitable primers for primer extension can be hybridised thereto.

Oligonucleotides are preferred as primers. These are nucleic acid molecules that are typically 6 to 60, e. However other molecules, e. The primers for use in sequencing preferably hybridise to the same sequences present in amplified nucleic acid molecules as do primers that were used to provide said amplified nucleic acids.

When primers are provided in solution and are annealed hybridised to nucleic acid molecules present in colonies to be sequenced, those primers which remain in solution or which do not anneal specifically can be removed after annealing.

Preferred annealing conditions temperature and buffer composition prevent non-specific hybridisation. These may be stringent conditions. Such conditions would typically be annealing temperatures close to a primer’s Tm melting temperature at a given salt concentration e.

Stringent conditions for a given system can be determined by a skilled person. They will depend on the base composition, GC content, the length of the primer used and the salt concentration.

Primers used for primer extension need not be provided in solution, since they can be provided in immobilised form. In this embodiment the primers should be provided in the vicinity of the immobilised molecules to which they are to be annealed.

Such primers may indeed already be present as excess immobilised primers that were not used in amplifying nucleic acid molecules during the formation of colonies. For example it can be synthesised artificially and can be added to a given molecule using a ligase.

Once a nucleic acid molecule annealed to a primer is provided, primer extension can be performed. DNA polymerases are however the enzymes of choice for preferred embodiments. Several of these are commercially available.

However it is not essential to use such polymerases. Any nucleotides may be used for primer extension reactions whether naturally occurring or normaturally occurring. A washing step is preferably incorporated after each primer extension step in order to remove unincorporated nucleotides that may interfere with subsequent steps.

The preferred washing solution should be compatible with polymerase activity and have a salt concentration that does not interfere with the annealing of primer molecules to the nucleic acid molecules to be sequenced.

In less preferred embodiments, the washing solution may interfere with polymerase activity. Here the washing solution would need to be removed before further primer extension.

Considering that many copies of molecules to be sequenced can be provided in a given colony, a combination of labelled and nonlabelled nucleotides can be used. In this case, even if a small proportion of the nucleotides are labelled e.

Thus in a further embodiment of the present invention there is provided a method for sequencing nucleic acid molecules present in a colony of the present invention, the method comprising the steps of:.

Steps b and c may be repeated one or more times. Preferably a plurality of different colonies are provided and several different sequences are determined simultaneously.

This further embodiment of the present invention can be used to reduce costs, since relatively few labelled nucleotides are needed. It can also be used to reduce quenching effects.

It is however also possible to use only labelled nucleotides for primer extension or to use a major portion thereof e. This can be done for example if labels are selected so as to prevent or reduce quenching effects.

Alternatively labels may be removed or neutralised at various stages should quenching effects become problematic e. However this can increase the number of steps required and it is therefore preferred that labels are not removed or at least that they are not removed after each nucleotide has been incorporated but are only removed periodically.

In other less preferred embodiments, the primer itself and its extension product may be removed and replaced with another primer. If required, several steps of sequential label-free nucleotide additions may be performed before actual sequencing in the presence of labelled nucleotides is resumed.

A further alternative is to use a different type of label from that used initially e. In preferred embodiments of the present invention a plurality of labelled bases are incorporated into an extended primer during sequencing.

This is advantageous in that it can speed up the sequencing procedure relative to methods in which, once a labelled base has been incorporated into an extended primer, the label must be removed before a further labelled base can be incorporated.

The plurality of labelled bases may be in the form of one or more contiguous stretches, although this is not essential. The present invention therefore also includes within its scope a method for sequencing nucleic acid molecules, comprising the steps of:.

Preferably the sequences of the nucleic acid molecules present at said first and said locations are different from one another—i. In view of the foregoing description it will be appreciated that a large number of different sequencing methods using colonies of the present invention can be used.

Various detection systems can be used to detect labels used in sequencing in these methods although in certain embodiments detection may be possible simply by eye, 50 that no detection system is needed.

A preferred detection system for fluorescent labels is a Charge-Coupled-Device CCD camera, which can optionally be coupled to a magnifying device. Any other device allowing detection and, preferably, also quantification of fluorescence on a surface may be used.

Devices such as fluorescent imagers or confocal microscopes may be chosen. In less preferred embodiments, the labels may be radioactive and a radioactivity detection device would then be required. Ideally such devices would be real-time radioactivity imaging systems.

Also less preferred are other devices relying on phosphor screens Moleculal Dynamics or autoradiography films for detection. Depending on the number of colonies to be monitored, a scanning system may be preferred for data collection.

Although an alternative is to provide a plurality of detectors to enable all colonies to be covered. Such a system allows a detector to move relative to a plurality of colonies to be analysed.

This is useful when all the colonies providing signals are not within the field of view of a detector. The detector may be maintained in a fixed position and colonies to be analysed may be moved into the field of view of the detector e.

Alternatively the colonies may be maintained in fixed position and the detection device may be moved to bring them into its field of view. The detection system is preferably used in combination with an analysis system in order to determine the number and preferably also the nature of bases incorporated by primer extension at each colony after each step.

This analysis may be performed immediately after each step or later on, using recorded data. The sequence of nucleic acid molecules present within a given colony can then be deduced from the number and type of nucleotides added after each step.

Preferably the detection system is part of an apparatus comprising other components. The present invention includes an apparatus comprising a plurality of labelled nucleotides, a nucleic acid polymerase and detection means for detecting labelled nucleotides when incorporated into a nucleic acid molecule by primer extension, the detection means being adapted to distinguish between signals provided by labelled nucleotides incorporated at different colonies.

The apparatus may also include temperature control, solvent delivery and washing means. It may be automated. Methods of apparatuses within the scope of the present invention can be used in the sequencing of:.

Both de novo sequencing and re-sequencing are discussed in greater detail later on see the following sections v and vi. For de novo sequencing applications, the order of nucleotides applied to a given location can be chosen as desired.

Generally a single order of four nucleotides would be repeated, although this is not essential. For re-sequencing applications, the order of nucleotides to be added at each step is preferably chosen according to a known sequence.

Re-sequencing may be of particular interest for the analysis of a large number of similar template molecules in order to detect and identify sequence differences e. Differences from a given sequence can be detected by the lack of incorporation of one or more nucleotides present in the given sequence at particular stages of primer extension.

In contrast to most commonly used techniques, the present method allows for detection of any type of mutation such as point mutations, insertions or deletions. Furthermore, not only known existing mutations, but also previously unidentified mutations can be characterised by the provision of sequence information.

In some embodiments of the present invention long nucleic acid molecules may have to be sequenced by several sequencing reactions, each one allowing for determination of part of the complete sequence.

These reactions may be carried out at different colonies where the different colonies are each provided with the same nucleic acid molecules to be sequenced but different primers, or in successive cycles applied at the same colony where between each cycles the primers and extension products are washed off and replaced by different primers.

This embodiment of the present invention aims to solve the problem of screening a large population for the identification of given features of given genes, such as the detection of single nucleotide polymorphisms.

In one preferred embodiment, it consists in generating tagged genomic DNA see section G ii supra. Thus each sample originating from a given individual sample has been labelled with a unique tag.

This tagged DNA can be used for generating primary colonies on an appropriate surface comprising immobilised primers. Several successive probe hybridisation assays to the colonies can then be performed.

Between each assay the preceding probe can be removed, e. Advantages of this embodiment of the present invention over other approaches for solving this problem are illustrated in the following example of a potential practical application.

It is intended to detect which part of a gene of, e. In order to obtain a representative array of sample, one might want to array randomly, colonies i. This is a much smaller surface than any other technology available at present time e.

The amount of reactants a great part of the cost will be proportional to the surface occupied by of the array of samples. Thus the present invention can provide an fold improvement over the presently available technology.

Thus, assuming that the bottleneck of the method is the time required to image the result of the assay, it takes of the order of 10 minutes to image the result of an assay on 50, samples, colonies.

This represents a 20 times improvement compared to the best method known at present time HySeq claims 30 days to achieve a comparable task. Improvements colony densities 10 times higher and imaging time of 1 second could allow for much higher throughput and finally the ultimately expected throughput could be about times faster than the best, not yet fully demonstrated, technology available at present time.

Another advantage of using the present invention lies in the fact that it overcomes the problem arising with individuals who have heterozygous mutations for a given gene.

While this problem may be addressed by existing sequencing methods to determine allelic polymorphisms, current high throughput mutation detection methods based on oligonucleotide probe hybridisation may lead to difficulties in the interpretation of results due to an unequal hybridisation of probes in cases of allelic polymorphisms and therefore errors can occur.

Version ditel 0121 micromax bit

Since enzymes are proteins, there is no possibility that dangerous chemical residue will be incorporated in the meat products, as happens with some antibiotics and anti-coccidiosis chemicals.

In one embodiment, the enzyme in question cleaves a linkage that effects release of a cell-surface protein. In another preferred embodiment, the enzyme is selected from the group consisting of esterases, cerebrosidases, and carbohydrases that cleave a linkage that effects release of a cell-surface protein or carbohydrate.

Alternatively, the enzyme is obtained by expressing the recombinant DNA coding for the enzyme in Bacillus megaterium. Thus, the composition can be an animal feed that contains no other anti-infection agent other than the enzyme.

The animal feed composition of the present invention further comprises grain material, such as corn, sorghum, wheat, barley or oats, a source of protein, such as beans or peas, and vitamins, amino acids, and minerals.

In addition, the method does not include administering an anti-infection agent other than the enzyme itself. The infection may be affected by a protozoan, such as Eimeria and Cryptosporidium, bacterial, such as Clostridium, fungal or yeast pathogen.

The detailed description and specific examples, while indicating preferred embodiments, are given for illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Further, the examples demonstrate the principle of the invention and cannot be expected to specifically illustrate the application of this invention to all the examples of infections where it will be obviously useful to those skilled in the prior art.

The enzyme class includes but is not limited to sphingpmyelinases and phospholipases of type C and D, and enzymes of like cleavage specificity. Exemplary of this class, therefore, are enzymes that cleave and release glycoproteins or carbohydrates that are membrane-anchored via linkage to phosphatidylinositol.

Thus, the enzyme phosphatidylinositol specific phospholipase C E. Low and Prasad, Proc. PI-PLC has been described from prokaryotic sources, including extracellular production by bacteria.

See Hendrickson et al. See Low, supra; Low and Saltiel, Science For example, the variant surface glycoproteins and other surface proteins and carbohydrates of several protozoan parasites are anchored by glycosyl phosphatidylinositol lipids GPI anchors and are sensitive to PI-PLC digestion and release.

The presence of GPI anchors in Giardia lamblia, considered a very primitive eukaryote, suggests that this kind of anchor evolved early in the eukaryotes. Consistent with this understanding is the discovery, in the archaebacteria, of a new phosphoglycerolipid GlcNalmyo-inositol-P-dialkylglycerol Nishihara et al.

In protozoa, the GPI anchorage system is used more heavily than in higher eukaryotes, and there is evidence that GPI-anchored structures are important for parasite survival in insect and mammalian hosts McConville and Ferguson, supra.

For example, the frequent shedding of variant surface glycoproteins may be a mechanism to avoid immune system attack. These are thought to be important for membrane attachment and subsequent infection.

See Gumett et al. The in vivo treatment of parasites with PI-PLC, if proven feasible, likely would help the host immune system and interfere with attachment and infection by pathogens entering the digestive tract.

Eimeria species are a widespread and costly problem for the poultry industry. Another protozoan parasite, Cryptosporidium parvum, is widespread and causes acute diarrheal disease in humans and many animals.

See Malaviya et al. See Lanzrein et al. In addition, Clostridium septicum alpha toxin and Aeromonas hydrophila aerolysin are both attached to the cell surface by means of a C-terminal GPI-anchor and can be removed from the cell surface by treatment with PI-PLC.

See Gordon et al. Also, the FimH binding to mast cells triggers an inflammation response. Thus, via this mechanism of decreasing inflammation and the underlying secretion of tumor necrosis factor, a phospholipase treatment, according to the present invention, should relieve symptoms characterizing conditions such as irritable bowel syndrome, colitis, and Crohn’s Disease.

See Mizutani et al. Conversely, pretreatment of cultured chick embryo fibroblasts with phospholipase C, isolated from Clostridium perfringens, markedly inhibited subsequent infection of the cells by Semliki Forest Virus.

See Friedman and Pastan, Proc. See Jacobs et al. There are several examples of host cell GPI-anchored proteins that are the binding sites for viruses. See Clarkson et al.

See Barbis and Parrish, Brazilian J. These include structures such as cholesterol esters Rostand and Esko, J. To this end, a key aspect of the present invention is the demonstration that an enzyme, which not only is active in cleaving cell surface components, can be administered orally as an anti-infection agent and be effective in vivo.

Notably, while a representative suitable enzyme, PI-PLC, has been available since the s, this approach has not been suggested heretofore. Feed preparations that contain an endo-1,4-D-mannanase are known, and some reports have proposed an antifuigal activity for mannanase.

In these instances, mannanase is combined with a recognized antibiotic, although the prospect of enzyme use in antibiotic-free feed has been discussed generally.

Adams, Feed Mix Special, at pages Further, the present invention contemplates a composition that contains such an enzyme, including a mannanase, but that contains no other anti-infection agent.

This result is unexpected because B. For example, see Stein and Logan, J. It is surprising, therefore, that an extracellular enzyme from a pathogen which causes gastroenteritis would have a curative effect, pursuant to the present invention, in relation to a disease caused by a bacterial infection.

In the aforementioned enzyme preparation, an extracellular phosphatidylinositol-specific phospholipase C [E. Enzyme treatment of the present invention worked effectively as a coccidiostat and antibiotic.

Therefore, it is an effective and commercially viable approach for the treatment of digestive tract infections particularly as currently used substances are banned. Surprisingly, addition of the PI-PLC to the animal feed without coating results in effective treatment of pathogenic infections.

Dried, shelf-stable formulations of enzymes that are suitable, pursuant to the present invention, for incorporation in tablets or capsules, for example, can be prepared by freeze-drying, spray drying in fluidized bed dryer with inert or carbohydrate carrier, or by using evaporative techniques in conjunction with glass-forming stabilizers.

See Franks et al. Another approach involves salt precipitation, for example, ammonium sulfate precipitate or solvent precipitate, as with acetone for powder formation, followed by drying and blending with a carrier.

Exemplary carbohydrates for use as carriers are xylose, fructose, glucose, sorbitol and maltotriose, among others, as described by Franks, supra. Choice of a carbohydrate carrier is based on compatibility with the enzyme, low hydroscopic tendency, and a favorable glass transition curve.

The stabilizer trehalose is particularly suitable for producing ambient temperature-stable biologics. Such solutions typically are sterile-filtered, prior to pharmaceutical use.

Feeds are composed mainly of grain material, a protein source, vitamins, amino acids, and minerals. The grain material typically includes corn, sorghum, wheat, barley, or oats. The source of proteins can be beans or peas, for example.

Exemplary minerals, amino acids and vitamins include B 12 , A, pantothenic acid, niacin, riboflavin, K, DL-methionine, L-lysine, choline chloride, folic acid, dicalcium phosphate, magnesium sulfonate, potassium sulfate, calcium carbonate, sodium chloride, sodium selenite, manganous oxide, calcium iodate, copper oxide, zinc oxide, and D-activated animal sterol.

Other preservatives for feed such as sodium benzoate, propylparaben, sodium or potassium sorbate, and ascorbyl palmitate are examples of approved chemical preservatives that can alsobe used to prevent potential spoilage by microbial growth in the product.

These preservatives can be applied to feeds by post-pelleting with a large dilution by automated spraying technology. See Fodge et al. Such liquid preparations may contain stabilizing carbohydrates such as sorbitol or glycerol, if compatible.

Materials that are desired components of feed, such as other enzymes or vitamins that are heat-labile, may be included for increased efficiency. Such dry enzyme concentrates are prepared by first concentrating the liquid enzyme preparation, using a 10 Kd NMWC or other suitable ultra-filter, to achieve a high percentage of enzyme content, and then by blending with a very dry carrier, such as corn grits, soy grits or even an inert material or insoluble salt that is approved for use in feeds.

It is well known that modifying protein structure, primarily through changing the encoding DNA sequence or, secondarily, through chemical modification can render enzymes more stable against inactivation.

One illustration in this regard is the use of chemical cross-linking of enzyme crystals. See Collins et al. Another approach to increasing the stability of enzyme for the present invention entails changing amino acids by mutagenesis of the gene that codes for the enzyme of interest, or obtaining genes or parts of genes for shuffling.

See Crameri et al. For example, see Giver et al. Thus, known protocols could be employed in this regard to make modified enzyme, for testing, according to the examples, to gauge suitability in the inventive treatment methodology.

The expression system Rygus and Hillen, Appl. Strains of this type, with amplified expression, provide a feasible means for producing commercially useful amounts of PI-PLC, to incorporate into animal feed in accordance with the present invention.

See Kamogashira et al. In the examples detailed below, the possibility of involving these two antibiotics was eliminated by excluding possible low molecular-weight antibiotics from the enzyme preparation.

The concentrate, containing protein larger than 10 Kd, was used for further processing. The small molecular-weight antibiotics, such as those described by Handelsman, supra, would pass through this filter.

Moreover, ammonium sulfate precipitation was employed to precipitate high molecular weight proteins, leaving low molecular weight materials in solution. After the ammonium sulfate precipitate was re-dissolved, the resultant enzyme solution was dialyzed against buffer, yet another treatment that removes low molecular-weight antibiotics.

Finally, the protein was precipitated a second time, again with ammonium sulfate, to relegate any remaining, low molecular-weight compounds to the solution. The fermentation broth also was tested, with an E.

When the culture density reached OD reading of 1. The initial batch volume was 9. The initial pH was adjusted to 7. The OD of the initial culture was 0.

At six hours, the fermentation was stopped with final OD of The fermentations were run without pH control and the final pH was 8. In this case the seed cultures were used at six hours with OD of 2.

The main fermentations were run for 6. Final pH was 8. Chilled broth was pumped through the filters with a peristaltic pump at about 2 liters per minute with recycling back to the holding reservoir.

The permeate containing the enzyme was collected in a reservoir chilled on ice. The initial cell-containing broth volume of about 9 liters was concentrated down to about 2 liters at which point diafiltation was started with 10 mMTris-HCl, pH 8.

After a total volume of about 14 liters of permeate was collected, the cell washing was terminated. The same pumping method with recycle of concentrate was used except the permeate was discarded.

Final concentrate with a volume of about mL from each fermentor was saved for the next processing step. The supernatant was discarded, and the precipitate was dissolved in a minimal volume of 10 mMTris-HCl, 0.

Table 1 below summarizes the assay data and predicts the approximate purity and total enzyme on a pure basis obtained for each of the four preparations. Ammonium sulfate g was added slowly and the solution was mixed on ice for several minutes.

The solution was centrifuged to collect the precipitate. The pellet fraction was dissolved in a minimal amount of 20 mM phosphate buffer pH 7. The protein concentration was measured at approximately See Kuppe et al.

The shake flask fermentation was used to evaluate the phosphatidylinositol specific phospholipase C production in Bacillus megaterium. At OD of about 0.

After three hours, supernatant was harvested by centrifugation. The phosphatidylinositol-specific phospholipase C activity was measured by a fluorescent substrate method Hendrickson, et al.

The pH was adjusted to 7. After growth to about 1. Two flasks were used to seed a L fermentor filled with L of the same steam sterilized medium. Tetracycline was sterile filtered 0.

The operating conditions for the first fermentor seed stage were as follows: The pH was controlled at 6. D reached, the contents were used to seed a L fermentor containing L of the same medium.

The second rate was held until all The fermentation was terminated at 20 hours. A test for the presence of antibiotic was conducted with E. The test was conducted as a. See Brantner, Pharmazie 52 1: No clearing zone indicating antibiotic activity was observed around the cylinders containing the enzyme samples.

The substrate 4-methylumbelliferyl-myo-inositolphosphate, N-methyl-morpholine salt was obtained from Biosynth Naperville, Ill. Enzyme dilutions if needed are made into 0.

The reaction at pH 8. Also, at pH 9. For the purpose of efficacy testing in animal feeding experiments, the units measured using this assay were converted to the equivalent Hendrickson Unit to facilitate comparison with the first tests before this assay was used.

The enzyme was extracted from feed test materials by weighing 4 g feed and adding it to 20 mL of 10 mM Tris-Cl pH 7. Appropriate dilutions of the extracts were made in 0.

Sample reactions should be foil-covered to protect the substrate from light. Finally, samples were centrifuged at 12, RPM in a microcentrifuge for 5 minutes. Background control levels were subtracted.

A rate of fluorescence units production per minute was calculated. Fluorescense units were converted to micromoles of reaction product and enzyme units extracted per original pound of feed was calculated.

Broiler Chicken Feeding Trial I. Water and feed were provided ad libitum throughout the 21 day test period. A randomized block design was used to allocate chicks to cages and cages to treatment groups.

All cages, feeders and waterers were sanitized prior to the beginning of the test. The surface will usually be planar, although this is not essential.

The present invention also provides an apparatus for providing a surface comprising colonies of the immobilised nucleic acid molecules discussed supra. Such an apparatus can include one or more of the following:.

Other apparatuses are within the scope of the present invention. These allow immobilised nucleic acids produced via the method of the present invention to be analysed.

They can include a source of reactants and detecting means for detecting a signal that may be generated once one or more reactants have been applied to the immobilised nucleic acid molecules.

They may also be provided with a surface comprising immobilised nucleic acid molecules in the form of colonies, as described supra. Desirably the means for detecting a signal has sufficient resolution to enable it to distinguish between signals generated from different colonies.

Apparatuses of the present invention of whatever nature are preferably provided in automated form so that once they are activated, individual process steps can be repeated automatically.

The present invention will now be described without limitation thereof in sections A to I below with reference to the accompanying drawings. It should be appreciated that procedures using DNA molecules referred to in these sections are applicable mutatis mutandis to RNA molecules, unless the context indicates otherwise.

It also illustrates the annealing, elongation and denaturing steps that are used to provide such colonies. Referring now to FIG. Each primer 1 is attached to the surface by a linkage indicated by a dark block.

This may be a covalent or a non-covalent linkage but should be sufficiently strong to keep a primer in place on the surface. In practice however longer sequences would generally be provided.

Between the two ends any sequence to be amplified or the complement of any sequence to be amplified can be provided. When primer extension is complete, as shown in FIG. The target molecule can then be separated from the extended immobilised strand e.

This separation step frees the extended, immobilised strand so that it can then be used to initiate a subsequent round of primer extension, as shown in FIGS. Primer extension is shown occurring in FIG.

Each of these strands can then themselves be used as templates in further rounds of primer extension initiated from new primers 3 and 4 , as shown in FIGS. Four single stranded, immobilised strands can be provided after two rounds of amplification followed by a strand separation step e.

Two of these have sequences corresponding to the sequence of the target molecule originally used as a template. The other two have sequences complementary to the sequence of the target molecule originally used as a template.

In practice a given immobilised strand and its immobilised complement may anneal once. It will therefore be appreciated that a given sequence and its complement can be provided in equal numbers in immobilised form and can be substantially homogeneously distributed within a colony.

Further rounds of amplification beyond those shown in FIG. Only a single template need be used to initiate each colony, although, if desired, a template can be reused to initiate several colonies.

It will be appreciated that the present invention allows very high densities of immobilised extended nucleic acid molecules to be provided. Within a colony each extended immobilised molecule will be located at a surface within one molecule length of another extended immobilised molecule.

Thus position 3 shown in FIG. A flat plate is shown schematically in plan view having primers immobilised thereon in a square grid pattern the primers are indicated by small dots. A regular grid is used solely for simplicity: At the position indicated by arrow X a template molecule has annealed to a primer and an initial bout of primer extension has occurred to provide an immobilised, extended nucleic acid strand.

Following strand separation, an end of that strand becomes free to anneal to further primers so that additional immobilised, extended nucleic acid strands can be produced.

This is shown having occurred sequentially at positions indicated by the letter Y. For simplicity, the primer chosen for annealing is positioned next to the primer carrying the nucleic acid strand: However, this primer will obviously be within a distance equal to the length of the nucleic acid strand.

It will be appreciated that annealing at only one rather than at all of these positions is required for colony cell growth to occur. After immobilised, extended, single-stranded nucleic acid molecules have been provided at the positions indicated by letter Y, the resultant molecules can themselves anneal to other primers and the process can be continued to provide a colony comprising a large number of immobilised nucleic acid molecules in a relatively small area.

It also depicts the typical observations that can be made, as can be seen on the examples shown in FIGS. The simultaneous amplification and immobilisation of nucleic acids using solid phase primers has been successfully achieved using the procedure described in Examples 1, 2 and 3 below:.

Microtitre wells with p57 or p58 were prepared as follows. The biotinylated probe was diluted in to a concentration of 2. The control reaction shows very few fluorescent spots, since the sequence of the flanking regions on the template do not correspond to the primer sequences grafted onto the well.

Referring now to FIGS. Here two different immobilised primers are used to provide primer extension. The embodiment shown in FIGS. The possibility of annealing occurring between both ends of an immobilised complement to a target molecule can also be avoided.

Several different concentrations of PCR template have been tested approximately 1, 0. These objects have an irregular shape, are 20 to micro-meters in size and have a thickness much larger than the field depth of the observation.

They present a circular shape, they are 1 to 5 micro meters in size and do not span the field of view. The number of spots depends on the concentration of the template used for initiating colony formation.

From the observed size of the colonies, one can estimate that more than 10, distinct colonies can be arrayed within 1 mm 2 of support. S1 and 52 are base pair and bp fragments, respectively, which have been cloned into pBlueScript Skminus plasmids and subsequently amplified through a PCR using P1 and P2 as primers.

A base pair fragment corresponding to the central sequence of the S1 fragment, but not including the Pl or P2 sequence was amplified by PCR as previously described. The biotinylated probes were hybridized to the samples in EasyHyb buffer Boehringer-Mannheim, Germany, using the following temperature scheme in the PTC thermocycler: The processing consisted in inversion and linear contrast enhancement, in order to provide a picture suitable for black and white print-out on a laser printer.

It can be seen from FIG. Amplified, single stranded nucleic acid molecules present in colonies provided by the present invention can themselves be used as templates to synthesise additional nucleic acid strands.

Colonies will usually comprise both a given nucleic acid strand and its complement in immobilised form FIG. Thus they can be used to provide additional copies not only of a given nucleic acid strand but also of its complement.

One way of doing this is to provide one or more primers primers TTA and TGG in solution that anneal to amplified, immobilised nucleic acid strands present in colonies FIG. These primers may be the same as primers initially used to provide the immobilised colonies, apart from being provided in free rather than immobilised form.

Primer extension, using AmpliTaq DNA polymerase and the four deoxyribonucleoside triphosphates labeled or unlabeled can then be used to synthesise complementary strands to immobilised nucleic acid strands or at least to parts thereof step iii.

Once newly formed strands FIG. The process can then be repeated if desired using the PCR reaction, to provide large number of such strands in solution FIG. Strands synthesised in this manner, after separation from the immobilised strands, can, if desired, be annealed to one another i.

Alternatively they can be separated from one another to provide homogenous populations of single-stranded nucleic acid molecules in solution. It should also be noted that once single-stranded molecules are provided in solution they can be used as templates for PCR or reverse PCR.

Therefore it is not essential to continue to use the immobilised nucleic acid strands to obtain further amplification of given strands or complementary strands thereto.

It should be noted that where a plurality of colonies are provided and nucleic acid strands in different colonies have different sequences, it is possible to select only certain colonies for use as templates in the synthesis of additional nucleic acid molecules.

This can be done by using primers for primer extension that are specific for molecules present in selected colonies. Alternatively primers can be provided to allow several or all of the colonies to be used as templates.

Such primers may be a mixture of many different primers e. Sl and S2 are base pair and b. The temperature control was performed in a PTC thermo-cycler. Reactions were stopped by rinsing the wells with TNT buffer.

PCR 25 cycles, 30 sec. P70 and P71 are suited for the amplification of both S1 and S2, since primer P70 contains the sequence of primer P1 and p7l contains P2. The pictures of the gels are presented in FIGS.

It is also possible to modify initially formed colonies to provide different colonies i. As a starting point, the primary colony FIG. A single-strand specific DNA exonuclease, might be used to remove all primers which have not been elongated.

Secondly and independently, the DNA molecules forming the colonies can be cleaved by using endonucleases. If desired, the enzymatically cleaved colony FIG. In any case, the secondary primers are available after denaturation e.

An important case is when the exonuclease digests only a few bases of the DNA molecule before being released in solution, and when digestion can proceed when another enzyme binds to the DNA molecule FIG.

In this case the exonuclease digestion will proceed until there remain only single stranded molecules which, on average, are half the length of the starting material, and are without any complementary parts which could form partial duplexes remaining in the single stranded molecules in a colony FIG.

In all cases, these treatments result in single-stranded fragments grafted onto a support which correspond to the sequence of the original template and that can be used for new DNA colony growing if an appropriate new template is provided for colony initiation FIG.

Templates useful for secondary colony growing may include molecules having known sequences or complements of such sequences. Alternatively templates may be derived from unsequenced molecules e.

In either event the templates should be provided with one or more regions for annealing with nucleic acid strands present in the primary colonies. Following a denaturing step ii, reannealing step iii and DNA polymerase step iv cycle, a replica of the original primary colony will be formed FIG.

The maximum size of a secondary colony provided by this embodiment of the present invention is restricted by the size of the primary colony onto which it grows. Several secondary growing processes can be used sequentially to create colonies for specific applications i.

The same procedure could be applied to a support covered with colonies or secondary primers as described in section E. Different immobilised primers are shown present in different regions of the support represented by squares.

Apparatuses of the present invention can be used for various procedures some of which will be described later on. Various preparation procedures are described below:. Here is described a method to prepare DNA originating from one biological sample or from a plurality of samples for amplification in the case where it is not necessary to keep track of the origin of the DNA when it is incorporated within a colony.

This can be done e. In order to standardise experimental conditions, the extracted and cut DNA fragments can be size-fractionated, e. Fragments obtained within a single fraction can be used in providing templates in order to reduce the variability in size of the templates.

Alternatively, the template DNA fragments can be inserted into a biological vector at a site that is flanked by the sequence of the primers that are grafted on the support. This cloned DNA can be amplified within a biological host and extracted.

Obviously, if one is working with a single primer grafted to the solid support for DNA colony formation, purifying fragments containing both PI and P2 primers does not pose a problem.

Hereafter, the DNA fragments obtained after such a suitable process are designated by the expression: Here it is described how to prepare DNA originating from a plurality of biological samples in the case where it is necessary to keep track of the origin of the DNA when it is incorporated within a colony.

The procedure is the same as that described in the previous section except that in this case, the oligonucleotide linkers used to tail the randomly cut genomic DNA fragments are now made of two parts; the sequence of the primers grafted onto the surface P1 and P2, FIG.

Note that for each sample, the tag may not be unique, but a plurality of tags could be used. This tagging procedure can be used for providing colonies carrying a means of identification which is independent from the sequence carried by the template itself.

This could also be useful when some colonies are to be recovered specifically using the procedure given in section D. This could also be useful in the case the recovered colonies are further processed, e.

The DNA of interest can first be extracted from a biological sample by any means known by those skilled in the art as mentioned supra. Hereafter, we will designate the DNA fragments obtained after such a suitable process by the expression: Note that for each sample, a plurality of tags might be used, as in ii supra.

Potential uses of tags are the same as in ii, supra. The procedure is similar to the procedures described for preparing DNA fragments in the previous sections except that the starting point is to extract mRNA by any means known to those skilled in the art e.

Certainly, the tags and primers described supra can be used in conjunction with the process of double-stranded cDNA synthesis to allow their incorporation into the templates.

Hereafter, we will designate the mRNA fragments obtained after such suitable processes by the expressions: In assay procedures of the present invention labels may be used to provide detectable signals.

Labels for use in the present invention are preferably attached. Staining agents can be used in the present invention. With certain staining agents the result can be observed with a suitable fluorescence imaging apparatus.

DNA colonies are first prepared for hybridisation. Then they are hybridised with a probe labelled or unlabelled. If required, the hybridised probed is assayed, and the result is observed.

This can be done with an apparatus of the present invention e. In a preferred embodiment of the present invention colonies are treated with a DNA restriction endonuclease which is specific either for a sequence provided by a double stranded form of one of the primers originally grafted onto the surface where colonies are formed or for another sequence present in a template DNA molecule see e.

After restriction enzyme digestion, the colonies can be heated to a temperature high enough for double stranded DNA molecules to be separated. After this thermal denaturing step, the colonies can be washed to remove the non-hybridised, detached single-stranded DNA strands, leaving a remaining attached single-strand DNA.

A further alternative is simply to heat denature DNA in the colonies. Single-stranded nucleic acid probes labelled or unlabelled can be hybridised to single-stranded DNA in colonies at the appropriate temperature and buffer conditions which depends on the sequence of each probe, and can be determined using protocols known to those skilled in the art.

A hybridised probe provided initially in unlabelled form can be used as a primer for the incorporation of the different or a subset of the different labelled or a mix of labelled and unlabelled deoxyribonucleoside triphosphates with a DNA polymerase.

The incorporated labelled nucleotides can then be detected as described supra. Firstly, the DNA colonies can be prepared for hybridisation by the methods described supra. Then they can be hybridised with a probe labelled or initially unlabelled.

If required, hybridised labelled probes are assayed and the result is observed with an apparatus as described previously. The probe may then be removed by heat denaturing and a probe specific for a second DNA sequence may be hybridised and detected.

These steps maybe repeated with new probes as many times as desired. Secondly, the probes can be assayed as described supra for unlabelled probes, except that only a subset preferably 1 only of the different labelled or unlabelled nucleotides are used at each cycle.

The colonies can then be assayed for monitoring the incorporation of the nucleotides. This second process can be repeated until a sequence of a desired length has been determined. DNA colonies can be generated from templates and primers, such that a RNA polymerase promoter sequence is positioned at one end of the double-stranded DNA in the colony.

The detection can be done non-specifically e. In another embodiment of the present invention, colonies can be analysed in order to determine sequences of nucleic acid molecules which form the colonies.

Since very large numbers of the same nucleic acid molecules can be provided within each colony the reliability of the sequencing data obtained is likely to be very high. The sequences determined may be full or partial.

Sequences can be determined for nucleic acids present in one or more colonies. A plurality of sequences may be determined at the same time. In some embodiments the sequence of a complementary strand to a nucleic acid strand to be sequenced or of a part thereof may be obtained initially.

However this sequence can be converted using base-pairing rules to provide the desired sequence or a part thereof. This conversion can be done via a computer or via a person. It can be done after each step of primer extension or can be done at a later stage.

Sequencing can be done by various methods. For example methods relying on sequential restriction endonuclease digestion and linker ligation can be used. This method comprises the steps of: However in a preferred method of the present invention, amplified nucleic acid molecules preferably in the form of colonies, as disclosed herein are sequenced by allowing primers to hybridise with the nucleic acid molecules, extending the primers and detecting the nucleotides used in primer extension.

Preferably, after extending a primer by a single nucleotide, the nucleotide is detected before a further nucleotide is used in primer extension step-by-step sequencing. One or more of the nucleotides used in primer extension may be labelled.

The use of labelled nucleotides during primer extension facilitates detection. Preferably the label is not present in naturally-occurring nucleotides. Ideally, labels are non-radioactive, such as fluorophores.

However radioactive labels can be used. Where nucleotides are provided in labelled form the labels may be the same for different nucleotides. If the same label is used each nucleotide incorporation can be used to provide a cumulative increase of the same signal e.

Alternatively different labels may be used for each type of nucleotide which may be detected at different wavelengths. In some embodiments of the present invention a mixture of labelled and unlabelled nucleotides may be provided, as will be described in greater detail later on.

In a preferred embodiment of the present invention the sequencing of nucleic acid molecules present in at least 2 different colonies is performed simultaneously.

More preferably, sequencing of nucleic acid molecules present in over 10, over, over or even over 1,, different colonies is performed simultaneously. Thus if colonies having different nucleic acids molecules are provided, many different sequences full or partial can be determined simultaneously—i.

If desired, controls may be provided, whereby a plurality of colonies comprising the same nucleic acid molecules are provided. By 50 determining whether or not the same sequences are obtained for nucleic acid molecules in these colonies it can be ascertained whether or not the sequencing procedure is reliable.

One sequencing method of the present invention is illustrated in FIG. In the example outlined in FIG. After several repetitions of the addition of single deoxyribonucleoside triphosphates, it will be possible to determine any sequence.

For example sequences of at least 10, at least 20, at least 50 or at least bases may be determined. If colonies are provided initially in a form comprising doublestranded molecules the colonies can be processed to provide single-stranded molecules for use in sequencing as described above.

It should however be noted that double stranded molecules can be used for sequencing without such processing. For example a double stranded DNA molecule can be provided with a promoter sequence and step-bystep sequencing can then be performed using an RNA polymerase and labeled ribonucleotides cf FIG.

Another alternative is for a nick to be introduced in a double stranded DNA molecule so that nick translation can be performed using labeled deoxyribonucleotides. One way of processing double-stranded molecules present in colonies to provide single-stranded colonies as described later with reference to FIG.

Here double-stranded immobilised molecules present in a colony which may be in the form of bridge-like structures are cleaved and this is followed by a denaturing step. Alternatively a denaturing step could be used initially and could be followed by a cleavage step.

Preferably cleavage is carried out enzymatically. However other means of cleavage are possible, such as chemical cleavage. An appropriate cleavage site can be provided in said molecule.

Denaturing can be performed by any suitable means. Once single-stranded molecules to be sequenced are provided, suitable primers for primer extension can be hybridised thereto.

Oligonucleotides are preferred as primers. These are nucleic acid molecules that are typically 6 to 60, e. However other molecules, e. The primers for use in sequencing preferably hybridise to the same sequences present in amplified nucleic acid molecules as do primers that were used to provide said amplified nucleic acids.

When primers are provided in solution and are annealed hybridised to nucleic acid molecules present in colonies to be sequenced, those primers which remain in solution or which do not anneal specifically can be removed after annealing.

Preferred annealing conditions temperature and buffer composition prevent non-specific hybridisation. These may be stringent conditions. Such conditions would typically be annealing temperatures close to a primer’s Tm melting temperature at a given salt concentration e.

Stringent conditions for a given system can be determined by a skilled person. They will depend on the base composition, GC content, the length of the primer used and the salt concentration.

Primers used for primer extension need not be provided in solution, since they can be provided in immobilised form. In this embodiment the primers should be provided in the vicinity of the immobilised molecules to which they are to be annealed.

Such primers may indeed already be present as excess immobilised primers that were not used in amplifying nucleic acid molecules during the formation of colonies. For example it can be synthesised artificially and can be added to a given molecule using a ligase.

Once a nucleic acid molecule annealed to a primer is provided, primer extension can be performed. DNA polymerases are however the enzymes of choice for preferred embodiments.

Several of these are commercially available. However it is not essential to use such polymerases. Any nucleotides may be used for primer extension reactions whether naturally occurring or normaturally occurring.

A washing step is preferably incorporated after each primer extension step in order to remove unincorporated nucleotides that may interfere with subsequent steps. The preferred washing solution should be compatible with polymerase activity and have a salt concentration that does not interfere with the annealing of primer molecules to the nucleic acid molecules to be sequenced.

In less preferred embodiments, the washing solution may interfere with polymerase activity. Here the washing solution would need to be removed before further primer extension.

Considering that many copies of molecules to be sequenced can be provided in a given colony, a combination of labelled and nonlabelled nucleotides can be used. In this case, even if a small proportion of the nucleotides are labelled e.

Thus in a further embodiment of the present invention there is provided a method for sequencing nucleic acid molecules present in a colony of the present invention, the method comprising the steps of:.

Steps b and c may be repeated one or more times. Preferably a plurality of different colonies are provided and several different sequences are determined simultaneously.

This further embodiment of the present invention can be used to reduce costs, since relatively few labelled nucleotides are needed. It can also be used to reduce quenching effects. It is however also possible to use only labelled nucleotides for primer extension or to use a major portion thereof e.

This can be done for example if labels are selected so as to prevent or reduce quenching effects. Alternatively labels may be removed or neutralised at various stages should quenching effects become problematic e.

However this can increase the number of steps required and it is therefore preferred that labels are not removed or at least that they are not removed after each nucleotide has been incorporated but are only removed periodically.

In other less preferred embodiments, the primer itself and its extension product may be removed and replaced with another primer. If required, several steps of sequential label-free nucleotide additions may be performed before actual sequencing in the presence of labelled nucleotides is resumed.

A further alternative is to use a different type of label from that used initially e. In preferred embodiments of the present invention a plurality of labelled bases are incorporated into an extended primer during sequencing.

This is advantageous in that it can speed up the sequencing procedure relative to methods in which, once a labelled base has been incorporated into an extended primer, the label must be removed before a further labelled base can be incorporated.

The plurality of labelled bases may be in the form of one or more contiguous stretches, although this is not essential. The present invention therefore also includes within its scope a method for sequencing nucleic acid molecules, comprising the steps of:.

Preferably the sequences of the nucleic acid molecules present at said first and said locations are different from one another—i. In view of the foregoing description it will be appreciated that a large number of different sequencing methods using colonies of the present invention can be used.

Various detection systems can be used to detect labels used in sequencing in these methods although in certain embodiments detection may be possible simply by eye, 50 that no detection system is needed.

A preferred detection system for fluorescent labels is a Charge-Coupled-Device CCD camera, which can optionally be coupled to a magnifying device. Any other device allowing detection and, preferably, also quantification of fluorescence on a surface may be used.

Devices such as fluorescent imagers or confocal microscopes may be chosen. In less preferred embodiments, the labels may be radioactive and a radioactivity detection device would then be required.

Ideally such devices would be real-time radioactivity imaging systems. Also less preferred are other devices relying on phosphor screens Moleculal Dynamics or autoradiography films for detection.

Depending on the number of colonies to be monitored, a scanning system may be preferred for data collection. Although an alternative is to provide a plurality of detectors to enable all colonies to be covered.

Such a system allows a detector to move relative to a plurality of colonies to be analysed. This is useful when all the colonies providing signals are not within the field of view of a detector.

The detector may be maintained in a fixed position and colonies to be analysed may be moved into the field of view of the detector e. Alternatively the colonies may be maintained in fixed position and the detection device may be moved to bring them into its field of view.

The detection system is preferably used in combination with an analysis system in order to determine the number and preferably also the nature of bases incorporated by primer extension at each colony after each step.

This analysis may be performed immediately after each step or later on, using recorded data. The sequence of nucleic acid molecules present within a given colony can then be deduced from the number and type of nucleotides added after each step.

Preferably the detection system is part of an apparatus comprising other components. The present invention includes an apparatus comprising a plurality of labelled nucleotides, a nucleic acid polymerase and detection means for detecting labelled nucleotides when incorporated into a nucleic acid molecule by primer extension, the detection means being adapted to distinguish between signals provided by labelled nucleotides incorporated at different colonies.

The apparatus may also include temperature control, solvent delivery and washing means. It may be automated. Methods of apparatuses within the scope of the present invention can be used in the sequencing of:.

Both de novo sequencing and re-sequencing are discussed in greater detail later on see the following sections v and vi. For de novo sequencing applications, the order of nucleotides applied to a given location can be chosen as desired.

Generally a single order of four nucleotides would be repeated, although this is not essential. For re-sequencing applications, the order of nucleotides to be added at each step is preferably chosen according to a known sequence.

Re-sequencing may be of particular interest for the analysis of a large number of similar template molecules in order to detect and identify sequence differences e.

Differences from a given sequence can be detected by the lack of incorporation of one or more nucleotides present in the given sequence at particular stages of primer extension.

In contrast to most commonly used techniques, the present method allows for detection of any type of mutation such as point mutations, insertions or deletions. Furthermore, not only known existing mutations, but also previously unidentified mutations can be characterised by the provision of sequence information.

In some embodiments of the present invention long nucleic acid molecules may have to be sequenced by several sequencing reactions, each one allowing for determination of part of the complete sequence.

These reactions may be carried out at different colonies where the different colonies are each provided with the same nucleic acid molecules to be sequenced but different primers, or in successive cycles applied at the same colony where between each cycles the primers and extension products are washed off and replaced by different primers.

This embodiment of the present invention aims to solve the problem of screening a large population for the identification of given features of given genes, such as the detection of single nucleotide polymorphisms.

In one preferred embodiment, it consists in generating tagged genomic DNA see section G ii supra. Thus each sample originating from a given individual sample has been labelled with a unique tag.

This tagged DNA can be used for generating primary colonies on an appropriate surface comprising immobilised primers. Several successive probe hybridisation assays to the colonies can then be performed.

Between each assay the preceding probe can be removed, e. Advantages of this embodiment of the present invention over other approaches for solving this problem are illustrated in the following example of a potential practical application.

It is intended to detect which part of a gene of, e. In order to obtain a representative array of sample, one might want to array randomly, colonies i. This is a much smaller surface than any other technology available at present time e.

The amount of reactants a great part of the cost will be proportional to the surface occupied by of the array of samples. Thus the present invention can provide an fold improvement over the presently available technology.

Thus, assuming that the bottleneck of the method is the time required to image the result of the assay, it takes of the order of 10 minutes to image the result of an assay on 50, samples, colonies.

This represents a 20 times improvement compared to the best method known at present time HySeq claims 30 days to achieve a comparable task. Improvements colony densities 10 times higher and imaging time of 1 second could allow for much higher throughput and finally the ultimately expected throughput could be about times faster than the best, not yet fully demonstrated, technology available at present time.

Another advantage of using the present invention lies in the fact that it overcomes the problem arising with individuals who have heterozygous mutations for a given gene. While this problem may be addressed by existing sequencing methods to determine allelic polymorphisms, current high throughput mutation detection methods based on oligonucleotide probe hybridisation may lead to difficulties in the interpretation of results due to an unequal hybridisation of probes in cases of allelic polymorphisms and therefore errors can occur.

In this embodiment of the present invention, each colony arises from a single copy of an amplified gene of interest. If an average of 10 colonies are generated for each individual locus, there will be an average of 5 colonies corresponding to one version of a gene and 5 colonies corresponding to the other version of the gene.

Thus heterozygotic mutations can be scored by the number of times a single allele is detected per individual genome sample. This embodiment of the present invention provides a solution to the problem of identifying and characterising novel allelic polymorphisms within known genes in a large population of biological samples.

In its preferred embodiment it consists in obtaining tagged DNA each sample originating from a given individual has been tagged with a unique tag—see section G iv. This encoded DNA can then be used for generating primary colonies on an appropriate surface comprising immobilised primers.

Several successive assays of probe hybridisation to the colonies can then be performed wherein between each cyclic assay the preceding probe can be removed by thermal denaturation and washing. The advantages of the present invention over other approaches for solving this problem are illustrated in the following example of potential practical application:.

It is desired to identify the variability of the sequence of a gene of, e. It is assumed that a reference sequence of the gene is known. For each individual, a PCR amplification can be performed to specifically amplify the gene of interest and link a tag and a colony generating primer.

In order to obtain a representative array of sample, one might want to array randomly 40 colonies i. If it is assumed that the bottleneck of the method is the time to image the result of the assay, it takes of the order of 15 minutes to image the result of an assay on 4 samples 40 colonies.

This can be compared to the most powerful systems operational at the present time. In this embodiment of the present invention with conservative assumptions colony density, imaging time, size of the CCD chip, a throughput of 3.

This embodiment of the present invention aims to solve the problem of sequencing novel genomes or parts thereof with low cost and in short time, where the sequence of the DNA is not known.

The DNA colonies can then be digested with a rare-cutting restriction enzyme, whose site is included in the linker, denatured and sequenced. These colonies are then digested with the corresponding restriction endonuclease and denatured to remove the non-attached DNA strand V, FIG.

Incorporation and detection of labeled nucleotides can then be carried out as previously described see section I iii, Methods of Sequencing. In this embodiment, the throughput obtainable can be at least times higher than presently available methods.

This embodiment of our invention means to solve the problem of monitoring the expression of a large number of genes simultaneously. Its preferred embodiment is depicted in FIG.

Firstly, primary colonies are prepared, as depicted in FIG. Secondly, the colonies are treated to turn then into supports i. At this stage FIG. Thirdly, step ii in FIG. If the template is mRNA, the priming step of colony regeneration will be performed with a reverse transcriptase.

After a given number of colony amplification cycles, preferably 1 to 50, the situation will be as depicted in FIG. Lastly, step iii in FIG. The relative levels of expression of the genes can be obtained by the following preferred methods:.

Firstly levels of expression can be monitored by following the rate of regeneration of the colonies i.

See Gordon et al. Also, the FimH binding to mast cells triggers an inflammation response. Thus, via this mechanism of decreasing inflammation and the underlying secretion of tumor necrosis factor, a phospholipase treatment, according to the present invention, should relieve symptoms characterizing conditions such as irritable bowel syndrome, colitis, and Crohn’s Disease.

See Mizutani et al. Conversely, pretreatment of cultured chick embryo fibroblasts with phospholipase C, isolated from Clostridium perfringens, markedly inhibited subsequent infection of the cells by Semliki Forest Virus.

See Friedman and Pastan, Proc. See Jacobs et al. There are several examples of host cell GPI-anchored proteins that are the binding sites for viruses. See Clarkson et al.

See Barbis and Parrish, Brazilian J. These include structures such as cholesterol esters Rostand and Esko, J. To this end, a key aspect of the present invention is the demonstration that an enzyme, which not only is active in cleaving cell surface components, can be administered orally as an anti-infection agent and be effective in vivo.

Notably, while a representative suitable enzyme, PI-PLC, has been available since the s, this approach has not been suggested heretofore. Feed preparations that contain an endo-1,4-D-mannanase are known, and some reports have proposed an antifuigal activity for mannanase.

In these instances, mannanase is combined with a recognized antibiotic, although the prospect of enzyme use in antibiotic-free feed has been discussed generally.

Adams, Feed Mix Special, at pages Further, the present invention contemplates a composition that contains such an enzyme, including a mannanase, but that contains no other anti-infection agent.

This result is unexpected because B. For example, see Stein and Logan, J. It is surprising, therefore, that an extracellular enzyme from a pathogen which causes gastroenteritis would have a curative effect, pursuant to the present invention, in relation to a disease caused by a bacterial infection.

In the aforementioned enzyme preparation, an extracellular phosphatidylinositol-specific phospholipase C [E. Enzyme treatment of the present invention worked effectively as a coccidiostat and antibiotic.

Therefore, it is an effective and commercially viable approach for the treatment of digestive tract infections particularly as currently used substances are banned.

Surprisingly, addition of the PI-PLC to the animal feed without coating results in effective treatment of pathogenic infections. Dried, shelf-stable formulations of enzymes that are suitable, pursuant to the present invention, for incorporation in tablets or capsules, for example, can be prepared by freeze-drying, spray drying in fluidized bed dryer with inert or carbohydrate carrier, or by using evaporative techniques in conjunction with glass-forming stabilizers.

See Franks et al. Another approach involves salt precipitation, for example, ammonium sulfate precipitate or solvent precipitate, as with acetone for powder formation, followed by drying and blending with a carrier.

Exemplary carbohydrates for use as carriers are xylose, fructose, glucose, sorbitol and maltotriose, among others, as described by Franks, supra. Choice of a carbohydrate carrier is based on compatibility with the enzyme, low hydroscopic tendency, and a favorable glass transition curve.

The stabilizer trehalose is particularly suitable for producing ambient temperature-stable biologics. Such solutions typically are sterile-filtered, prior to pharmaceutical use. Feeds are composed mainly of grain material, a protein source, vitamins, amino acids, and minerals.

The grain material typically includes corn, sorghum, wheat, barley, or oats. The source of proteins can be beans or peas, for example. Exemplary minerals, amino acids and vitamins include B 12 , A, pantothenic acid, niacin, riboflavin, K, DL-methionine, L-lysine, choline chloride, folic acid, dicalcium phosphate, magnesium sulfonate, potassium sulfate, calcium carbonate, sodium chloride, sodium selenite, manganous oxide, calcium iodate, copper oxide, zinc oxide, and D-activated animal sterol.

Other preservatives for feed such as sodium benzoate, propylparaben, sodium or potassium sorbate, and ascorbyl palmitate are examples of approved chemical preservatives that can alsobe used to prevent potential spoilage by microbial growth in the product.

These preservatives can be applied to feeds by post-pelleting with a large dilution by automated spraying technology. See Fodge et al. Such liquid preparations may contain stabilizing carbohydrates such as sorbitol or glycerol, if compatible.

Materials that are desired components of feed, such as other enzymes or vitamins that are heat-labile, may be included for increased efficiency. Such dry enzyme concentrates are prepared by first concentrating the liquid enzyme preparation, using a 10 Kd NMWC or other suitable ultra-filter, to achieve a high percentage of enzyme content, and then by blending with a very dry carrier, such as corn grits, soy grits or even an inert material or insoluble salt that is approved for use in feeds.

It is well known that modifying protein structure, primarily through changing the encoding DNA sequence or, secondarily, through chemical modification can render enzymes more stable against inactivation.

One illustration in this regard is the use of chemical cross-linking of enzyme crystals. See Collins et al. Another approach to increasing the stability of enzyme for the present invention entails changing amino acids by mutagenesis of the gene that codes for the enzyme of interest, or obtaining genes or parts of genes for shuffling.

See Crameri et al. For example, see Giver et al. Thus, known protocols could be employed in this regard to make modified enzyme, for testing, according to the examples, to gauge suitability in the inventive treatment methodology.

The expression system Rygus and Hillen, Appl. Strains of this type, with amplified expression, provide a feasible means for producing commercially useful amounts of PI-PLC, to incorporate into animal feed in accordance with the present invention.

See Kamogashira et al. In the examples detailed below, the possibility of involving these two antibiotics was eliminated by excluding possible low molecular-weight antibiotics from the enzyme preparation.

The concentrate, containing protein larger than 10 Kd, was used for further processing. The small molecular-weight antibiotics, such as those described by Handelsman, supra, would pass through this filter.

Moreover, ammonium sulfate precipitation was employed to precipitate high molecular weight proteins, leaving low molecular weight materials in solution. After the ammonium sulfate precipitate was re-dissolved, the resultant enzyme solution was dialyzed against buffer, yet another treatment that removes low molecular-weight antibiotics.

Finally, the protein was precipitated a second time, again with ammonium sulfate, to relegate any remaining, low molecular-weight compounds to the solution. The fermentation broth also was tested, with an E.

When the culture density reached OD reading of 1. The initial batch volume was 9. The initial pH was adjusted to 7. The OD of the initial culture was 0. At six hours, the fermentation was stopped with final OD of The fermentations were run without pH control and the final pH was 8.

In this case the seed cultures were used at six hours with OD of 2. The main fermentations were run for 6. Final pH was 8. Chilled broth was pumped through the filters with a peristaltic pump at about 2 liters per minute with recycling back to the holding reservoir.

The permeate containing the enzyme was collected in a reservoir chilled on ice. The initial cell-containing broth volume of about 9 liters was concentrated down to about 2 liters at which point diafiltation was started with 10 mMTris-HCl, pH 8.

After a total volume of about 14 liters of permeate was collected, the cell washing was terminated. The same pumping method with recycle of concentrate was used except the permeate was discarded.

Final concentrate with a volume of about mL from each fermentor was saved for the next processing step. The supernatant was discarded, and the precipitate was dissolved in a minimal volume of 10 mMTris-HCl, 0.

Table 1 below summarizes the assay data and predicts the approximate purity and total enzyme on a pure basis obtained for each of the four preparations. Ammonium sulfate g was added slowly and the solution was mixed on ice for several minutes.

The solution was centrifuged to collect the precipitate. The pellet fraction was dissolved in a minimal amount of 20 mM phosphate buffer pH 7. The protein concentration was measured at approximately See Kuppe et al.

The shake flask fermentation was used to evaluate the phosphatidylinositol specific phospholipase C production in Bacillus megaterium. At OD of about 0. After three hours, supernatant was harvested by centrifugation.

The phosphatidylinositol-specific phospholipase C activity was measured by a fluorescent substrate method Hendrickson, et al. The pH was adjusted to 7. After growth to about 1. Two flasks were used to seed a L fermentor filled with L of the same steam sterilized medium.

Tetracycline was sterile filtered 0. The operating conditions for the first fermentor seed stage were as follows: The pH was controlled at 6. D reached, the contents were used to seed a L fermentor containing L of the same medium.

The second rate was held until all The fermentation was terminated at 20 hours. A test for the presence of antibiotic was conducted with E. The test was conducted as a. See Brantner, Pharmazie 52 1: No clearing zone indicating antibiotic activity was observed around the cylinders containing the enzyme samples.

The substrate 4-methylumbelliferyl-myo-inositolphosphate, N-methyl-morpholine salt was obtained from Biosynth Naperville, Ill. Enzyme dilutions if needed are made into 0. The reaction at pH 8. Also, at pH 9.

For the purpose of efficacy testing in animal feeding experiments, the units measured using this assay were converted to the equivalent Hendrickson Unit to facilitate comparison with the first tests before this assay was used.

The enzyme was extracted from feed test materials by weighing 4 g feed and adding it to 20 mL of 10 mM Tris-Cl pH 7. Appropriate dilutions of the extracts were made in 0. Sample reactions should be foil-covered to protect the substrate from light.

Finally, samples were centrifuged at 12, RPM in a microcentrifuge for 5 minutes. Background control levels were subtracted. A rate of fluorescence units production per minute was calculated. Fluorescense units were converted to micromoles of reaction product and enzyme units extracted per original pound of feed was calculated.

Broiler Chicken Feeding Trial I. Water and feed were provided ad libitum throughout the 21 day test period. A randomized block design was used to allocate chicks to cages and cages to treatment groups.

All cages, feeders and waterers were sanitized prior to the beginning of the test. Lighting was continuous 24 hour per day with incandescent lamps. Body weights were determined at day one and day.

Feed consumption was measured at day On day 7, all birds were further infected with, Clostridium perfringens through the water supply. In the negative control T1 there was no treatment for infection.

In the positive control T2 a coccidiostat and antibiotic were added to the feed. The study was done in randomized battery cages, on a blinded basis, to test the effect of PI-PLC made from the natural source, Bacillus cereus wild type PI-PLC, or a recombinant Bacillus megaterium on male broiler performance reared to 21 days of age.

The natural source also contains other extracellular enzymes but the PI-PLC prepared from Bacillus megaterium is highly purified and was further purified by ultrafiltration using a Kd NMWC membrane.

Birds were challenged at 8 days of age with Avian coccidia, E. Each of the nine treatments Table 6 had 10 replications or cages. Dead birds, if present, were not replaced after the 8 th day.

Feed was fed in mash form on an ad libitum basis throughout the entire trial test period day 0 to day Thereafler, nine treated diets, in mash form, were fed from days of age.

Birds were challenged at 8 days of age with avian coccidia 75, E acervulina oocysts and 1, E. Dead birds were removed from the cages when they were detected and were not replaced. Feed was fed in MASH form on an ad libitum basis throughout the entire trial test period day 0 to day In the case of E.

The results are shown in Table 9. Birds were challenged at 8 days of age with coccidia 75, E acervulina oocysts and 1, E. Each of the treatments had 8 replications or cages. Birds were not replaced.

Feed and water was fed ad libitutim throughout the entire trial test period Days 0 to Day Diets were fed in MASH from days of age. Importantly, this study shows that either the PI-PLC or mannanase when combined with salinomycin, but without the antibiotic BMD Tests 6 and 5 restores the performance to basically the level of the uninfected tests No.

This provides evidence for antibacterial function. For cell pretreatment, cell cultures were overlaid With dilutions of enzymes as described in Table 11, and examined for morphological changes From 5 to 45 min post overlay.

After 45 minutes, the monolayer cultures were washed twice, then inoculated with untreated E. For application during infection, sporozoites were suspended in the appropriate dilution of the enzyme and inoculated immediately into the cell cultures.

After 45 minutes incubation, cultures were fixed, stained, and the invasion was quantified. At the enzyme levels used in these experiments, no morphological change was noted.

Both a given strand and its complementary strand can be provided in amplified form in a single colony. Colonies of any particular size can be provided. Desirably a majority of the colonies present on a surface i.

Colonies can be arranged in a predetermined manner or can be randomly arranged. Two or three dimensional colony configurations are possible. The configurations may be regular e.

Colonies can be provided at high densities. Where many different nucleic acid molecules are to be amplified, many different primers may be provided. Universal primers can be used where the molecules to be amplified comprise first and second sequences, as described previously.

Synthetic oligodeoxynucleotide primers are available commercially from many suppliers e. Microsynth, Switzerland, Eurogentech, Belgium. Grafting of primers onto silanized glass or quartz and grafting of primers onto silicon wafers or gold surface has been described Maskos, U.

M Southern, Oligonucleotide hybridizations on glass supports: Direct-detection of nucleic-acid hybridization on the surface of a charge-coupled-device, Nucleic Acids Research 22 Grafting biotinylated primers to supports covered with streptavidin is another alternative.

This grafting method is commonly used for bio-macromolecules in general. Non-covalent grafting of primers at the interface between an aqueous phase and a hydrophobic phase through an hydrophobic anchor is also possible for the present invention.

Such anchoring is commonly used for bio-macromolecules in general S. Protein binding to supported lipid membranes, Langmuir 9, Preferred forms of such interfaces would be liposomes, lipidic vesicles, emulsions, patterned bilayers, Langmuir or Langmuir-Blodgett films.

The patterns may be obtained by directed pattering on templates, e. Grier, Like charge attractions in metastable colloidal crystallites, Nature, In the above methods, one, two or more different primers can be grafted onto a surface.

The primers can be grafted homogeneously and simultaneously over the surface. Using microlithographic methods it is possible to provide immobilised primers in a controlled manner.

Comparison of methods for photochemical phosphoramidite-based DNA-synthesis. Journal Of Organic Chemistry 60 These may be provided in distinct areas that may correspond in configuration to colonies to be formed, e.

Within each area, only a single type of primer oligonucleotide need be provided. Alternatively a mixture comprising a plurality of different primers may be provided. In either case, primers can be homogeneously distributed within each area.

They may be provided in the form of a regular array. Where areas initially comprise only one type of immobilised primer they may be modified, if desired, to carry two or more different types of primer.

By providing a mixture of templates with different sequences from one another, primer extension of one type of primer using the mixture of such templates followed by strand separation will result in different modified primers.

One, two or more different types of extended primer can be provided in this manner at any area where primary primers are initially located. Substantially equal portions of different templates can be used, if desired, in order to provide substantially equal proportions of different types of immobilised extended primer over a given area.

If different proportions of different immobilised extended primers are desired, then this can be achieved by adjusting the proportions of different template molecules initially used accordingly.

A restriction endonuclease cleavage site may be located within the primer. A primer may also be provided with a restriction endonuclease recognition site which directs DNA cleavage several bases distant Type II restriction endonucleases.

In any event, restriction endonucleases can be useful in allowing an immobilised nucleic acid molecule within a colony to be cleaved so as to release at least a part thereof.

As an alternative to using other restriction endonucleases, ribozymes can be used to release at least parts of nucleic acid molecules from a surface when such molecules are RNA molecules.

Other methods are possible. For example if a covalent bond is used to link a primer to a surface this bond may be broken e. Primers for use in the present invention are preferably at least five bases long.

Normally they will be less than or less than 50 bases long. However this is not essential. A target molecule when in single-stranded form comprises a first part having a sequence that can anneal with a first primer and a second part having a sequence complementary to a sequence that can anneal with a second primer.

In a preferred embodiment the second part has the same sequence as the second primer. The second primer may have a sequence that is the same as, or different from, the sequence of the first primer.

The target molecule will usually also comprise a third part located between the first and second parts. This part of the molecule comprises a particular sequence to be replicated. It may be derived from random fractionation by mechanical means or by limited restriction enzyme digestion of a nucleic acid sample, for example.

Further parts of the target molecules may be provided if desired. For example parts designed to act as tags may be provided. Whatever parts are present, target nucleic acid molecules can be provided by techniques known to those skilled in the art of nucleic acid manipulation.

For example, two or more parts can be joined together by ligation. If necessary, prior to ligation appropriate modifications can be made to provide molecules in a form ready for ligation.

For example if blunt end ligation is desired then a single-strand specific exonuclease such as Sl nuclease could be used to remove single stranded portions of molecules prior to ligation. Techniques useful for nucleic acid manipulation are disclosed in Sambrook et al, Molecular Cloning, 2 nd Edition, Cold Spring Harbor Laboratory Press, for example.

Once a template molecule has been synthesised it can be cloned into a vector and can be amplified in a suitable host before being used in the present invention. It may alternatively be amplified by PCR.

As a further alternative, batches of template molecules can be synthesised chemically using automated DNA synthesisers e. It is however important to note that the present invention allows large numbers of nucleic acid molecules identical in sequence to be provided in a colony arising from a single molecule of template.

Furthermore, the template can be re-used to generate further colonies. Thus it is not essential to provide large numbers of template molecules to be used in colony formation.

The template can be of any desired length provided that it can participate in the method of the present invention. Preferably it is at least 10, more preferably at least 20 bases long.

More preferably it is at least or at least bases long. Turning now to reaction conditions suitable for the method of the present invention, it will be appreciated that the present invention uses repeated steps of annealing of primers to templates, primer extension and separation of extended primers from templates.

These steps can generally be performed using reagents and conditions known to those skilled in PCR or reverse transcriptase plus PCR techniques. Excess deoxyribonucleoside triphosphates are desirably provided.

Preferred deoxyribonucleoside triphosphates are abbreviated; dTTP deoxythymidine nucleoside triphosphate, dATP deoxyadenosine nucleoside triphosphate, dCTP deoxycytosine nucleoside triphosphate and dGTP deoxyguanosine nucleoside triphosphate.

However alternatives are possible. These may be naturally or non-naturally occurring. A buffer of the type generally used in PCR reactions may also be provided. A nucleic acid polymerase used to incorporate nucleotides during primer extension is preferably stable under the pertaining reaction conditions in order that it can be used several times.

This is particularly useful in automated amplification procedures. Thus, where heating is used to separate a newly synthesised nucleic acid strand from its template, the nucleic acid polymerase is preferably heat stable at the temperature used.

Such heat stable polymerases are known to those skilled in the art. They are obtainable from thermophilic micro-organisms. The nucleic acid polymerase need not however be DNA dependent.

It may be RNA dependent. Thus it may be a reverse transcriptase—i. Such a temperature range will normally be maintained during primer extension. Once sufficient time has elapsed to allow annealing and also to allow a desired degree of primer extension to occur, the temperature can be increased, if desired, to allow strand separation.

They can be used to control the timing of colony initiation, e. Following strand separation e. The washing step can be omitted between initial rounds of annealing, primer extension and strand separation, if it is desired to maintain the same templates in the vicinity of immobilised primers.

This allows templates to be used several times to initiate colony formation. It is preferable to provide a high concentration of template molecules initially so that many colonies are initiated at one stage.

The size of colonies can be controlled, e. Other factors which affect the size of colonies can also be controlled. Once colonies have been formed they can be used for any desired purpose.

A surface comprising immobilised nucleic acid strands in the form of colonies of single stranded nucleic acid molecules is also within the scope of the present invention.

Normally each immobilised nucleic acid strand within a colony will be located on the surface so that an immobilised and complementary nucleic acid strand thereto is located on the surface within a distance of the length of said immobilised nucleic acid strand i.

This allows very high densities of nucleic acid strands and their complements to be provided in immobilised form. Preferably there will be substantially equal proportions of a given nucleic acid strand and its complement within a colony.

A nucleic acid strand and its complement will preferably be substantially homogeneously distributed within the colony. It is also possible to provide a surface comprising single stranded nucleic acid strands in the form of colonies, where in each colony, the sense and anti-sense single strands are provided in a form such that the two strands are no longer at all complementary, or simply partially complementary.

Such surfaces are also within the scope of the present invention. Normally, such surfaces are obtained after treating primary colonies, e. Once single stranded colonies have been provided they can be used to provide double stranded molecules.

Thus surfaces comprising colonies of non-bridged double stranded nucleic acid molecules are also within the scope of the present invention. Using the present invention, small colonies can be provided that contain large numbers of nucleic acid molecules whether single or double stranded.

Many colonies can therefore be located on a surface having a small area. Colony densities that can be obtained may therefore be very high, as discussed supra. Different colonies will generally be comprised of different amplified nucleic acid strands and amplified complementary strands thereto.

Thus the present invention allows many different populations of amplified nucleic acid molecules and their complements to be located on a single surface having a relatively small surface area.

The surface will usually be planar, although this is not essential. The present invention also provides an apparatus for providing a surface comprising colonies of the immobilised nucleic acid molecules discussed supra.

Such an apparatus can include one or more of the following:. Other apparatuses are within the scope of the present invention. These allow immobilised nucleic acids produced via the method of the present invention to be analysed.

They can include a source of reactants and detecting means for detecting a signal that may be generated once one or more reactants have been applied to the immobilised nucleic acid molecules.

They may also be provided with a surface comprising immobilised nucleic acid molecules in the form of colonies, as described supra. Desirably the means for detecting a signal has sufficient resolution to enable it to distinguish between signals generated from different colonies.

Apparatuses of the present invention of whatever nature are preferably provided in automated form so that once they are activated, individual process steps can be repeated automatically. The present invention will now be described without limitation thereof in sections A to I below with reference to the accompanying drawings.

It should be appreciated that procedures using DNA molecules referred to in these sections are applicable mutatis mutandis to RNA molecules, unless the context indicates otherwise. It also illustrates the annealing, elongation and denaturing steps that are used to provide such colonies.

Referring now to FIG. Each primer 1 is attached to the surface by a linkage indicated by a dark block. This may be a covalent or a non-covalent linkage but should be sufficiently strong to keep a primer in place on the surface.

In practice however longer sequences would generally be provided. Between the two ends any sequence to be amplified or the complement of any sequence to be amplified can be provided.

When primer extension is complete, as shown in FIG. The target molecule can then be separated from the extended immobilised strand e. This separation step frees the extended, immobilised strand so that it can then be used to initiate a subsequent round of primer extension, as shown in FIGS.

Primer extension is shown occurring in FIG. Each of these strands can then themselves be used as templates in further rounds of primer extension initiated from new primers 3 and 4 , as shown in FIGS.

Four single stranded, immobilised strands can be provided after two rounds of amplification followed by a strand separation step e. Two of these have sequences corresponding to the sequence of the target molecule originally used as a template.

The other two have sequences complementary to the sequence of the target molecule originally used as a template. In practice a given immobilised strand and its immobilised complement may anneal once.

It will therefore be appreciated that a given sequence and its complement can be provided in equal numbers in immobilised form and can be substantially homogeneously distributed within a colony.

Further rounds of amplification beyond those shown in FIG. Only a single template need be used to initiate each colony, although, if desired, a template can be reused to initiate several colonies.

It will be appreciated that the present invention allows very high densities of immobilised extended nucleic acid molecules to be provided. Within a colony each extended immobilised molecule will be located at a surface within one molecule length of another extended immobilised molecule.

Thus position 3 shown in FIG. A flat plate is shown schematically in plan view having primers immobilised thereon in a square grid pattern the primers are indicated by small dots. A regular grid is used solely for simplicity: At the position indicated by arrow X a template molecule has annealed to a primer and an initial bout of primer extension has occurred to provide an immobilised, extended nucleic acid strand.

Following strand separation, an end of that strand becomes free to anneal to further primers so that additional immobilised, extended nucleic acid strands can be produced.

This is shown having occurred sequentially at positions indicated by the letter Y. For simplicity, the primer chosen for annealing is positioned next to the primer carrying the nucleic acid strand: However, this primer will obviously be within a distance equal to the length of the nucleic acid strand.

It will be appreciated that annealing at only one rather than at all of these positions is required for colony cell growth to occur. After immobilised, extended, single-stranded nucleic acid molecules have been provided at the positions indicated by letter Y, the resultant molecules can themselves anneal to other primers and the process can be continued to provide a colony comprising a large number of immobilised nucleic acid molecules in a relatively small area.

It also depicts the typical observations that can be made, as can be seen on the examples shown in FIGS. The simultaneous amplification and immobilisation of nucleic acids using solid phase primers has been successfully achieved using the procedure described in Examples 1, 2 and 3 below:.

Microtitre wells with p57 or p58 were prepared as follows. The biotinylated probe was diluted in to a concentration of 2. The control reaction shows very few fluorescent spots, since the sequence of the flanking regions on the template do not correspond to the primer sequences grafted onto the well.

Referring now to FIGS. Here two different immobilised primers are used to provide primer extension. The embodiment shown in FIGS. The possibility of annealing occurring between both ends of an immobilised complement to a target molecule can also be avoided.

Several different concentrations of PCR template have been tested approximately 1, 0. These objects have an irregular shape, are 20 to micro-meters in size and have a thickness much larger than the field depth of the observation.

They present a circular shape, they are 1 to 5 micro meters in size and do not span the field of view. The number of spots depends on the concentration of the template used for initiating colony formation.

From the observed size of the colonies, one can estimate that more than 10, distinct colonies can be arrayed within 1 mm 2 of support. S1 and 52 are base pair and bp fragments, respectively, which have been cloned into pBlueScript Skminus plasmids and subsequently amplified through a PCR using P1 and P2 as primers.

A base pair fragment corresponding to the central sequence of the S1 fragment, but not including the Pl or P2 sequence was amplified by PCR as previously described. The biotinylated probes were hybridized to the samples in EasyHyb buffer Boehringer-Mannheim, Germany, using the following temperature scheme in the PTC thermocycler: The processing consisted in inversion and linear contrast enhancement, in order to provide a picture suitable for black and white print-out on a laser printer.

It can be seen from FIG. Amplified, single stranded nucleic acid molecules present in colonies provided by the present invention can themselves be used as templates to synthesise additional nucleic acid strands.

Colonies will usually comprise both a given nucleic acid strand and its complement in immobilised form FIG. Thus they can be used to provide additional copies not only of a given nucleic acid strand but also of its complement.

One way of doing this is to provide one or more primers primers TTA and TGG in solution that anneal to amplified, immobilised nucleic acid strands present in colonies FIG. These primers may be the same as primers initially used to provide the immobilised colonies, apart from being provided in free rather than immobilised form.

Primer extension, using AmpliTaq DNA polymerase and the four deoxyribonucleoside triphosphates labeled or unlabeled can then be used to synthesise complementary strands to immobilised nucleic acid strands or at least to parts thereof step iii.

Once newly formed strands FIG. The process can then be repeated if desired using the PCR reaction, to provide large number of such strands in solution FIG. Strands synthesised in this manner, after separation from the immobilised strands, can, if desired, be annealed to one another i.

Alternatively they can be separated from one another to provide homogenous populations of single-stranded nucleic acid molecules in solution. It should also be noted that once single-stranded molecules are provided in solution they can be used as templates for PCR or reverse PCR.

Therefore it is not essential to continue to use the immobilised nucleic acid strands to obtain further amplification of given strands or complementary strands thereto.

It should be noted that where a plurality of colonies are provided and nucleic acid strands in different colonies have different sequences, it is possible to select only certain colonies for use as templates in the synthesis of additional nucleic acid molecules.

This can be done by using primers for primer extension that are specific for molecules present in selected colonies. Alternatively primers can be provided to allow several or all of the colonies to be used as templates.

Such primers may be a mixture of many different primers e. Sl and S2 are base pair and b. The temperature control was performed in a PTC thermo-cycler. Reactions were stopped by rinsing the wells with TNT buffer.

PCR 25 cycles, 30 sec. P70 and P71 are suited for the amplification of both S1 and S2, since primer P70 contains the sequence of primer P1 and p7l contains P2. The pictures of the gels are presented in FIGS.

It is also possible to modify initially formed colonies to provide different colonies i. As a starting point, the primary colony FIG. A single-strand specific DNA exonuclease, might be used to remove all primers which have not been elongated.

Secondly and independently, the DNA molecules forming the colonies can be cleaved by using endonucleases. If desired, the enzymatically cleaved colony FIG. In any case, the secondary primers are available after denaturation e.

An important case is when the exonuclease digests only a few bases of the DNA molecule before being released in solution, and when digestion can proceed when another enzyme binds to the DNA molecule FIG.

In this case the exonuclease digestion will proceed until there remain only single stranded molecules which, on average, are half the length of the starting material, and are without any complementary parts which could form partial duplexes remaining in the single stranded molecules in a colony FIG.

In all cases, these treatments result in single-stranded fragments grafted onto a support which correspond to the sequence of the original template and that can be used for new DNA colony growing if an appropriate new template is provided for colony initiation FIG.

Templates useful for secondary colony growing may include molecules having known sequences or complements of such sequences. Alternatively templates may be derived from unsequenced molecules e.

In either event the templates should be provided with one or more regions for annealing with nucleic acid strands present in the primary colonies. Following a denaturing step ii, reannealing step iii and DNA polymerase step iv cycle, a replica of the original primary colony will be formed FIG.

The maximum size of a secondary colony provided by this embodiment of the present invention is restricted by the size of the primary colony onto which it grows. Several secondary growing processes can be used sequentially to create colonies for specific applications i.

The same procedure could be applied to a support covered with colonies or secondary primers as described in section E. Different immobilised primers are shown present in different regions of the support represented by squares.

Apparatuses of the present invention can be used for various procedures some of which will be described later on. Various preparation procedures are described below:.

Here is described a method to prepare DNA originating from one biological sample or from a plurality of samples for amplification in the case where it is not necessary to keep track of the origin of the DNA when it is incorporated within a colony.

This can be done e. In order to standardise experimental conditions, the extracted and cut DNA fragments can be size-fractionated, e. Fragments obtained within a single fraction can be used in providing templates in order to reduce the variability in size of the templates.

Alternatively, the template DNA fragments can be inserted into a biological vector at a site that is flanked by the sequence of the primers that are grafted on the support. This cloned DNA can be amplified within a biological host and extracted.

Obviously, if one is working with a single primer grafted to the solid support for DNA colony formation, purifying fragments containing both PI and P2 primers does not pose a problem.

Hereafter, the DNA fragments obtained after such a suitable process are designated by the expression: Here it is described how to prepare DNA originating from a plurality of biological samples in the case where it is necessary to keep track of the origin of the DNA when it is incorporated within a colony.

The procedure is the same as that described in the previous section except that in this case, the oligonucleotide linkers used to tail the randomly cut genomic DNA fragments are now made of two parts; the sequence of the primers grafted onto the surface P1 and P2, FIG.

Note that for each sample, the tag may not be unique, but a plurality of tags could be used. This tagging procedure can be used for providing colonies carrying a means of identification which is independent from the sequence carried by the template itself.

This could also be useful when some colonies are to be recovered specifically using the procedure given in section D. This could also be useful in the case the recovered colonies are further processed, e.

The DNA of interest can first be extracted from a biological sample by any means known by those skilled in the art as mentioned supra. Hereafter, we will designate the DNA fragments obtained after such a suitable process by the expression: Note that for each sample, a plurality of tags might be used, as in ii supra.

Potential uses of tags are the same as in ii, supra. The procedure is similar to the procedures described for preparing DNA fragments in the previous sections except that the starting point is to extract mRNA by any means known to those skilled in the art e.

Certainly, the tags and primers described supra can be used in conjunction with the process of double-stranded cDNA synthesis to allow their incorporation into the templates.

Hereafter, we will designate the mRNA fragments obtained after such suitable processes by the expressions: In assay procedures of the present invention labels may be used to provide detectable signals.

Labels for use in the present invention are preferably attached. Staining agents can be used in the present invention. With certain staining agents the result can be observed with a suitable fluorescence imaging apparatus.

DNA colonies are first prepared for hybridisation. Then they are hybridised with a probe labelled or unlabelled. If required, the hybridised probed is assayed, and the result is observed.

This can be done with an apparatus of the present invention e. In a preferred embodiment of the present invention colonies are treated with a DNA restriction endonuclease which is specific either for a sequence provided by a double stranded form of one of the primers originally grafted onto the surface where colonies are formed or for another sequence present in a template DNA molecule see e.

After restriction enzyme digestion, the colonies can be heated to a temperature high enough for double stranded DNA molecules to be separated. After this thermal denaturing step, the colonies can be washed to remove the non-hybridised, detached single-stranded DNA strands, leaving a remaining attached single-strand DNA.

A further alternative is simply to heat denature DNA in the colonies. Single-stranded nucleic acid probes labelled or unlabelled can be hybridised to single-stranded DNA in colonies at the appropriate temperature and buffer conditions which depends on the sequence of each probe, and can be determined using protocols known to those skilled in the art.

A hybridised probe provided initially in unlabelled form can be used as a primer for the incorporation of the different or a subset of the different labelled or a mix of labelled and unlabelled deoxyribonucleoside triphosphates with a DNA polymerase.

The incorporated labelled nucleotides can then be detected as described supra. Firstly, the DNA colonies can be prepared for hybridisation by the methods described supra.

Then they can be hybridised with a probe labelled or initially unlabelled. If required, hybridised labelled probes are assayed and the result is observed with an apparatus as described previously.

The probe may then be removed by heat denaturing and a probe specific for a second DNA sequence may be hybridised and detected. These steps maybe repeated with new probes as many times as desired.

Secondly, the probes can be assayed as described supra for unlabelled probes, except that only a subset preferably 1 only of the different labelled or unlabelled nucleotides are used at each cycle.

The colonies can then be assayed for monitoring the incorporation of the nucleotides. This second process can be repeated until a sequence of a desired length has been determined.

DNA colonies can be generated from templates and primers, such that a RNA polymerase promoter sequence is positioned at one end of the double-stranded DNA in the colony. The detection can be done non-specifically e.

In another embodiment of the present invention, colonies can be analysed in order to determine sequences of nucleic acid molecules which form the colonies. Since very large numbers of the same nucleic acid molecules can be provided within each colony the reliability of the sequencing data obtained is likely to be very high.

The sequences determined may be full or partial. Sequences can be determined for nucleic acids present in one or more colonies. A plurality of sequences may be determined at the same time.

In some embodiments the sequence of a complementary strand to a nucleic acid strand to be sequenced or of a part thereof may be obtained initially. However this sequence can be converted using base-pairing rules to provide the desired sequence or a part thereof.

This conversion can be done via a computer or via a person. It can be done after each step of primer extension or can be done at a later stage. Sequencing can be done by various methods. For example methods relying on sequential restriction endonuclease digestion and linker ligation can be used.

This method comprises the steps of: However in a preferred method of the present invention, amplified nucleic acid molecules preferably in the form of colonies, as disclosed herein are sequenced by allowing primers to hybridise with the nucleic acid molecules, extending the primers and detecting the nucleotides used in primer extension.

Preferably, after extending a primer by a single nucleotide, the nucleotide is detected before a further nucleotide is used in primer extension step-by-step sequencing.

One or more of the nucleotides used in primer extension may be labelled. The use of labelled nucleotides during primer extension facilitates detection. Preferably the label is not present in naturally-occurring nucleotides.

Ideally, labels are non-radioactive, such as fluorophores. However radioactive labels can be used. Where nucleotides are provided in labelled form the labels may be the same for different nucleotides.

If the same label is used each nucleotide incorporation can be used to provide a cumulative increase of the same signal e. Alternatively different labels may be used for each type of nucleotide which may be detected at different wavelengths.

In some embodiments of the present invention a mixture of labelled and unlabelled nucleotides may be provided, as will be described in greater detail later on. In a preferred embodiment of the present invention the sequencing of nucleic acid molecules present in at least 2 different colonies is performed simultaneously.

More preferably, sequencing of nucleic acid molecules present in over 10, over, over or even over 1,, different colonies is performed simultaneously. Thus if colonies having different nucleic acids molecules are provided, many different sequences full or partial can be determined simultaneously—i.

If desired, controls may be provided, whereby a plurality of colonies comprising the same nucleic acid molecules are provided. By 50 determining whether or not the same sequences are obtained for nucleic acid molecules in these colonies it can be ascertained whether or not the sequencing procedure is reliable.

One sequencing method of the present invention is illustrated in FIG. In the example outlined in FIG. After several repetitions of the addition of single deoxyribonucleoside triphosphates, it will be possible to determine any sequence.

For example sequences of at least 10, at least 20, at least 50 or at least bases may be determined. If colonies are provided initially in a form comprising doublestranded molecules the colonies can be processed to provide single-stranded molecules for use in sequencing as described above.

It should however be noted that double stranded molecules can be used for sequencing without such processing. For example a double stranded DNA molecule can be provided with a promoter sequence and step-bystep sequencing can then be performed using an RNA polymerase and labeled ribonucleotides cf FIG.

Another alternative is for a nick to be introduced in a double stranded DNA molecule so that nick translation can be performed using labeled deoxyribonucleotides. One way of processing double-stranded molecules present in colonies to provide single-stranded colonies as described later with reference to FIG.

Here double-stranded immobilised molecules present in a colony which may be in the form of bridge-like structures are cleaved and this is followed by a denaturing step.

Alternatively a denaturing step could be used initially and could be followed by a cleavage step. Preferably cleavage is carried out enzymatically. However other means of cleavage are possible, such as chemical cleavage.

An appropriate cleavage site can be provided in said molecule. Denaturing can be performed by any suitable means. Once single-stranded molecules to be sequenced are provided, suitable primers for primer extension can be hybridised thereto.

Oligonucleotides are preferred as primers. These are nucleic acid molecules that are typically 6 to 60, e. However other molecules, e. The primers for use in sequencing preferably hybridise to the same sequences present in amplified nucleic acid molecules as do primers that were used to provide said amplified nucleic acids.

When primers are provided in solution and are annealed hybridised to nucleic acid molecules present in colonies to be sequenced, those primers which remain in solution or which do not anneal specifically can be removed after annealing.

Preferred annealing conditions temperature and buffer composition prevent non-specific hybridisation. These may be stringent conditions. Such conditions would typically be annealing temperatures close to a primer’s Tm melting temperature at a given salt concentration e.

Stringent conditions for a given system can be determined by a skilled person. They will depend on the base composition, GC content, the length of the primer used and the salt concentration.

Primers used for primer extension need not be provided in solution, since they can be provided in immobilised form. In this embodiment the primers should be provided in the vicinity of the immobilised molecules to which they are to be annealed.

Such primers may indeed already be present as excess immobilised primers that were not used in amplifying nucleic acid molecules during the formation of colonies. For example it can be synthesised artificially and can be added to a given molecule using a ligase.

Quran micromax ditel 0121 netflix para

Novel oligonucleotide arrays and their use for sorting, isolating, sequencing, and manipulating nucleic acids. For de novo sequencing applications, the order of nucleotides applied to a given location can be chosen as desired. This can be of practical interest because with coding, the same codes thus the same oligonucleotides involved in assaying the code can be used for any set of genes, whereas without code, a different set of specific oligonucleotides has to be used for each set of genes. The process of claim 28 wherein said complementary oligonucleotides act as primers in amplifying said solid support bound nucleic acid fragments. Reduced representation bisulfite sequencing using uracil n-glycosylase ung and endonuclease iv. A regular grid is used solely for simplicity: Enzyme dilutions if needed are made into 0.

For example, avian coccidiosis is a disease that is only managed, but not really under control. Feed consumption was measured at day In preferred embodiments of the present invention a plurality of labelled bases are incorporated into an extended primer during sequencing.

This could, mean that when significant antibiotic resistant pathogens develop, there may be no new antibiotics available to treat the infections. The process of claim 14 wherein the adapter sequences are ligated to the nucleic acid fragments in step b. See Fodge et al. Only the regenerated colonies are present in a double stranded form, and are digested.

See…

491 492 493 494 495

 

Gto ditel 0121 micromax app

Diets were fed in MASH from days of age. Amplification occurs during the PCR cycling, which includes a step during which double stranded DNA molecules are denatured typically a reaction mix is heated, e. The template can be of any desired length provided that it can participate in the method of the present invention. It should also be noted that once single-stranded molecules are provided in solution they can be used as templates for PCR or reverse PCR. See…

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