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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6858—Allele-specific amplification
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to compositions and methods for detecting and analyzing genomic
• nucleic acid polymorphisms variation, and, more particularly, for detecting and analyzing nucleic acid polymorphisms.
• nucleic acid sequences within a complex genome are within a complex genome.
• immobilization techniques such as those described by Southern, J. Mol. Biol.
• Restriction enzymes are endonucleases which
• DNA-binding membranes such as nitrocellulose
• a short single-stranded DNA sequence that is complementary to a target sequence in the fragments i.e. a probe, can be labelled and used to detect the target sequence by hybridization.
• restriction site differences has been a favored means for characterizing genomic variation.
• the procedure uses the techniques developed by
• RFLP Restriction Fragment Length Polymorphism
• RFLP analysis provides a means for following the segregation of genes derived from each parent.
• RFLP loci can be mapped genetically by determining the frequencies of recombination between different RFLP loci. By correlating the segregation of the RFLP loci with the segregation of known genetic markers, the genetic maps of RFLP markers can be merged with genetic maps of other types of
• markers e.g., morphological or biochemical markers.
• nucleic acid amplification For example, J.S. Chamberlain et al., Nucleic Acids Research 16:11141 (1988) have described an amplification method to amplify more than a single nucleic acid sequence.
• the approach involves using more than one pair of primers in a Polymerase Chain Reaction.
• PCR Polymerase Chain Reaction
• PCR is a procedure for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification by introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase.
• the two primers are selected complementary to flanking sequences on respective strands of the double-stranded target sequence.
• the mixture is denatured and the primers are then allowed to anneal to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form
• the steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e. denaturation, annealing and extension constitute one "cycle;” there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
• the length of the amplified segment of the desired target sequence is determined by the relative
• PCR Polymerase Chain Reaction
• the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR-amplified”.
• the PCR procedure uses two primers with the intent of amplifying a single sequence whose length is defined by the position of the primers.
• J. S. Chamberlain et al., supra used more than one pair of primers in order to produce multiple sequences.
• the use of multiple pairs of primers (so-called "multiplex genomic DNA amplification") for analysis of genomic variation also has distinct disadvantages.
• minisatellite loci with the intent of amplifying the entire minisatellite.
• the procedure has a number of disadvantages. First, the procedure requires that the hypervariable "minisatellite” regions first be identified. See A.J. Jeffreys et al., Nature 314:67 (1985). Second, the procedure requires specific polynucleotide probes. See A.J. Jeffreys patents: U.K. Patent 2166445 and EPC 0238 329. Thirdly, minisatellite PCR must be terminated before the yield of product reaches chemical amounts because of production of a heterodisperse smear. Thus, a signal generation step like hybridization must be used to identify the products. D. L. Nelson et al., Proc. Nat. Acad. Sci. USA 86:6686 (1989) describe a procedure where PCR is used to produce multiple sequences. See also S.A.
• this procedure uses single primers directed at the known sequences of short interspersed repeats believed to exist in great number (approximately 900,000 in the haploid human genome). This procedure also has a number of disadvantages. First, this procedure, like the minisatellite PCR procedure, requires that the primers and the sequences to be amplified be known. Secondly, while PCR can be run to yield chemical amounts of discrete product, this is only possible if less than the entire genome is used. When the entire genome is used, there is again the production of a smear.
• the present invention involves a more desirable means of amplifying more than a single sequence.
• the present invention provides a method of obtaining information as important and useful as RFLP data, but without the accompanying labor, time and expense.
• the present invention does not require the use of restriction enzymes or nucleic acid transfer to perform an analysis of genomic variation.
• the invention allows for amplification in chemical amounts utilizing nucleic acid representing the entire genome.
• the present invention relates to compositions and methods for detecting and analyzing genomic variation and, in particular, nucleic acid
• nucleic acid sample comprising providing a nucleic acid sample comprising nucleic acid sequences of a distinct nucleic acid source, providing a primer comprising an oligonucleotide sequence of at least eleven nucleotides which is capable of hybridizing to at least a portion of the nucleic acid sequences and generating a plurality of amplification products therefrom in an amplification system, thereby amplifying at least one portion of the nucleic acid sequences, so that discrete portions of the nucleic acid sequences are detectable.
• a method for amplifying a plurality of sequences found in a nucleic acid sample comprises providing a nucleic acid sample comprising single-stranded nucleic acid sequences from a distinct nucleic acid source, bringing the sample together with a primer comprising an
• nucleotides capable of hybridizing to a plurality of regions within the nucleic acid sequences, under conditions which synthesize an extension product which is complementary to a portion of the nucleic acid sequences, and wherein the extension product, when separated from the nucleic acid sequence, is also capable of hybridizing to the oligonucleotide primer, separating the primer extension products from the nucleic acid sequences to which they were
• oligonucleotide primer under conditions which synthesize a primer extension product from the primers hybridized to the templates, thereby
• a method for detecting variation between nucleic acid samples comprising providing at least two nucleic acid samples each comprising nucleic acid sequences representing the entire genome of a distinct nucleic acid source; providing a single primer comprising an oligonucleotide sequence of at least eleven nucleotides capable of hybridizing to at least a portion of the nucleic acid sequences from each source and capable of generating a plurality of amplification products therefrom in an amplification system; and bringing together each nucleic acid sample with the primer in a separate amplification system, thereby amplifying at least one portion of the nucleic acid sequences from each source, so that discrete portions of the nucleic acid sequences are detectable.
• Certain embodiments of this aspect of the invention also provide that the amplified portions of nucleic acid sequences from each nucleic acid source so amplified are compared to determine the degree of homology between the nucleic acid sources.
• Additional aspects of the invention provide primers which find use in the present method, and a mixture of nucleic acid sequences comprising a plurality of double-stranded products comprising a first and a second single-stranded polynucleotides each having a 5'-terminal region sequence and a 3'-terminal region sequence which is substantially the inverted complement thereof, and an internal region.
• Figure 1A is a photograph of an ethidium bromide stained gel of electrophoresed SPAR-amplified
• Figure 1B is a photograph of an autoradiograph of the electrophoresed SPAR-amplified and PCR-amplified sequences of Figure 1 following transfer to
• Figure 2 is a photograph of an ethidium bromide stained gel of the electrophoresed SPAR-amplified sequences from chicken genomic DNA amplified primers of the present invention.
• Figure 3 is a photograph of an ethidium bromide stained gel of the electrophoresed SPAR-amplified sequences from chicken genomic DNA under three annealing temperatures ( Figure 3A, 3B and 3C).
• Figure 4 is a photograph of an ethidium bromide stained gel of the electrophoresed, SPAR-amplified sequences from chicken genomic DNA derived from different sources, i.e. different individual
• Figures 5A and 5B are (direct print) photographs of ethidium bromide stained gels of the
• the present invention relates to compositions and methods for detecting and analyzing genomic variation and, in particular, nucleic acid
• a method for amplifying a plurality of nucleic acid sequences found in a nucleic acid sample.
• a nucleic acid sample is employed which comprises nucleic acid sequences of a distinct nucleic acid source. Any source of nucleic acid, in purified or non-purified form, can be utilized as the source for the nucleic acid sample, provided the sample is from a distinct nucleic acid source which is suspected of harboring polymorphisms useful for detecting genomic variation.
• the method may employ any nucleic acid, for example DNA or RNA, including messenger RNA, which may be single-stranded or double-stranded.
• RNA messenger RNA
• a DNA-RNA hybrid which contains one strand of each distinct nucleic acid may be utilized. It is also possible to utilize a mixture of any one or more of such nucleic acids provided they are from a source appropriate to facilitate the analysis of genomic variation.
• the nucleic acid sample does not need to be provided in pure form; it may be a fraction of a more complex mixture, e.g., it may constitute only a minor fraction of a particular sample of biological origin.
• the present invention contemplates that the nucleic acid is derived from a microorganismal source, such as virus, bacteria, fungi, yeast, algae, mycoplasma and protozoa.
• a microorganismal source such as virus, bacteria, fungi, yeast, algae, mycoplasma and protozoa.
• the present invention contemplates that the nucleic acid provided is derived from a plant.
• plants which serve as nucleic acid sources include the angiosperms as well as the gymnosperms.
• angiosperms include both monocotyledons and dicotyledons, such as corn, barley, wheat, apple, alfalfa, soybean, oil rape, tobacco and tomato.
• gymnosperms examples include cycads and conifers, such as loblolly pine.
• the present invention employs nucleic acid samples derived from animal sources, including both vertebrates and
• vertebrates include mammals, birds, reptiles and amphibians, such as human, horse, dog, cow, chicken, mouse, rat and salmon.
• invertebrates include arthropods, moHusks,
• a primer capable of hybridizing to at least a portion of the nucleic acid sequences in the nucleic acid sample and generating a plurality of amplification products from the nucleic acid sequences in an appropriate amplification system.
• primer refers to an oligonucleotide, whether occurring naturally (e.g., as a component of a purified restriction enzyme digest product) or constructed synthetically.
• oligonucleotide is defined as a molecule
• nucleotides e.g., two or more nucleotides
• primers which find use in the present invention will form extension products in an
• the present invention be limited by the mechanism(s) whereby the primers of the present invention form extension products.
• the primer may be substantially complementary to at least a portion of the nucleic acid sequences contained in the nucleic acid sample, so that the primer is capable of
• an extension product is the collection of generated nucleic acid sequences which contain a particular primer sequence together with an additional sequence complementary to the nucleic acid sequence bordering the template.
• a primer will comprise an oligonucleotide capable of hybridizing to at least a portion of the nucleic acid sequences and capable of generating a plurality of amplification products therefrom in an amplification system.
• the selected primer will be capable of hybridizing to a plurality of regions within nucleic acid sequences in the nucleic acid sample, and will be capable of hybridizing to an extension product which contains the oligonucleotide sequence and is substantially complementary to a portion of the nucleic acid sequences.
• oligodeoxyribo-nucleotides generally in accordance with the formula:
• X 1 is desX 1 or an oligonucleotide of from 3 to 11 bases selected from the group consisting of A-G-A-G, G-A-C-C-A-A-C-T-G-G-T, and C-C-C;
• X 2 is desX 2 or an oligonucleotide of 3 bases selected from the group consisting of A-A-T and C-C-C;
• X 3 is desX 3 or an oligonucleotide of from 1 to 3 bases selected from the group consisting of T-C-T, G-G-T, A-A-C, and G;
• X 4 is an oligonucleotide of 3 bases selected from the group consisting of C-A-A, A-G-C and A-C-A;
• Y 1 is a base selected from the group consisting of C and A;
• Y 2 is an oligonucleotide of 3 bases selected from the group consisting of G-G-C, C-C-T, G-A-C, and T-A-C;
• Y 3 is desX 3 or an oligonucleotide of from 5 to 13 bases selected from the group consisting of A-A-C-A-G-G, G-G-G-C-C-T-G-G-T-C-G-A-T,
• the term "des-" is taken to mean that the particular base or oligonucleotide fragment may not be present in selected primer sequences within the scope of the formulae.
• W 1 is an oligonucleotide of from 7 to 12 bases selected from the group consisting of
• W 2 is an oligonucleotide of 2 bases selected from the group consisting of A-T and T-C;
• W 3 is a base selected from the group consisting of T, G, and A;
• Z 1 is an oligonucleotide of from 3 to 7 bases selected from the group consisting of G-G-G, A-G-C-C-T-C-G and T-C-C; with the provisos that at least approximately fifty percent (50%) of the deoxyribonucleotides of the primer are deoxyguanylic acid and deoxycytidylic acid.
• primers in accordance with formula IV will include sequences having the formula: 5'-W 1 -A-T-W 3 -T-G-G-T-(T/G)-G-G-(C/G)-Z 1 -3' (V) wherein W 1 , W 3 , and Z 1 are as previously defined.
• primers of formula V are sequences having the formula:
• primers other than those disclosed in the above formulae (I) to (Va) will be capable of hybridizing to at least a portion of the nucleic acid sequences in a nucleic acid sample and generating a plurality of amplification products therefrom in an amplification system.
• primers other than those disclosed in the above formulae (I) to (Va) will be capable of hybridizing to at least a portion of the nucleic acid sequences in a nucleic acid sample and generating a plurality of amplification products therefrom in an amplification system.
• products can be produced by exposing the complexes to an amplification system which generates extension products from each hybridized primer.
• a representative example of such an amplification system for a nucleic acid such as DNA will typically contain a pool of deoxyribonucleotides and a catalyst such as a DNA polymerase in a suitable buffer and under suitable reaction conditions (e.g., time, temperature, volume, etc.).
• a catalyst such as a DNA polymerase
• suitable reaction conditions e.g., time, temperature, volume, etc.
• the deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP will be added to the amplification system mixture in a buffered aqueous solution, preferably adjusted to a pH of between 7 and 9, most preferably of approximately pH 8.
• an excess of at least 1,000:1 (primer: sequence), and preferably an excess of at least 1,000,000:1 (primer: sequence), will generally be used for most nucleic acid samples.
• a large molar excess will generally be preferred in order to improve the efficiency of the amplification process.
• Suitable catalysts for this purpose include, for example, enzymes such as one or more of E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, reverse transcriptases, and other enzymes, including heat stable enzymes which will facilitate combination of the nucleotides in the proper manner to form the primer extension products.
• a presently preferred catalyst is the DNA polymerase from Thermus aguaticus (Tag), as described in U.S. Patent No. 4,889,818, the entire disclosure of which is incorporated herein by this reference.
• the temperature of the reaction mixture will range from, e.g., room temperature up to a
• the selected temperature will depend in part on the particular catalyst used. For example, some DNA polymerases are used at a temperature generally no greater than about 40°C (e.g. Klenow). Other DNA polymerases can be used at much higher temperatures (e.g., Tag polymerase is generally used at 72°C).
• the PCR procedure uses two primers with the intent of amplifying a single sequence whose length is defined by the position of the primers.
• sequences are amplified so that genomic variations in the sequences may be identified and correlated to genetically related individuals or groups.
• primers from the chicken ⁇ -globin sequence can be employed.
• the sequence [from J.B. Dodgson and J.D. Engel, J. Bio. Chem. 258:4623 (1983)] is provided in Table 1.
• Primer 226A corresponds to positions 64-88 in the 5'-end flanking region of the ⁇ -globin sequence.
• Primer 227 corresponds to positions 578-603 (opposite strand) in the Exon 2 region of the ⁇ -globin
• primers 226 and 227 can be used for amplification of a 540 bp product.
• primers 226 and 227 were used, both separately and together, in nine separate amplification reactions (Table 2).
• Table 2 The products of the reactions were electrophoresed and a photograph of the ethidium bromide stained gel is shown as Figure 1A.
• Molecular weight markers were provided in Lanes 1 and 12.
• Lanes 2-10 correspond to the nine amplification reactions (see Table 2).
• primer 227 was used alone (lane 2), no amplification bands are observed. Where primers 226 and 227 are used in equal amounts (lane 3), a single PCR-product band is observed running at approximately the expected molecular weight for a 540 bp product. Where the ratio of primer 226 to primer 227 is 2:1 (lane 4) or 4:1 (lane 5), the same single product band is observed but with some increased background. On the other hand, where the ratio is 10:1 (lane 6) or greater (lanes 7-9), a host of new product bands are apparent. Surprisingly, the new product bands can be generated using primer 226 alone (lane 10). Interestingly, the single product band observed clearly in lanes 3-5 becomes less apparent as the concentration of primer 227 decreases until it disappears (lane 10).
• SPAR Amplification Reaction
• the primer is used in conjunction with a nucleic acid sample comprising nucleic acid sequences of a distinct nucleic acid source.
• the selected primer will be capable of hybridizing to at least a portion of the nucleic acid sequences. However, it is not necessary that the sequences are known.
• the primer comprises an oligonucleotide capable of hybridizing to a plurality of regions (templates) within the nucleic acid sequences, under conditions which the primer serves as the initiating sequence for the synthesis of an extension product which is
• the primer contemplated by the present invention is complementary to a portion of the nucleic acid sequences, and wherein the extension product, when separated from the nucleic acid sequence, is also capable of hybridizing to the primer.
• the primer contemplated by the present invention is also capable of hybridizing to the primer.
• inventions will detect inverted repeat nucleic acid sequences in the nucleic acid sample.
• n-mers for a given n, where an n-mer is an oligodeoxynucleotide of length n bases.
• the probability of two with a specified sequence appearing by chance within 3kb of each other, and with the 3'-ends pointing towards each other is less than one in a genome of four billion bases.
• primer 246, a 16-mer, is completely homologous to an internal sequence of primer 226A, but lacks five nucleotides on both the 3'-end and 5'-end.
• SPAR was carried out as in Figure 1, except that annealing was performed at two temperatures (45°C and 55°C) for two (2) minutes. SPAR cycles were carried out in this manner for 35 cycles followed by 15 minutes at 70°C in the final cycle. The products were electrophoresed, the gel stained, and the stained gel photographed ( Figure 2).
• Figure 2 shows that primers differing by a single nucleotide may produce a multiple band pattern which is almost completely different (e.g. compare lanes 2 and 4), while in other cases, primers that differ by a single nucleotide will produce almost the same multiple band patterns (e.g., compare lanes 6 and 7).
• Figure 2 also shows that the annealing
• temperature can change the multiple band pattern generated by SPAR (e.g. compare lanes 3 and 13).
• SPAR e.g. compare lanes 3 and 13
• primers 226A and 235 were used.
• Primer 235 corresponds to positions 671-695 (opposite strand) of the ⁇ -globin sequence (see Table 1).
• primers 226A and 235 can be used together to define a 581 bp region between their annealing boundaries and allow for amplification of a 632 bp product.
• Figure 3A shows the expected single PCR-product band (arrow) as well as a multiple band pattern.
• the single PCR-product band in Figure 3A gradually decreases in intensity (see lanes 2-8) and disappears when primer 226A is used alone (lane 9). New bands, however, appear as the ratio of 226A primer to 235 primer increases.
• Figure 3B shows the results when the same amplifications are carried out using a 60°C annealing temperature. Again the single product band expected for PCR (arrow) decreases in intensity as the ratio of 226A primer to 235 primer increases (see lanes
• Figure 3C shows the results when the same amplifications are carried out using a 65°C annealing temperature.
• the single product band expected for PCR decreases in intensity as the ratio of 226A primer to 235 primer increases (see lanes 2-7).
• a weak multiband pattern is evident, although the bands are not intense as this ratio changes (see in particular lanes 5 and 6).
• no signal is apparent at this annealing temperature when primer 226A is used alone in a SPAR reaction (see lane 9).
• primers capable of producing amplification products according to the present invention recognize template sequences which are not randomly distributed throughout the genome.
• the primer is believed to permit the synthesis of an extension product which is complementary to a portion of the nucleic acid sequences, and wherein the extension product, when separated from the nucleic acid sequence, is also capable of hybridizing to the primer.
• the multiple sequences generated by SPAR provide a rich source of markers for detection and analysis of genomic variation.
• Certain embodiments of the present invention contemplate comparisons between the nucleic acid extension products from at least two sources (e.g. different individuals within a species, individuals from different species, etc.). It is desired that the amplified nucleic acid sequences from each nucleic acid source be thereafter compared to
• the degree of "size homology" of the amplification products is measured, e.g., the amplified nucleic acid sequences from each nucleic acid source are electrophoresed side-by-side in a gel.
• the various amplification products are, in this manner, separated by size, appearing as bands in the gel. Where there are a plurality of amplification products from one nucleic acid source, a distinct migration pattern of bands will be evident.
• the different migration patterns of bands evident between two sources of nucleic acid is a measure of the degree of "size homology" because different size products indicate different sequences.
• the present invention contemplates measuring the degree of homology of the amplified nucleic acid sequences by other methods as well. While not required, the present invention also contemplates measuring the degree of "sequence homology" of the amplified nucleic acid sequences by a) hybridization, b) restriction digestion and/or c) sequencing. If hybridization is used, the size-separated
• amplification products are transferred to a suitable blotting medium (e.g., nitrocellulose) and
• hybridization can be carried out in the manner of Southern et al. described above. Where restriction digestion is used, the amplified nucleic acid sample is digested with one or more restriction enzymes prior to or after electrophoresis. Where sequencing is used, the individual nucleic acid bands of the gel are recovered and sequenced.
• nucleic acid in genomic DNA it is possible to amplify nucleic acid in genomic DNA to a level detectable by several different methodologies (e.g. hybridization with a labelled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32 P-labelled CTP or ATP into the amplified segment).
• amplification it is generally preferred that amplification proceed to allow chemical amounts of amplified nucleic acid to be created. This allows for detection by simple means such as ethidium bromide staining.
• MW molecular weight
• OD optical density
• EDTA ethylenediaminetetraacetic acid
• dNTP deoxyribonucleoside 5'-triphosphate
• TAE buffer (0.06 M Tris-acetate, pH 8.3; 0.003 M EDTA); Tag buffer (50 mM KCl, 2.5 mM MgCl 2 , 10 mM Tris, pH 8.5, 200 ⁇ g/ml gelatin); PAGE
• V voltage
• W watts
• mA milliamps
• bp base pair
• CPM counts per minute
• Tag polymerase approximately 8 ⁇ l dNTPs (each at 2.5 ⁇ M, totaling 10 ⁇ M) and 100 ng to 1 ⁇ g of a selected primer.
• Tag polymerase approximately 0.5 ⁇ l; 5 Units/ ⁇ l, Bethesda Research Laboratories,
• the SPAR will typically be performed in a programmable thermal cycler (ERICOMP, Inc., San Diego, CA); a small amount of mineral oil or paraffin oil in each well can be used for maximum efficiency of heat exchange.
• ERICOMP programmable thermal cycler
• denaturation will be at 95°C for one minute; annealing at 55°C for two minutes; and extension at 70°C for three to eight minutes.
• SPAR cycles will normally be carried out in this manner for 25-35 cycles (approximately 12 minutes per cycle) followed by approximately 15 minutes at 70°C.
• primer 127 is derived from the ⁇ gt11 ⁇ -galactosidase gene near the EcoRI site.
• T.V. Huynh et al. describe a lambda vector ⁇ gt11 [In: DNA Cloning: A practical approach, IRL Press, 1:49 (1985)], which carries a portion of the E. coli ⁇ -galactosidase gene, including the upstream elements. There is, within the carboxy-a
• DNA can be inserted.
• the ⁇ gt series of insertion vectors was designed to express cDNA as
• DNA fragments (up to 7.2 kb) are cloned into the unique EcoRI site located in lacZ, allowing expression of a fusion protein if the cloned sequence is properly in-frame.
• the primers (see Table 5) will be synthesized, for example on a CYCLONETM DNA
• the multiple band pattern generated by SPAR is investigated with different sources of genomic nucleic acid.
• Two different primers are used (226 and 216) separately in each amplification with each nucleic acid source.
• Primer 226 (see Table 5) has been fully described above.
• Primer 216 a 16-mer, was synthesized having the repeating unit -GACA- (see Table 5).
• Genomic DNA was extracted from eight individual chickens and used to provide nucleic acid samples in SPAR. The amplified products were evaluated by electrophoresis.
• Figure 4 is a photograph of the ethidium bromide stained gel. Lane 1 contains molecular weight markers. Lanes 2-9 show that the amplification products for the individual chicken nucleic acid samples using primer 216 comprise a multi-band pattern in every case. While a number of prominent bands are seen to be shared, primer 216 also produces amplification product bands associated with only a few of the individual chickens (arrows), indicating polymorphisms. Lane 10 is a control lane (no nucleic acid sample).
• Lanes 11-17 show that the amplification products for the nucleic acid samples using primer 226 also comprise a multiband pattern in every case. Again, certain prominent bands found in each pattern, while other bands are associated only with particular individuals (arrows), indicating polymorphisms.
• Genomic DNA was extracted from twenty-four (24) different corn plants, each from a different inbred corn line, and used as samples in SPAR.
• Amplification was carried out using 10 ⁇ l Tag buffer (10X), 0.3 ⁇ l of each dNTP (100 mM), 2 ⁇ l gelatin (10 mg/ml), 0.5 ⁇ l Taq polymerase and 1 ⁇ l of spermidine (100 mM) in a total volume of 100 ⁇ l (brought up in water). Denaturation was at 94°C for one (1) minute; annealing was at 48°C for two (2) minutes; and extension at 72°C for three (3) minutes. The SPAR was carried out in this manner for 24 cycles, then extension was carried out for ten (10) minutes in a final cycle. The products were electrophoresed as described above, the gel stained, and the stained gel photographed ( Figure 5A and 5B). Molecular weight markers were provided in the end lanes.
• amplification products for the individual corn lines using primer 89 comprise a multi-band pattern in all but one case. While a number of prominent bands are found in each pattern, primer 89 also produces amplification product bands associated with only a few of the individual corn lines (arrows), indicating polymorphisms. Similarly, lanes 2-12 of Figure 5B show that the amplification products for the
• the present invention provides a method offering information as important and useful as RFLP data without the
• the present invention does not require the use of restriction enzymes or gel transfer.
• the present invention provides primers as well as a single primer
• sequences provide a rich source of markers for analysis of genomic variation.

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• Plant Pathology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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• 1991
  • 1991-11-05 EP EP19920900859 patent/EP0509089A4/en not_active Withdrawn
  • 1991-11-05 CA CA002073184A patent/CA2073184A1/en not_active Abandoned
  • 1991-11-05 AU AU89539/91A patent/AU8953991A/en not_active Abandoned
  • 1991-11-05 JP JP92500936A patent/JPH05505311A/ja active Pending
  • 1991-11-05 WO PCT/US1991/008233 patent/WO1992007948A1/en not_active Application Discontinuation

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