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WO2006094360A1 - Procede destine a amplifier les acides nucleiques - Google Patents

Procede destine a amplifier les acides nucleiques Download PDF

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Publication number
WO2006094360A1
WO2006094360A1 PCT/AU2006/000318 AU2006000318W WO2006094360A1 WO 2006094360 A1 WO2006094360 A1 WO 2006094360A1 AU 2006000318 W AU2006000318 W AU 2006000318W WO 2006094360 A1 WO2006094360 A1 WO 2006094360A1
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Prior art keywords
primer
nucleic acid
primers
pcr
amplification
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Matthew James Hayden
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Molecular Plant Breeding Nominees Ltd
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Molecular Plant Breeding Nominees Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to methods for amplifying nucleic acids, such as by polymerase chain reaction (PCR), and applications therefor.
  • PCR polymerase chain reaction
  • nucleotide and amino acid sequence information prepared using Patentln Version 3.3.
  • Each nucleotide sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (e.g.
  • nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:
  • sequence identifier eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as ⁇ 400>l.
  • nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine,
  • C Cytosine
  • G represents Guanine
  • T represents thymine
  • Y represents a pyrimidine residue
  • R represents a purine residue
  • M represents Adenine or Cytosine
  • K represents Guanine or Thymine
  • S represents Guanine or Cytosine
  • W represents
  • H represents a nucleotide other than Guanine
  • B represents a nucleotide other than Adenine
  • V represents a nucleotide other than Thymine
  • D represents a nucleotide other than Cytosine
  • N represents any nucleotide residue.
  • derived from shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
  • the present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference: i. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of VoIs I, II, and ffl; ii. DNA Cloning: A Practical Approach, VoIs. I and II (D. N. Glover, ed., 1985),
  • nucleic acid amplification techniques have become key tools for gene cloning, gene expression analysis, diagnosis, identification of samples (e.g., for animal breeding or identification of a crop plant), identification of individuals (e.g., forensic identification, maternity testing, paternity testing, marker assisted breeding and other breeding programs) and gene mapping.
  • these amplification techniques are used to detect a nucleotide sequence variation that forms a basis for genetic diversity between organisms and that contributes to a phenotype of an organism (e.g., a quantitative trait).
  • amplification techniques are used to detect a genetic marker such as, for example, a single nucleotide polymorphism (SNP) or a simple sequence repeat (SSR) (e.g., a dinucleotide repeat or a trinucleotide repeat) that is associated with a phenotype of interest.
  • a genetic marker such as, for example, a single nucleotide polymorphism (SNP) or a simple sequence repeat (SSR) (e.g., a dinucleotide repeat or a trinucleotide repeat) that is associated with a phenotype of interest.
  • SNP single nucleotide polymorphism
  • SSR simple sequence repeat
  • the number of known/characterised genetic markers is rapidly expanding. For example, as at January 2005 there were about 5x10 6 validated SNPs in the human genome, about 4xlO 4 SNPs in the wheat genome and about 2xlO 4 SNPs in the rice genome. With this increase in number of markers there is an associated increase in the number of markers that are associated with a particular phenotype. As a consequence, this has lead to a demand for assays that enable rapid and inexpensive detection of a large number of genetic markers.
  • a polymerase such as, for example, a DNA polymerase and/or a RNA polymerase
  • PCR polymerase chain reaction
  • a polymerase mediated replication technique uses a primer (e.g., a short oligonucleotide) capable of selectively annealing to a nucleic acid template to provide the binding site for the polymerase to initiate replication.
  • a primer e.g., a short oligonucleotide
  • the nucleic acid is amplified.
  • a standard PCR involves annealing paired oligonucleotides to opposite strands of a double stranded nucleic acid to thereby define the limits of the region to be amplified.
  • oligonucleotides provide the site of binding for a polymerase and initiate replication of the defined region.
  • the nucleic acid template is amplified by sequential rounds of primer annealing and polymerase-mediated replication.
  • PCR provides an advantage in that it facilitates exponential amplification of a nucleic acid by virtue of the ability of the primers to anneal to and initiate amplification of previously replicated nucleic acid.
  • RNA e.g., using a reverse transcriptase
  • nucleic acid corresponding to RNA is amplified.
  • Additional PCR variants include, for example, allele specific PCR, competitive PCR and nested PCR.
  • PCR is suitable for the amplification and analysis of genetic markers and in applications related thereto such as, for example, identification of individuals, paternity testing, maternity testing, plant breeding, animal breeding, diagnostic, prognostic and therapeutic applications.
  • multiplex PCR is a PCR wherein a plurality of primer sets is employed, each set capable of amplifying nucleic acid from a different locus. Primer sets that are likely to anneal to and initiate amplification of their respective target nucleic acids under similar conditions are selected for use in a multiplex reaction to ensure that each nucleic acid template is amplified. The PCR is then performed under a single set of conditions and nucleic acid from each locus amplified.
  • primers that are capable of use in a multiplex reaction anneal to their nucleic acid template at approximately the same temperature, i.e., the melting temperature (Tm) of each primer is the same or about the same.
  • Tm melting temperature
  • Methods for determining the Tm of a given nucleic acid sequence include, for example, the Wallace Rule that relies on the G+C and A+T content of the nucleic acid being analyzed (Wallace et al, Nucleic Acids Res. 6, 3543, 1979).
  • the nearest neighbour method is used to estimate the Tm of a primer (Howley, et al, J. Biol. Chem. 254, 4876, Santa Lucia, Proc. Natl. Acad.
  • the primer comprise one or more nucleotide analogues, such as, for example, 2-aminoglycine (peptide nucleic acid - PNA) the method of Giesen et al, (Nucl. Acids Res., 26: 5004-5006) is useful for determining the Tm of the primer. Those primers having the same or similar Tm are selected for further analysis to determine their suitability for use in a multiplex reaction.
  • nucleotide analogues such as, for example, 2-aminoglycine (peptide nucleic acid - PNA)
  • Giesen et al Nucl. Acids Res., 26: 5004-5006
  • Primers that are unlikely to self dimerize or do not comprise a region of self- complementarity are also selected.
  • a region of self-complementarity results in formation of secondary structure in the single stranded primer, or enables a primer to anneal to another copy of itself such that it forms a primer dimer that is able to extend during amplification. In either case, the ability of the primer to selectively anneal to a nucleic acid template is reduced.
  • To determine whether or not a primer comprises regions of self complementarity the sequence of the primer is incrementally overlapped and the presence of base-pairing determined. Those primers with a reduced probability of self-dimerization are selected.
  • a similar screen is used to determine a primer that does not bind to other primers used in a multiplex reaction. Such binding of primers in an amplification reaction leads to the formation of primer dimers thereby reducing the ability of each primer to selectively anneal to a target nucleic acid.
  • Primer length is also a consideration in determining a primer suitable for a multiplex reaction. Primers useful for multiplex must be of sufficient length to anneal to a nucleic acid template. While shorter primers are known to anneal to a plurality of sites in a nucleic acid due to non-specific annealing, increasing primer length does not indefinitely increase primer specificity (Burpo, Biochemistry, 218: 1 -11, 2001). Rather, primers of approximately 18 to 30 nucleotides in length are generally considered to be of sufficient length to enable selective annealing to a target nucleic acid.
  • each primer must also be tested empirically to ensure its suitability for use in a multiplex reaction.
  • multiplex PCR is frequently complicated by the occurrence of artefacts, such as, the amplification of spurious products resulting from non-specific annealing of the primers to template nucleic acid.
  • competition between primers for polymerase binding and nucleic acid amplification has been shown to reduce the yield of PCR ⁇ roduct(s) (Edwards et al, PCR Methods Applic. 3: S65-S75).
  • a multiplex PCR generally requires a significant level of time-consuming and costly optimization. Once optimized, only about 10 markers are amplified in any single multiplex PCR (Edwards et al, supra).
  • reaction components e.g., Mg 2+ , dNTP and/or polymerase type or concentration
  • amplification conditions e.g., annealing temperature and extension time
  • Belgrader et al Genome Science and Technology 1: 77-85, 1996, discloses the use of combinations of chimeric primers and universal primers (i.e., primers consisting of the sequence of the universal tag).
  • a plurality of sets of chimeric primers is used to amplify alleles of interest in a multiplex reaction and amplification products then amplified further in a separate reaction vessel using the universal primers.
  • the second reaction is effectively a nested PCR in which the primary amplification products are amplified in a "singleplex" reaction. This method is believed to reduce background associated with previous multiplex reaction formats.
  • the method of Belgrader et al suffers from several disadvantages associated with using multiple distinct PCR reactions, such as, for example, the risk of contamination due to increased sample handling (as a result of the use of two distinct reactions).
  • additional reaction components e.g., Mg 2+ , dNTP and/or polymerase
  • the different reactions generally require different amplification conditions (e.g., annealing temperature and extension time) and optimization.
  • MSTP Multiplexing Short Tandem Repeat Polymorphisms with Tailed Primers
  • An initial reaction is performed wherein the chimeric primer and the second primer amplify the nucleic acid template and the annealing temperature is then reduced to permit the amplification product to be further amplified using the universal primer and the second primer.
  • Significant optimization of primer concentration, magnesium concentrations and annealing temperatures is required. Accordingly, this method is not suitable for simple or rapid application to a new multiplex reaction.
  • non-specific amplification may occur frequently in the second stage of the amplification reaction, because the second primer is required to anneal at a temperature substantially lower than its Tm.
  • the present inventors sought to produce a multiplex PCR assay that facilitates the specific amplification of a number of nucleic acids of interest in a single closed-tube reaction, thereby reducing costs and risks of contamination associated with the use of multiple distinct reactions.
  • closed-tube is meant that reagents for all amplification reactions or stages, e.g., primers, enzyme, buffers, are present throughout said reactions or stages. Such a process would have utility as a low-cost high-throughput process.
  • the present inventors have found that in a single closed-tube PCR it is possible, in a first round, to specifically amplify nucleic acid at a locus of interest using an amount of tagged locus-specific primers suitable for performing exhaustive PCR (i.e., such that there is little or substantially no primer remaining after amplification).
  • the first round amplification product is then amplified using tag primers having lower Tm than the tagged locus-specific primers and annealed to the incorporated tag sequence in said first round amplification product at a lower annealing temperature than used in the first round.
  • the inventors have demonstrated the efficacy of this method for multiplex PCR, by showing that it is possible to detect simple sequence repeats in nucleic acid from wheat, barley, apricot, cherry, cattle, sheep and a fungus.
  • the present inventors have also demonstrated the robust nature of this amplification method by amplifying nucleic acid from DNA isolated using a harsh method likely to produce degraded DNA.
  • the method produced by the inventors facilitates amplification of nucleic acid from various concentrations of template DNA.
  • the present invention provides a method for amplifying nucleic acid comprising:
  • PCR Chain Reaction
  • first primer comprises a locus-specific sequence capable of annealing to the nucleic acid template at a first temperature and a tag sequence that does not anneal to the nucleic acid template
  • second primer or set of second primers wherein each second primer comprises a sequence capable of annealing to a nucleic acid comprising a sequence complementary to a portion of a first primer comprising the tag sequence and wherein each second primer has a melting temperature (Tm) lower than the Tm of the first primer and is not capable of annealing substantially to the first primer or nucleic acid template at the first temperature;
  • Tm melting temperature
  • PCR or “polymerase chain reaction” shall be taken to mean an amplification reaction employing multiple cycles of (i) denaturation of double- stranded nucleic acid such as a nucleic acid “template” to be amplified or a hybrid between a "template” and a complementary “primer”; (ii) annealing of a primer to its complementary sequence in the single-stranded “template”; and (iii) extension of the primer in the 5'- to 3'- direction by a polymerase activity e.g., an activity of a thermostable polymerase, such as, Taq, to thereby produce a double-stranded nucleic acid comprising a newly-synthesized strand complementary to the single-stranded template.
  • a polymerase activity e.g., an activity of a thermostable polymerase, such as, Taq
  • PCR reverse-transcriptase mediated PCR
  • TULIP touch-up and loop incorporated primers
  • RC-PCR rapid competitive PCR
  • the amplification reaction is a multiplex PCR.
  • annealing or similar term shall be taken to mean that a primer and a nucleic acid to be amplified (i.e., template or primary amplification product) are base-paired to each other to form a double-stranded or partially double- stranded nucleic acid, using a temperature or other reaction condition known in the art to promote or permit base-pairing between complementary nucleotide residues.
  • the ability to form a duplex and/or the stability of a formed duplex depend on one or more factors including the length of a region of complementarity between the primer and nucleic acid to be amplified, the percentage content of adenine and thymine in a region of complementarity (i.e., "A+T content"), the incubation temperature relative to the melting temperature (Tm) of a duplex, and the salt concentration of a buffer or other solution in which the amplification is performed.
  • A+T content percentage content of adenine and thymine in a region of complementarity
  • Tm melting temperature
  • the salt concentration of a buffer or other solution in which the amplification is performed Generally, to promote annealing, the primers and nucleic acid to be amplified are incubated at a temperature that is at least about 1-5°C below a primer Tm that is predicted from its A+T content and length.
  • Duplex formation can also be enhanced or stabilized by increasing the amount of a salt (e.g., NaCl, MgCl 2 , KCl, sodium citrate, etc) in the reaction buffer, or by increasing the time period of the incubation, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press ; Hames and Higgins, Nucleic Acid Hybridization: A Practical Approach, IRL Press, Oxford (1985); Berger and Kimmel, Guide to Molecular Cloning Techniques, In: Methods in Enzymology, VoI 152, Academic Press, San Diego CA (1987); or Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience, ISBN 047150338 (1992).
  • a salt e.g., NaCl, MgCl 2 , KCl, sodium citrate, etc
  • the "template” may comprise DNA, RNA or RNA/DNA with or without any nucleotide analogs therein including single-stranded or double-stranded genomic DNA, mRNA or cDNA.
  • the present invention is not limited by the nature or source of the template nucleic acid.
  • the template nucleic acid can be derived directly or indirectly from an organism, in a tissue or cellular sample obtained previously from an organism, or present in an aqueous or non-aqueous extract of a tissue or cellular sample.
  • the present invention is particularly useful for amplifying genetic markers.
  • the nucleic acid template comprises one or more genetic markers, such as, for example, a SNP, a simple nucleotide polymorphism, a short tandem repeat or a polynucleotide repeat (e.g., a trinucleotide repeat or a pentanucleotide repeat). Additional suitable nucleic acid templates are described herein.
  • a “primer” is a nucleic acid molecule comprising any combination of ribonucleotides, deoxyribonucleotides and analogs thereof such that it comprises DNA, RNA or DNA/RNA with one or more ribonucleotide or deoxyribonucleotide analogs contained therein, and capable of annealing to a nucleic acid template to act as a binding site for an enzyme, e.g., DNA or RNA polymerase, thereby providing a site for initiation of replication of a specific nucleic acid in the 5' to 3' direction.
  • an enzyme e.g., DNA or RNA polymerase
  • the nucleotide sequence of a primer is generally substantially complementary to the nucleotide sequence of a template nucleic acid to be amplified, or at least comprises a region of complementarity sufficient for annealing to occur and extension in the 5' to 3' direction there from.
  • a degree of non-complementarity will not significantly adversely affect the ability of a primer to initiate extension.
  • Primers are generally, but not necessarily, short synthetic nucleic acids of about 12-50 nucleotides in length.
  • the first primer or each primer of the set of first primers comprises at least about 12-15 nucleotides in length capable of annealing to a strand of the nucleic acid template.
  • Primers may also comprise at least about 20 or 25 or 30 nucleotides in length capable of annealing to a strand of the template.
  • set with reference to a “set of first primers” or a “set of second primers” " or more generally to a “set of primers” shall be taken to mean a number of primers having different, albeit not necessarily entirely different, sequences.
  • a preferred set of primers will comprise primers that are capable of annealing to and priming the amplification of different amplicons from one or more template molecules.
  • amplicon is meant an amplified sequence, which may be nucleic acid comprising a short tandem repeat sequence, single nucleotide polymorphism (SNP), microsatellite marker, intron, promoter, open reading frame, or whole gene.
  • SNP single nucleotide polymorphism
  • a set of first primers may be distinct from a second primer or set of second primers by virtue of their different Tm values and the prohibition on the second primer or set of second primers participating in the first round of amplification.
  • Preferred sets of primers will comprise at least 2-5 primers, or 5-10 primers or 10-15 primers or 15-20 primers or 20-50 primers or even as many as 100 primers in a single reaction vessel.
  • the amount of a first primer or set of first primers sufficient to permit amplification of a nucleic acid template without substantial residual unincorporated primer is generally determined empirically without undue experimentation. For example, the amount of residual unincorporated primer is determined by performing a series of reactions with different primer concentrations and otherwise identical reaction conditions and determining a primer concentration for which there is little or substantially no residual unincorporated primer. It will be understood in the art that "residual unincorporated primer” means the amount of primer remaining following an amplification reaction comprising a predetermined set of conditions e.g., number of cycles, duration of extension reaction, nucleotide concentration, etc.
  • without substantial residual unincorporated primer means that the amount of residual unincorporated primer is not sufficient to promote primer amplification in a subsequent round of amplification that is detectable using a detection means, e.g., an amplification reaction, e.g., an amplification reaction performed using the second primer or set of second primers. Accordingly, any unincorporated first primer(s), if present on termination of the first round of amplification, is not in an amount sufficient to be amplified to a detectable level using the second primer(s).
  • a detection means e.g., an amplification reaction, e.g., an amplification reaction performed using the second primer or set of second primers. Accordingly, any unincorporated first primer(s), if present on termination of the first round of amplification, is not in an amount sufficient to be amplified to a detectable level using the second primer(s).
  • a preferred amount or concentration of first primer is between about 7.5nM and about 25OnM (e.g., about 23nM or about 3OnM or about 4OnM or about 8OnM), more preferably between about 2OnM and about 20OnM, even more preferably between about 5OnM and about 15OnM and still more preferably between about 75nM and about 10OnM.
  • a limiting amount of a primer is about 23nM of primer or about 3OnM of primer or about 4OnM of primer or about 8OnM of primer.
  • each first primer and each second primer is present in an amount suitable for performing exhaustive PCR, i.e., an amount of primer(s) and number of amplification cycles sufficient for the primer(s) to be substantially incorporated into the amplification product with (or "leaving") little or no residual unincorporated primer.
  • amounts of both primer sets produce approximately equivalent levels of PCR product in the first and second rounds thereby facilitating detection of the amplified products using a standard detection means, e.g., a semi-automated DNA fragment analyser.
  • each second primer is also present in an amount suitable for performing exhaustive PCR, e.g., to control product formation
  • a second primer is present at a concentration of about 75nM or 10OnM, 20OnM, 30OnM or 40OnM.
  • locus-specific sequence is meant a nucleotide sequence complementary to a template nucleic acid being amplified, irrespective of the nature or formulation of said nucleic acid (e.g., it may be intact or fragmented genomic DNA, RNA or a pool thereof, or a library of DNA or cDNA fragments, etc).
  • a locus-specific sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 15 to 54 or 57 to 5345.
  • a locus specific sequence useful for amplifying nucleic acid from wheat and/or barley comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 57 to 5056; a locus specific sequence useful for amplifying nucleic acid from a Prunus spp., e.g., apricot or cherry, comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 5057 to 5264; a locus specific sequence useful for amplifying nucleic acid from a mammal, e.g., cattle or a sheep comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 5265 to 5294; or a locus specific sequence useful for amplifying nucleic acid from a fungus, e.g., Rhynchosporium secalis comprises a nucleotide sequence set forth in SEQ ID NOs: 5295 to 5342.
  • tag sequence is meant a sequence other than a locus-specific sequence.
  • the tag region provides for enhanced specificity of the first primer or set thereof, and a template within the amplicon of the first amplification round to which the second primer or set of second primers anneals. Accordingly, it is preferable that the tag region comprises sufficient nucleotides for a primer to selectively anneal to and produce an amplification product in an amplification reaction.
  • the tag region is at least about 12 nucleotides in length, preferably at least about 15 nucleotides in length, more preferably at least about 17 nucleotides in length and still more preferably at least about 19 nucleotides in length. In an exemplification of the invention, the inventors have used a tag sequence of about 19 or 20 nucleotides in length.
  • a tag sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 14.
  • the tag sequence comprises a nucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14.
  • tags are employed, the present invention clearly encompasses the use of the same or different tags on each of the primers, e.g., use of the same or a different set of primers.
  • a primer annealing to a strand of the nucleic acid template may comprises one tag and a primer annealing to the other strand of the nucleic acid template may comprise a different tag.
  • tags are employed on first and second primers or sets thereof to monitor first and second round amplifications, respectively.
  • first primers annealing to one strand of the template that each comprise one tag
  • first primers annealing to the other stand of the template that each comprise another tag
  • second primers to thereby provide substrates for annealing of a set of second primers.
  • tags is employed and specific first round amplicons are amplified using tag specific primer/s.
  • the "portion" of the first primer to which the second primer(s) anneal will comprise a sequence complementary to all or part of the tag sequence in the first primer, and optionally one or more nucleotides complementary to the locus-specific sequence.
  • the present invention is not to be limited by the precise alignment of the first and second primers with respect to the tag sequence provided that the second primer is capable of priming the amplification of an amplicon torn the product of the first amplification round (i.e., the product amplified by priming with a first primer).
  • a second primer comprises a nucleotide sequence comprising at least 10 consecutive nucleotides of a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 14.
  • the second primer comprises a nucleotide sequence comprising at least 11 nucleotides of a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 14.
  • the second primer comprises a nucleotide sequence comprising at least 12 nucleotides of a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 14.
  • the second primer or set thereof comprises a nucleotide sequence set forth in SEQ ID NO: 55 or 56.
  • a second set of primers comprises a primer comprising a nucleotide sequence set forth in SEQ ID NO: 55 and a primer comprising a nucleotide sequence set forth in SEQ ID NO: 56.
  • one or more primers is labelled with a detectable ligand, such as, for example a radioactive label, fluorescent molecule, dye, etc.
  • a detectable ligand such as, for example a radioactive label, fluorescent molecule, dye, etc.
  • one or more of the second primers is labelled with a detectable ligand.
  • a detectable ligand for example, by incorporating a dye-label into one of the tag primers, a second round amplification product is labelled thereby enabling rapid detection of amplified nucleic acid e.g., using a DNA fragment analyzer. Such rapid detection is clearly useful for high throughput analyses.
  • the absence of detectable annealing of the second primer or set of second primers to the template is determined empirically e.g., by the appearance of a correct amplification product following a first round amplification or alternatively by the absence of detectable amplification of template using a second primer or set thereof.
  • This selectivity is partially attributed to the fact that the first primer or set thereof has a greater predicted melting temperature (Tm) than the second primer or set thereof.
  • Tm predicted melting temperature
  • the first primer or set thereof has a Tm at least about 1O 0 C greater than that of the second primer or set thereof. More preferably, the first primer or set thereof has a Tm at least about 15 0 C or 18 0 C or 2O 0 C greater than that of the second primer or set thereof.
  • the difference between the first temperature and the temperature at which the second primer(s) is(are) annealed is at least about 8 0 C or 9 0 C or 1O 0 C or 15 0 C.
  • a temperature of 63 0 C can be employed to anneal a first primer and a temperature of 54 0 C employed to anneal a second primer (i.e., a difference of 9 0 C).
  • the plurality of amplification reactions are performed in a reaction vessel (i.e., the samples are not transferred to a separate vessel between the amplification reactions).
  • reaction vessel shall be construed in its broadest context to include any standard vessel suitable for performing a PCR, such as, for example, a reaction tube (such as, for example, an Eppendorf tube, a polypropylene tube, a glass tube or a glass/plastic composite tube), capillary, microtitre well, or a solid substrate such as a glass slide, microarray matrix, or tissue slice.
  • a reaction tube such as, for example, an Eppendorf tube, a polypropylene tube, a glass tube or a glass/plastic composite tube
  • capillary microtitre well
  • a solid substrate such as a glass slide, microarray matrix, or tissue slice.
  • the term "providing in a reaction vessel” shall be taken to include the supply of one or more reaction vessels with reagents therein, or alternatively, the provision of a reaction vessel with any number of reagents therein, and separately one or more reagents, with instructions for their combination.
  • at least the primers are provided in a reaction vessel, or alternatively, provided separately with instructions for their combination.
  • reagents suitable for performing PCR such as, for example, primers, template nucleic acid, ribonucleotide triphosphates and/or deoxyribonucleotide triphosphates or analogs thereof, an appropriate reaction buffer, and a polymerase enzyme (e.g., a thermostable polymerase).
  • primers for example, primers, template nucleic acid, ribonucleotide triphosphates and/or deoxyribonucleotide triphosphates or analogs thereof
  • ribonucleotide triphosphates and/or deoxyribonucleotide triphosphates or analogs thereof an appropriate reaction buffer
  • a polymerase enzyme e.g., a thermostable polymerase
  • deoxyribonucleotide is an art-recognized term referring to those bases of DNA each comprising phosphate, deoxyribose and a purine or pyrimidine base selected from the group consisting of adenine (A), cytidine (C), guanine (G) and thymine (T).
  • deoxyribonucleotide triphosphates e.g., dATP, dCTP, dGTP and dTTP, are capable of being incorporated into DNA by an enzyme of DNA synthesis e.g., a DNA polymerase.
  • ribonucleotide is an art-recognized term referring to those bases of RNA each comprising a purine or pyrimidine base selected from the group consisting of adenine (A), cytidine (C), guanine (G) and uracil (U) linked to ribose. Ribonucleotides are capable of being incorporated into RNA by an enzyme of RNA synthesis e.g., an RNA polymerase.
  • analog means a compound having a physical structure that is related to a ribonucleotide or deoxyribonucleotide and preferably is capable of forming a hydrogen bond with a ribonucleotide or deoxyribonucleotide residue or an analog thereof (i.e., it is able to anneal with the ribonucleotide or deoxyribonucleotide residue or an analog thereof to form a base-pair).
  • Such analogs may possess different chemical and biological properties to the ribonucleotide or deoxyribonucleotide residue to which they are structurally related. Methylated, iodinated, brominated or biotinylated residues are particularly preferred, however other analogs may also be used.
  • reaction vessel Preferably, no additional components are added to the reaction vessel after amplification of the template has commenced and the reaction volume is not modified by the addition or subtraction of any reagents after this point.
  • This feature of the invention avoids or reduces contamination problems associated with excessive sample handling.
  • the present invention further comprises combining reagents suitable for performing PCR in a reaction vessel, and more particularly, combining an amount of a first primer or set of first primers sufficient to permit amplification of a nucleic acid template without substantial residual unincorporated primer, and a second primer or set of second primers in a reaction vessel.
  • the present invention may further comprise providing the nucleic acid template, for example, in the form of a biological sample derived from a subject.
  • the biological sample is isolated previously from the subject being characterised.
  • the present invention further comprises producing and/or synthesizing a first primer and/or a second primer or set thereof.
  • Methods for the production and/or synthesis of primers are well-known in the art and/or described herein.
  • the present invention further comprises detecting the amplified nucleic acid using a detection means e.g., electophoresis or mass spectrometry.
  • a detection means include hybridization (e.g., hybridization of a nucleic acid, peptide nucleic acid (PNA) or locked nucleic acid (LNA) probe), or amplification (e.g., allele specific PCR, a ligase chain reaction, a rolling circle amplification, a transcription mediated amplification (TMA), a nucleic acid sequence based amplification (NASBA) or a Q-beta replicase mediated amplification).
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • amplification e.g., allele specific PCR, a ligase chain reaction, a rolling circle amplification, a transcription mediated amplification (TMA), a nucleic acid sequence based amplification (NASBA) or a Q-beta replica
  • the present invention is useful for amplifying a single nucleic acid template or, alternatively, a plurality of nucleic acids, e.g., in a multiplex format.
  • the present invention provides a method for amplifying nucleic acid comprising:
  • PCR Polymerase Chain Reaction
  • each first primer comprises a locus-specific sequence capable of annealing to the nucleic acid template at a first temperature and a tag sequence comprising a nucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14; and (b) a second primer or set of second primers wherein each second primer comprises a nucleotide sequence set forth in SEQ ID NO: 55 or SEQ ID NO: 56, wherein each second primer has a melting temperature (Tm) lower than the Tm of the first primer and is not capable of annealing substantially to the first primer or nucleic acid template at the first temperature;
  • Tm melting temperature
  • the present invention provides a method for multiplex amplification of nucleic acid comprising performing the method described herein according to any embodiment using amount(s) of sets of first primers and set(s) of second primers and performing the PCR under conditions sufficient to amplify a plurality of nucleic acids.
  • the present invention provides a method for multiplex amplification of nucleic acid comprising:
  • PCR Polymerase Chain Reaction
  • each first primer comprises a locus-specific sequence capable of annealing to a nucleic acid template at a first temperature and a tag sequence that does not anneal to the nucleic acid template
  • each second primer comprises a sequence capable of annealing to a nucleic acid comprising a sequence complementary to a portion of a first primer comprising the tag sequence and wherein each member of the set of second primers has a melting temperature (Tm) lower than the Tm of each member of the set of first primers and is not capable of annealing substantially to members of the set of first primers or the plurality of nucleic acid templates at the first temperature;
  • Tm melting temperature
  • the subject method additionally comprises performing PCR under conditions sufficient to amplify nucleic acid prior to (ii) wherein said conditions comprise an annealing temperature suitable for members of both the first and second sets of primers to anneal to a nucleic acid template.
  • a temperature of about 5O 0 C may be employed and a small number of amplification cycles (e.g., 1 to 5 cycles) carried out.
  • Such amplification is useful for ensuring amplification of all template nucleic acids despite using first primers each with a different Tm.
  • a plurality of primers in the first set of primers comprises the same tag sequence.
  • a subset of the first set of primers comprises the same tag sequence.
  • a subset of the first set of primers in the first set of primers comprises a first tag sequence and a subset of primers in the first set of primers comprises a second tag sequence.
  • each primer in the first set of primers or all primers in the first set of primers comprises the same tag sequence.
  • the present invention additionally provides a method for the multiplex amplification of nucleic acid comprising:
  • PCR Polymerase Chain Reaction
  • each first primer comprises a locus-specific sequence capable of annealing to a nucleic acid template at a first temperature and a tag sequence comprising a nucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14; and (b) a second primer or set of second primers wherein each second primer comprises a nucleotide sequence set forth in SEQ ID NO: 55 or SEQ ID NO: 56, wherein each member of the set of second primers has a melting temperature (Tm) lower than the Tm of each member of the set of first primers and is not capable of annealing substantially to members of the set of first primers or nucleic acid template at the first temperature; (ii) performing PCR under conditions
  • the method described herein additionally comprises determining one or more first primer(s) or set(s) of first primers by a process comprising predicting the lengths of amplification products produced by one or more candidate first primers or candidate set(s) of first primers and determining one or more first primer(s) or set(s) of first primers that are suitable for producing predicted amplification products or groups of predicted amplification products of sufficiently different lengths to permit their resolution by a means that fractionates nucleic acid according to length.
  • the method comprises selecting predicted amplification products of sufficiently different length to permit resolution by a means that fractionates nucleic acid according to length.
  • the means that fractionates nucleic acid according to length comprises electrophoresis, e.g., polyacrylamide gel electrophoresis or capillary electrophoresis.
  • the means that fractionates nucleic acid according to its length comprises mass spectrometry.
  • the method of the invention additionally comprises grouping the predicted amplification products such that the predicted amplification products in each group are of sufficiently different length to permit their resolution by a means that fractionates nucleic acid according to length and selecting one or more groups of predicted amplification products.
  • the method of determining one or more first primer(s) or set(s) of first primers comprises using a computer-based algorithm to determine the lengths of the predicted amplification products.
  • the method of determining one or more first primer(s) or set(s) of first primers additionally comprises producing a database of allele length data from the lengths of the predicted amplification products.
  • the method comprises predicting and/or grouping and/or selecting the predicted amplification products and/or groups of predicted amplification products using a database of allele length data for the predicted amplification products to thereby provide the lengths of said predicted amplification products.
  • the method additionally comprises retrieving data pertaining to allele length(s) and/or predicted amplification products and/or groups of predicted amplification products in a computer-readable format.
  • the method additionally comprises selecting first primer(s) or set(s) of first primers or one or more groups of first primers.
  • the selected first primer(s) or set(s) of first primers are predicted to produce predicted amplification products having at least 5 nucleotides difference in length.
  • the method additionally comprises providing, producing or synthesizing the one or more first primer(s) or set(s) of first primers.
  • the detection of one or more nucleic acids is useful for, for example, determining relationships between one or more individuals, isolates of an organism, cultivars of an organism, species or genera.
  • the method of the invention is used to detect one or more nucleic acids that are polymorphic between two or more individuals, isolates of an organism, cultivars of an organism, species or genera.
  • the present invention additionally provides a method comprising performing a method described herein to detect one or more polymorphic nucleic acid/s in an individual, isolate of an organism, cultivar of an organism, species or genus wherein the polymorphic nucleic acid detected characterizes the individual, isolate of an organism, cultivar of an organism, species or genus.
  • the present invention is useful for typing an organism within or between groups, or for differentiating between individuals or groups (e.g., for identification of a specific plant variety).
  • the skilled artisan will appreciate that the method of the invention is applicable to, for example, the analysis of a sample (e.g., a food sample) to identify the presence of a foreign agent (e.g., a genetically modified plant).
  • a sample e.g., a food sample
  • a foreign agent e.g., a genetically modified plant.
  • the present invention has clear applicability, for example, in the identification/diagnosis of a disease or disorder, for example, by detection and/or identification of the infectious agent that causes the disease or disorder. Such methods are clearly contemplated by the present invention.
  • the present invention additionally contemplates methods of screening an animal species for the purpose of animal husbandry, for example, for the selection of a desired trait (e.g., marbled beef from cattle, or enhanced milk quality from cattle, enhanced speed or stamina in horses or enhanced meat quality from pigs).
  • a desired trait e.g., marbled beef from cattle, or enhanced milk quality from cattle, enhanced speed or stamina in horses or enhanced meat quality from pigs.
  • Such screening involves the detection of one or more genetic markers associated with a trait of interest in a sample from a non-human animal and selecting those animals comprising the marker/s, for example, for breeding.
  • the present invention provides a process of characterising or identifying one or more individuals, isolates of an organism, cultivars of an organism, species or genera said process comprising: (i) providing in a reaction vessel reagents suitable for performing Polymerase Chain Reaction (PCR) comprising: (a) an amount of a first primer or set of first primers sufficient to permit amplification of a nucleic acid template without substantial residual unincorporated primer, wherein each first primer comprises a locus-specific sequence capable of annealing to the nucleic acid template at a first temperature and a tag sequence that does not anneal to the nucleic acid template; and (b) a second primer or set of second primers wherein each second primer comprises a sequence capable of annealing to a nucleic acid comprising a sequence complementary to a portion of a first primer comprising the tag sequence and wherein each second primer has a melting temperature (Tm) lower than the Tm of the first primer and is not capable of annealing substantially to the first primer
  • Tm melting temperature
  • one or more cultivar(s) of wheat is (are) characterised or identified or one or more cultivar(s) of barley is (are) characterised or identified.
  • one or more species or genera of plants is characterised or identified, for example, the plant is wheat; or the plant is barley; or the plant is Prunus spp, e.g., apricot or cherry.
  • one or more species or genera of animals is characterised or identified. Suitable animals will be apparent to the skilled artisan and include, for example, a bovine animal or an ovine animal.
  • fungus is characterised or identified.
  • the fungus is Rhynchosporium secalis
  • the detected, amplified nucleic acid from the individual, isolate of an organism, cultivar of an organism, species or genus is compared to the detected, amplified nucleic acid from a reference sample to thereby characterise or identify the individual, isolate of an organism, cultivar of an organism, species or genus
  • nucleic acid template or reference sample is selected from the group consisting of:
  • nucleic acid from an individual, isolate, cultivar, species or genus (i) nucleic acid from an individual, isolate, cultivar, species or genus; (ii) nucleic acid from a plurality of individuals, isolates, cultivars, species or genera;
  • the nucleic acid template or reference sample is nucleic acid from a cultivar of wheat.
  • the nucleic acid template or reference sample is nucleic acid from barley.
  • the nucleic acid template or reference sample is nucleic acid from one or more species or genera of plant, e.g., wheat or barley.
  • the plant is Prunus spp, e.g., apricot or cherry.
  • the nucleic acid template or reference sample is nucleic acid from one or more species or genera of animal, e.g., a bovine animal or an ovine animal.
  • the nucleic acid template or reference sample is nucleic acid from one or more species or genera of fungus, e.g., Rhynchosporium secalis.
  • the present invention contemplates a method for differentiating between or identifying differences between related organisms.
  • the method may be used to differentiate between members of a population of related (or inbred) organisms that are used to map the site of a gene.
  • the present invention additionally provides a process of differentiating between two or more related organisms or individuals said process comprising: (i) providing in a reaction vessel reagents suitable for performing Polymerase Chain Reaction (PCR) comprising: (a) an amount of a first primer or set of first primers sufficient to permit amplification of a nucleic acid template without substantial residual unincorporated primer, wherein each first primer comprises a locus-specific sequence capable of annealing to the nucleic acid template at a first temperature and a tag sequence that does not anneal to the nucleic acid template; and (b) a second primer or set of second primers wherein each second primer comprises a sequence capable of annealing to a nucleic acid comprising a sequence complementary to a portion of PCR
  • the detected, amplified nucleic acid from the organism or individual is compared to the detected, amplified nucleic acid from one or more related organism(s) or individual(s) to thereby differentiate between the two or more related organisms or individuals.
  • the present invention is useful for differentiating between any individuals and/or organisms.
  • the present inventors have used the method of the present invention to amplify genetic markers that facilitate differentiation between wheat plants, barley plants, Primus plants, bovine animals, ovine animals or fungi.
  • the present invention is also useful for detecting or diagnosing an infection in a subject.
  • the present invention provides a process for detecting an infection or diagnosing an infection in a subject, said process comprising:
  • PCR Polymerase Chain Reaction
  • a reaction vessel reagents suitable for performing Polymerase Chain Reaction (PCR) comprising: (a) an amount of a first primer or set of first primers sufficient to permit amplification of a nucleic acid template from an infectious organism without substantial residual unincorporated primer, wherein each first primer comprises a locus-specific sequence capable of annealing to the nucleic acid template at a first temperature and a tag sequence that does not anneal to the nucleic acid template; and (b) a second primer or set of second primers wherein each second primer comprises a sequence capable of annealing to a nucleic acid comprising a sequence complementary to a portion of a first primer comprising the tag sequence and wherein each second primer has a melting temperature (Tm) lower than the Tm of the first primer and is not capable of annealing substantially to the first primer or nucleic acid template at the first temperature;
  • Tm melting temperature
  • the method additionally comprises providing the sample from the subject.
  • the sample is isolated previously from a subject.
  • Suitable samples will be apparent to the skilled artisan and will depend on the subject from which the sample is isolated and the infectious organism.
  • an infection in a human may be detected by performing the method of the present embodiment to detect nucleic acid from an infectious organism in a blood sample or a sample derived therefrom.
  • nucleic acid from an infectious fungus is detected in leaf tissue from a plant.
  • the present invention is useful for diagnosing a variety of infections and/or diseases.
  • the present inventors have detected the presence of the infectious organism Rhynchosporium secalis in plant tissue.
  • the present invention additionally provides a primer comprising a locus-specific sequence comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 15 to 54 or 57 to 5345 and a tag sequence.
  • the tag sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 14.
  • the tag sequence comprises a nucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14.
  • the present invention also provides a primer (i.e., a second primer) comprising a sequence capable of annealing to a nucleic acid comprising a sequence complementary to the tag sequence of a primer described herein according to any embodiment.
  • the primer comprises the nucleotide sequence set forth in SEQ ID NO: 55 or SEQ ID NO: 56.
  • the primer is labelled with a detectable marker, e.g., a fluorescent marker.
  • the present invention additionally provides for the use of a primer comprising a locus- specific sequence capable of annealing to a nucleic acid template and a tag sequence in the method or process described herein according to any embodiment.
  • the locus specific sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 15 to 54 or 57 to 5345.
  • tag sequences include a tag sequence comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 14.
  • the present invention also provides for the use of a primer comprising a nucleotide sequence set forth in SEQ ID NO: 55 or SEQ ID NO: 56 in the method or process described herein according to any embodiment..
  • the present invention additionally provides a kit comprising (a) one or more first primers or set(s) of first primers wherein each first primer comprises a locus-specific sequence and a tag sequence; and (b) a second primer or set of second primers wherein each second primer is capable of annealing to a nucleic acid comprising a sequence complementary to all or a part of said tag sequence, wherein each of said first primers are capable of annealing to a nucleic acid template at a first temperature and wherein each of said second primers is capable of annealing to all or part of said tag sequence at a temperature lower than the first temperature but not at the first temperature.
  • the kit additionally comprises allele length data comprising the lengths of predicted amplification products of the first primer(s) or set(s) of first primers.
  • the kit additionally comprises an algorithm for determining the lengths of predicted amplification products of the first primer(s) or set(s) of first primers.
  • the kit additionally comprises groups of allele length data wherein each group of allele length data comprises the lengths of predicted amplification products of the first primer(s) or set(s) of first primers such that said predicted amplification products in each group are of sufficiently different length to permit their resolution by a means that fractionates nucleic acid according to length.
  • the kit additionally comprises an algorithm for determining groups of predicted amplification products of the first primer(s) or set(s) of first primers such that said predicted amplification products in each group are of sufficiently different length to permit their resolution by a means that fractionates nucleic acid according to length.
  • the kit additionally comprises an algorithm for determining or selecting first primer(s) or set(s) of first primer(s) or groups thereof from the allele length data or lengths of predicted amplification products that are capable of producing predicted amplification products of sufficiently different length to permit their resolution by a means that fractionates nucleic acid according to length.
  • the kit additionally comprises sufficient information to access allele length data comprising the lengths of predicted amplification products of the first primer(s) or set(s) of first primers and/or an algorithm for determining the lengths of said predicted amplification products and/or for grouping said predicted amplification products such that the predicted amplification products in each group are of sufficiently different length to permit their resolution by a means that fractionates nucleic acid according to length.
  • the kit additionally comprises sufficient information to access an algorithm for determining or selecting first primer(s) or set(s) of first primers or groups thereof from the allele length data or lengths of predicted amplification products.
  • the information comprises an access code to a computer-readable medium.
  • the allele length data or algorithm in the kit is in a computer-readable medium.
  • the computer-readable medium comprises a computer database and/or is implemented using a computer programme.
  • the computer-readable medium is a web-based database or programme.
  • the computer-readable medium comprises a computer diskette or CD-ROM.
  • the kit is packaged with instructions for use.
  • the kit is packaged with instructions for use in the method or process described herein according to any embodiment.
  • the first primer comprises a locus specific sequence comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 15 to 54 or 57 to 5345.
  • the first primer comprises a tag sequence comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 14.
  • the second primer comprises a nucleotide sequence set forth in SEQ ID NO: 55 or SEQ ID NO: 56.
  • the first primer or set of first primers and the second primer or set of second primers are contained within a reaction vessel.
  • the first primer or set of first primers in the reaction vessel are in an amount sufficient to permit amplification of a nucleic acid template without substantial residual unincorporated primer.
  • the second primer or set of second primers in the reaction vessel are in an amount sufficient to permit amplification of a nucleic acid template without substantial residual unincorporated primer.
  • the kit additionally comprises means for amplifying a nucleic acid.
  • Suitable means will be apparent to the skilled artisan and include, for example, a polymerase and/or a suitable buffer and/or dNTPs. Suitable means are described herein and are to be taken to apply mutatis mutandis to the present embodiment of the invention.
  • the present invention also provides the kit as described herein according to any embodiment when used in the method as described herein according to any embodiment.
  • the present invention additionally provides for the use of the kit described herein according to any embodiment for amplifying nucleic acid.
  • the kit is used to amplify nucleic acid using a method described herein according to any embodiment.
  • Figure Ia is a copy of a photographic representation showing the effect of DNA concentration on the specificity and efficiency of the assay of the invention.
  • Increasing concentrations of nucleic acid from various strains of wheat was used as a template for a PCR of the invention.
  • This analysis was performed with primer sets capable of amplifying different genetic markers.
  • the primers used in this assay amplified the marker cfd55 or barc55 as shown at the base of the Figure.
  • the marker designations relate to the primers set forth in Table 6, 7 12 or 16. Template source and concentrations used for each reaction are set forth in Table 1.
  • Figure Ib is a copy of a photographic representation showing the effect of DNA quality on the specificity and efficiency of the assay of the invention.
  • Nucleic acid was extracted from different strains of barley using two different methods (a salt based method that preserved the quality of the nucleic acid or a sodium hydroxide based method that produced low-quality nucleic acid).
  • the effect of DNA quality on an amplification method of the invention was determined using two different primer sets.
  • the primers used in this assay amplified the marker bmag ⁇ or gmsl as shown at the base of the Figure.
  • the marker designations relate to the primers set forth in Table 6, 7 12 or 16.
  • the template used for each amplification reaction is set forth in Table 2.
  • Figure 2 is a copy of a photographic representation showing the effect of primer concentration on the specificity and efficiency of an assay of the invention.
  • PCR reactions according to an embodiment of the invention were performed using decreasing concentrations of primers to determine the effect of primer concentration on specificity of amplification.
  • the arrows at the bottom of the gel images indicate decreasing concentration of locus-specific primer.
  • the primers used in this assay amplified the marker gwm642, gdm77, gwmlO2 5 gwml94 or gwml74 as shown at the base of the Figure.
  • the marker designations relate to the primers set forth in Table 6, 7 12 or 16. Template, primers and primer concentrations are set forth in Table 3.
  • Figure 3 is a copy of a photographical representation showing amplification products produced using a multiplex reaction of the invention performed to amplify six markers simultaneously.
  • Nucleic acid was amplified from a variety of sources using primers specific for a variety of different marker.
  • the template used for each reaction is set forth in Table 4 and the markers amplified set forth in Table 5.
  • Figure 4 is a copy of a photographic representation showing a comparison of nucleic acid amplified using conventional techniques (conv) and nucleic acid amplified using the assay of the invention (Mpx Rdy).
  • the primers used in this assay amplified the marker gwm276, gwm301, gwm340, gwm368, gwm389 or gwm513 as indicated at the base of the Figure.
  • the marker designations relate to the primers set forth in Table 6, 7 12 or 16.
  • Figure 5 is a graphical representation showing the frequency distribution of mean fluorescence peak heights for 366 single-locus barley and wheat multiplex-ready SSRs. Amplification was performed for 366 markers and the approximate level of nucleic acid amplified determined. The average fluorescence peak height for each marker was calculated from the fluorescence peak heights observed for eight genetically diverse barley (or wheat) cultivars, indicating that the method used amplified approximately equal levels of nucleic acid in a large number of reactions.
  • Figure 6 is a graphical representation showing the frequency distribution of mean fluorescence peak heights for 1070 single-locus barley and wheat multiplex-ready SSRs detected using an ABI3730 instrument. Amplification was performed for 1070 markers and the approximate level of nucleic acid amplified determined. The average fluorescence peak height for each marker was calculated from the fluorescence peak heights observed for eight genetically diverse barley (or wheat) cultivars, indicating that the method used amplified approximately equal levels of nucleic acid in a large number of reactions.
  • Figure 7 is a graphical representation showing the frequency distribution of mean fluorescence peak heights for 64 single-locus Prunus spp. multiplex-ready SSRs detected using an ABI3730 instrument. Amplification was performed for 64 markers and the approximate level of nucleic acid amplified determined. The average fluorescence peak height for each marker was calculated from the fluorescence peak heights observed for six apricot varieties and six cherry varieties, indicating that the method used amplified approximately equal levels of nucleic acid in a large number of reactions.
  • Figure 8 is a copy of a photographic representation showing amplicons produced using a multiplex reaction of the invention and separated by electrophoresis on a 4% polyacrylamide gel.
  • Six-plex reactions were performed with nucleic acid from one of six apricot varieties as set forth in Table 8.
  • the markers amplified are set forth in Table 9 and the primers used to amplify the markers in Table 7.
  • Figure 9 is a copy of a photographic representation showing amplicons produced using a multiplex reaction of the invention and separated by electrophoresis on a 4% polyacrylamide gel. Ten-plex reactions were performed with nucleic acid from one of two apricot varieties or one of two cherry varieties as set forth in Table 10. The markers amplified are set forth in Table 11 and the primers used to amplify the markers in Table 7.
  • Figure 10a is a copy of a photographic representation showing the effect of locus specific primer concentration on the amplification of a marker (HMHlR) using nucleic acid from cattle or sheep.
  • Nucleic acid from the sources set forth in Table 13 was amplified with a primer pair set forth in Table 12 and separated by electrophoresis using a 4% polyacrylamide gel. The concentration of primer used is indicated in Table 13.
  • Figure 10b is a copy of a photographic representation showing the effect of locus specific primer concentration on the amplification of a marker (BM2113) using nucleic acid from cattle or sheep.
  • Nucleic acid from the sources set forth in Table 13 was amplified with a primer pair set forth in Table 12 and separated by electrophoresis using a 4% polyacrylamide gel. The concentration of primer used is indicated in Table 13.
  • Figure 11 is a copy of a photographic representation showing the effect of template DNA concentration on the amplification of a marker (BM2113) using nucleic acid from cattle or sheep.
  • Nucleic acid from the sources set forth in Table 14 was amplified with a primer pair set forth in Table 12 and separated by electrophoresis using a 4% polyacrylamide gel. The concentration of nucleic acid used is indicated in Table 14.
  • Figure 12 is a copy of a photographic representation showing amplicons produced using a multiplex reaction of the invention and separated by electrophoresis on a 4% polyacrylamide gel. Three-plex reactions were performed with nucleic acid from cattle or sheep as set forth in Table 15. The markers amplified are set forth in Table 16 and the primers used to amplify the markers in Table 12. Detailed description of the preferred embodiments Primer design
  • a primer is designed such that it comprises a sequence having at least about 80% identity overall to a strand of a template nucleic acid. More preferably, the degree of sequence identity is at least about 85% or 90% or 95% or 98% or 99%.
  • the primer or a region of a primer may comprise a sequence having at least about 80% identity to a strand of a locus of interest. Accordingly, this provides the "locus specific" region of the first primer or set of primers.
  • a primer of the invention (or more specifically, the locus-specific region of a first primer of the invention) will depend upon the sequence of the nucleic acid of interest. Accordingly, the sequence of the locus-specific region of a first primer of the invention is not to be taken to be limited to a particular sequence. Rather the sequence need only be sufficient to allow for annealing of the first primer to a template nucleic acid and initiation of an amplification reaction.
  • a primer is generally extended in the 5'- to 3'- direction it is preferred that at least the 3 '-terminal nucleotide is complementary to the relevant nucleotide in the template nucleic acid. More preferably, at least the 3 or 4 or 6 or 8 or 10 contiguous nucleotides at the 3'- terminus of the primer are complementary to the relevant nucleotides in the template nucleic acid. The complementarity of the 3' terminus of the primer ensures that the extending end of the primer is capable of initiating amplification of the template nucleic acid, for example, by a polymerase.
  • a primer of the invention does not comprise multiple contiguous nucleotides that are not identical to a strand of the template nucleic acid.
  • the primer comprises no more than 6 or 5 or 4 or 3 or 2 contiguous nucleotides that are not identical to a strand of the template nucleic acid. More preferably, any nucleotides that are not identical to a strand of the template nucleic acid are non-contiguous.
  • nucleotide sequences may be aligned and their identity calculated using the BESTFIT program or other appropriate program of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, Nucl. Acids Res. 12, 387-395, 1984).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST 2 Sequences a tool that is used for direct pairwise comparison of two nucleotide sequences.
  • NCBI Network Codebook
  • a primer comprises or consists of at least about 10 nucleotides, more preferably at least about 12 nucleotides or at least about 15 or 20 nucleotides that anneal to a nucleic acid template or are complementary to the nucleic acid template.
  • primers are also used in PCR reactions, for example, reactions in which a long region of nucleic acid (e.g., greater than lOOObp) is amplified.
  • the present invention additionally contemplates a primer comprising at least about 25 or 30 or 35 nucleotides that anneal to a nucleic acid template or are complementary to the nucleic acid template.
  • a primer comprising one or modified bases need only comprise a region of at least about 8 nucleotides that anneal to a nucleic acid template or are complementary to the nucleic acid template.
  • the complementary nucleotides are contiguous.
  • the number of nucleotides capable of annealing to a nucleic acid template is related to the stringency under which the primer will anneal.
  • a primer of the invention anneals to a nucleic acid template under moderate to high stringency conditions.
  • the stringency under which a primer of the invention anneals to a template nucleic acid is determined empirically. Generally, such a method requires performance of an amplification reaction using one or more primers under various conditions and determining the level of specific amplification produced.
  • a primer of the invention is labelled with a detectable marker (e.g., a radionucleotide or a fluorescent marker) and the level of primer that has annealed to a target nucleic acid under suitably stringent conditions is determined.
  • a detectable marker e.g., a radionucleotide or a fluorescent marker
  • a moderate stringency annealing conditions will generally be achieved using a condition selected from the group consisting of: (i) an incubation temperature between about 42°C and about 55°C; (ii) an incubation temperature between about 15 0 C and 1O 0 C less than the predicted
  • Tm for a primer for a primer
  • Mg 2+ concentration of between about 2mM and 3mM.
  • High stringency annealing conditions will generally be achieved using a condition selected from the group consisting of:
  • a reagent such as, for example, glycerol (5-10%), DMSO (2-10%), formamide (1 - 5%), Betaine (0.5 - 2M) or tetramethylammonium chloride (TMAC, >50mM) are known to alter the annealing temperature of a primer and a nucleic acid ternplate(Sarkar et ah, Nucl. Acids Res. 18: 7465; 1990, Baskaran et al. Genome Res. 6: 633-638, 1996; and Frackman et al, Promega Notes 65: 27, 1998).
  • the conditions under which a primer anneals to a nucleic acid template are determined in silico.
  • methods for determining the predicted melting temperature (or Tm) of a primer or the temperature at which a primer denatures from a specific nucleic acid are known in the art.
  • the method of Wallace et al estimates the Tm of a primer based on the G, C, T and A content.
  • the described method uses the formula 2(A + G) + 4(G + C) to estimate the Tm of a probe or primer.
  • the nearest neighbour method described by Breslauer et al, Proc. Natl. Acad. ScI USA, 83:3746-3750, 1986 is useful for determining the Tm of a primer.
  • the nearest neighbour method uses the formula:
  • ⁇ H° is standard enthalpy for helix formation
  • ⁇ S° is standard entropy for helix formation
  • Q is the total strand concentration
  • n reflects the symmetry factor, which is 1 in the case of self-complementary strands and 4 in the case of non-self- complementary strands
  • R is the gas constant (1.987).
  • dH enthalpy for helix formation
  • dS entropy for helix formation
  • R molar gas constant (1.987cal/°C mol)
  • c is the nucleic acid molar concentration (determined empirically, W.Rychlik et.al, supra), (default value is 0.2 ⁇ M for unified thermodynamic parameters)
  • [K + ] is salt molar concentration (default value is 50 mM).
  • Suitable software for determimng the Tm of an oligonucleotide using the nearest neighbour method is known in the art and available from, for example, US Department of Commerce, Northwest Fisheries Service Center and Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine.
  • M is the molarity of Na+ and % form is the percentage of formamide (set to 50%)
  • Tm is determined using the formula (described by Giesen et al, Nucl. Acids Res., 26: 5004-5006):
  • T n U 1n D NA is the melting temperature as calculated using a nearest neighbour model for the corresponding DNA/DNA duplex applying AH 0 and AS 0 values as described by SantaLucia et al. Biochemistry, 35: 3555-3562, 1995.
  • ⁇ S+MEMa3 ⁇ £C/4r 7 3 A suitable program for determining the Tm of a primer comprising LNA is available from, for example, Exiqon, Vedbaek, Germany.
  • Medium stringency is to be considered to be within 1O 0 C to 2O 0 C or 1O 0 C to 15 0 C of the calculated Tm of the probe or primer.
  • a primer of the invention selectively anneals to a target nucleic acid.
  • selective anneals means that the probe is used under conditions where a target nucleic acid, anneals to the probe to produce a signal that is significantly above background (i.e., a high signal-to-noise ratio).
  • the level of specificity of annealing is determined, for example, by performing an amplification reaction using the primer and detecting the number of different amplicons produced.
  • different amplicons is meant that amplified nucleic acids of differing nucleotide sequence and/or molecular weight are produced. Clearly, amplicons that differ in molecular weight are readily identified, for example, using gel electrophoresis.
  • a primer that selectively anneals to a target nucleic acid produces an amplicon at a level greater than any other amplicon. Preferably only one amplicon is produced at a detectable level.
  • An alternative technique to determine the selective annealing of a primer of the invention comprises performing a search of known nucleotide sequences from the sample being assayed (e.g., a database of known sequences from an organism or cell from which the template nucleic acid is derived). Using this technique a sequence similar to or complementary to the sequence of the primer is identified. While such a technique does not ensure selective annealing it is useful for determining a primer (or set of primers) capable of annealing to a plurality of sites in a nucleic acid and possibly producing multiple amplicons (i.e., non-selective annealing).
  • a primers or primer sequence that is predicted to be or shown to be capable of selectively annealing to a nucleic acid template is also optionally analyzed for one or more additional characteristics that make it suitable for use as a primer in the method of the invention.
  • a primer is analyzed to ensure that it is unlikely to form secondary structures (i.e., the primer does not comprise regions of self- complementarity).
  • all primers may be assessed to determine their ability to anneal to one another and form "primer dimers".
  • a primer satisfies the following criteria:
  • the primer comprises a region that is to anneal to a target sequence having at least about 17-28 bases in length; (ii). the primer comprises about 50-60% (G+C); (iii) the 3'-terminus of the primer is a G or C, or CG or GC (this prevents "breathing" of ends and increases efficiency of initiation of amplification); (iv) preferably, the primer has a Tm between about 55 and about 8O 0 C; (v) the primer does not comprise three or more contiguous Cs and/or Gs at the 3'- ends of primers (as this may promote mispriming at G or C-rich sequences due to the stability of annealing);
  • the 3'-end of a primer should not be complementary with another primer in a reaction; and (vii) the primer does not comprise a region of self-complementarity.
  • the composition of the template nucleic acid is considered (i.e. the nucleotide sequence) as is the type of amplification reaction to be used.
  • the 3 1 nucleotide of one of the primers used in such a reaction corresponds to the site of an allele of interest, such as, for example a SNP. In this manner only in the presence of a nucleotide that is complementary to that in the primer does annealing occur and amplification achieved.
  • a primer or pair of primers is designed that anneals to nucleic acid adjacent (albeit, not necessarily immediately adjacent) to the site of the simple repeat.
  • the primer be used in a multiplex reaction it is preferred that the amplification product produced is not similar in molecular weight to that produced using another primer or set thereof thereby rendering detection difficult. Accordingly, it is preferred that there is sufficient difference in molecular weight in amplified products to enable detection using a technique known in the art, such as, for example, gel electrophoresis or mass spectrometry.
  • each of the nucleic acids amplified using the method of the invention is different molecular weight.
  • the tag sequence in a first primer of the invention serves the dual purpose of enhancing the specificity annealing of the first primer and providing a site within the amplicon produced in the first amplification reaction to which a second primer of the invention anneals.
  • the tag sequence comprises a sequence of nucleotides that produce a site or template suitable for annealing of another primer when used in an amplification reaction. Methods for determining a sequence suitable for annealing of a primer are known in the art and/or described hereinabove.
  • the length of the tag sequence depends on the number of nucleotides required by the second primer to anneal and initiate nucleic acid amplification in an amplification reaction.
  • the second primer is capable of annealing to the amplicon produced in the first reaction under moderate to high stringency conditions.
  • the second primer comprise one or more ribonucleotides and/or one or more PNA residues and/or one or more LNA residues the number of nucleotides required for annealing is fewer than in the case of a primer comprising only deoxyribonucleotides .
  • probes comprising LNA and/or PNA are capable of selectively annealing to a target comprising as few as 8 nucleotides. Accordingly, it is preferred that the tag sequence comprises at least about 8 nucleotides. More preferably, the tag region comprises at least about 10 nucleotides, more preferably about 12 nucleotides, even more preferably, at least about 15 nucleotides, still more preferably, at least about 17 nucleotides and even more preferable at least about 19 nucleotides.
  • a tag sequence that is unable to anneal to the template nucleic acid is selected to ensure that it does not cause non-specific annealing of the first primer in the first amplification reaction and the amplification of non-template nucleic acid.
  • the tag sequence is unable to anneal to a nucleic acid in a sample being assayed to such a degree as to amplify nucleic acid to a detectable level (i.e. background amplification).
  • the requirement that the tag sequence not anneal to a template nucleic acid does not require that the tag sequence not anneal under any conditions. Rather, it is preferred that the tag sequence is not capable of annealing to the template nucleic acid under conditions sufficient for annealing of the locus specific sequence to the template nucleic acid.
  • the tag sequence may anneal to the template nucleic acid under low stringency conditions.
  • the tag comprises a sequence of nucleotides that does not naturally occur in a sample being assayed. Methods for determining a sequence that is not present in a sample being assayed will be apparent to the skilled artisan.
  • the nucleotide sequence of the tag sequence is analyzed using a program, such as, for example, BLAST to determine whether or not that sequence (or its complement) occurs naturally in an organism being assayed.
  • a nucleotide sequence is selected from an organism different to that from which a sample being assayed is derived.
  • the tag is derived from, for example, an unrelated mammal or plant or a virus or a bacteria or a fungus that is not a pathogen of the mammal or plant.
  • a tag sequence is selected from a bacterial page gene, e.g., tag comprises a sequence from M13 phage GTAAACGACGGCCAGT (SEQ ID NO: 1) or a sequence from T7 phage TAATACGACTCACTATAGGG (SEQ ID NO: 2).
  • Such a tag is useful as, for example, a tag sequence for a primer used to amplify a sequence from a mammal or a plant or a fungus.
  • an artificial sequence is used for a tag.
  • a tag sequence described by Heath et al, Med Genet 37:272-280, 2000 is used (i.e., a tag sequence comprises a nucleotide sequence selected from the group consisting of:
  • the tag sequence comprises a nucleotide sequence selected from the group consisting of: (i) GCTAAATCGGACTAGCTACC (SEQ ID NO: 7); and (ii) TAATCCAGCTACGCTGCATC (SEQ ID NO: 8).
  • a zip-code sequence is used as a tag sequence.
  • a tag sequence comprises the nucleotide sequence GGAGCACGCTATCCCGTTAGAC (SEQ ID NO: 9) or CGCTGCCAACTACCGCACATG (SEQ ID NO: 10) or CCTCGTGCGAGGCGTATTCTG (SEQ ID NO: 11) or
  • a zip code sequence is generally a sequence of nucleotides that has been produced synthetically and is predicted not to occur in a nucleic acid derived from a specific organism.
  • the present inventors have used a tag sequence that is artificially produced and predicted not to anneal to amplify nucleic acid under conditions suitable for annealing of the locus specific sequence of a first primer of the invention.
  • the tag region of the first primer or set of primers comprises a nucleotide sequence CACGACGTTGTAAAACGAC (SEQ ID NO: . 13) or GTACATTAAGTTCCCATTAC (SEQ ID NO: 14).
  • the tag comprises or consists of the sequence of the second primer of the invention.
  • the tag sequence need not only comprise a region that provides a suitable template for annealing of a second primer to an amplicon produced in the first amplification.
  • the tag comprises additional sequence to facilitate binding of a polymerase to enable replication of amplified nucleic acid.
  • the tag comprises a spacer sequence between the locus specific sequence and the tag sequence.
  • a spacer region is rich in adenosine and/or thymine rather than cytosine and/or guanine. This is because a spacer region rich in cytosine and/or guanine increases the Tm of the first primer more than a spacer region rich in adenosine and/or thymine. Accordingly, a CG rich spacer may cause background by non-specific amplification of nucleic acids.
  • the tag comprises the nucleotide sequence of one or more restriction endonuclease sites.
  • a restriction endonuclease site is located at, for example, the 5' end or the 3' end or both the 5' and 3' ends or within the primer.
  • the use of such a restriction endonuclease site enables, for example, cloning of a nucleic acid amplified using the method of the invention.
  • the primer may be advantageous to include sufficient additional nucleic acid to ensure binding and cleavage of the site. Suitable sequences to ensure such cleavage are known in the art and described, for example, in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995).
  • the tag comprises additional sequence to enable for transcription and/or translation of a nucleic acid amplified by the method of the invention.
  • the tag comprises the sequence of a promoter, such as, for example, the T7 promoter.
  • a promoter enables the production of, for example, a riboprobe, for use in, for example, in situ hybridization and/or Northern or Southern hybridization.
  • the method of the invention enables not only the amplification and/or detection of a template nucleic acid but also the production of a probe that allows detection of a cell, tissue or organism that comprises or expresses the template nucleic acid.
  • a second primer of the invention is capable of annealing to and initiating amplification of an amplicon produced in a first reaction using a first primer of the invention.
  • the second primer is capable of annealing to a strand of template nucleic acid comprising the tag sequence of the first primer. This is not to say that the second primer anneals to the entire tag sequence of the first primer. Nor does the second primer only anneal to the tag sequence of the first primer. Rather, the second primer anneals to a nucleic acid comprising a tag sequence, or to a nucleic acid within a tag sequence.
  • the degree of nucleotide sequence identity between the portion of the first primer comprising the tag sequence (or a portion thereof) and the second primer is at sufficient to enable selective annealing of the second primer to an amplicon produced using the first primer.
  • the second primer is at least about 80% or 90% identical to a region of the first primer.
  • the degree of identity is at least about 95%, more preferable 97% or 98% or 99%.
  • the portion of the first primer comprising the tag sequence includes a sufficient number of nucleotides to enable the second primer to selectively anneal to an amplicon produced using a first primer of the invention.
  • the number of nucleotides required for annealing of a primer to a template nucleic acid is dependent on the composition of the primer.
  • a primer consisting of deoxynucleotides comprises at least about 12 nucleotides, or 14 nucleotides or 16 nucleotides. Suitable criteria for the design of a primer (for example, primer length) are described supra and are taken to apply mutatis mutandis to this embodiment of the invention.
  • a second primer comprises 14 nucleotides or 16 nucleotides identical to a portion of a first primer comprising a tag sequence.
  • the region of the first primer is also of sufficient nucleotide composition to enable annealing of the second primer or set thereof to an amplification product produced using the first primer and initiation of nucleic acid replication mediated by a polymerase.
  • a second primer of the invention has a Tm lower than that of the first primer and is not capable of annealing substantially to its template nucleic acid at the temperature at which the first primer anneals to its template in the first reaction.
  • the first primer anneals to target nucleic acid at a temperature at which there is no detectable annealing of the second primer to target nucleic acid.
  • the difference in the Tm of the first and second primers is sufficient that the second primer is unable to anneal to a detectable level under conditions sufficient for selective annealing of the first primer. Accordingly, the second primer is unable to anneal under moderate to high stringency conditions as determined for the first primer.
  • the difference in Tm between the first and second primers is at least about 1O 0 C or at least about H 0 C or at least about 12 0 C or at least about 15 0 C or at least about 2O 0 C.
  • the first primer (or set thereof) has a Tm at least about 15 0 C to 18 0 C greater than that of the second primer. This is not to say that all primers of a set of primers must have the same Tm, rather the Tm of each primer of a first set of primers is at least about 1O 0 C greater than that of a second primer or set thereof.
  • the second primer has a Tm of at least about 35 0 C, for example, at least about 38 0 C, preferably, at least about 4O 0 C, more preferably, at least about 42 0 C.
  • a second primer may be analyzed in silico to predict whether or not it will anneal under conditions sufficient for selective annealing of the first primer
  • the presence or absence of detectable annealing of the second primer should be determined or confirmed by empirical means.
  • an amplification reaction is performed using the second primer and a suitable template under conditions used for annealing the first primer.
  • the first primer (or set thereof) anneals to its target at a temperature at least about 5 0 C greater than the temperature at which the second primer anneals to its target, more preferably, at least about 6 0 C, more preferably, at least about 7 0 C greater and even more preferably, at least about 9 0 C greater.
  • a second primer used in the method of the present invention comprises the nucleotide sequence of a tag region described supra or a region thereof.
  • the second primer comprises a nucleotide sequence within a nucleotide sequence set forth in any one of SEQ ID NOs: 1-13.
  • the second primer comprises a sequence of at least 10 consecutive nucleotides of a nucleotide sequence set forth in any one of SEQ ID NOs: 1-13.
  • the second primer comprises a sequence of at least 12 consecutive nucleotides of a nucleotide sequence set forth in any one of SEQ ID NOs: 1-13.
  • the second primer comprises a sequence of at least 15 consecutive nucleotides of a nucleotide sequence set forth in any one of SEQ ID NOs: 1-13.
  • the second primer comprises a nucleotide sequence set forth in SEQ ID NO: 55 or 56.
  • the present invention provides a method amenable to multiplex amplification or detection of nucleic acids.
  • multiplex amplification will be understood by the skilled artisan to mean that a plurality of distinct nucleic acids are amplified in a single reaction, e.g., a plurality of nucleic acids are amplified using the method of the present invention.
  • multiplex detection of nucleic acids shall be taken to mean that a plurality of nucleic acids is detected in a single detection reaction.
  • each of the nucleic acids may be amplified individually using the method described herein according to any embodiment.
  • a plurality of nucleic acids e.g., a set of the nucleic acids to be detected is amplified in a multiplex reaction.
  • the present invention contemplates amplifying one or more nucleic acids using the method of the invention and pooling the amplified nucleic acid with additional amplified nucleic acid prior to detecting the amplified nucleic acids.
  • a multiplex assay is performed in which each of the amplification products produced may be detected using a detection means such as, for example, electrophoresis, e.g., polyacrylamide gel electrophoresis or capillary electrophoresis or mass spectrometry. Suitable fractionation methods will be apparent to the skilled artisan and/or described herein.
  • the length of an amplification product produced by a primer or a set of primers is determined empirically and/or predicted from in silico information.
  • suitable primers are determined from a database comprising nucleotide sequence information, such as, for example, the database of the National Center for Biotechnology Information or the Ensembl database available from the European Biotechnology Institute and/or the Wellcome Trust Sanger Institute.
  • amplification products having sufficiently different lengths are determined from a database of allele length data, e.g., , information concerning the length of amplification products produced by amplifying specific alleles or markers, e.g., a SSR.
  • Suitable databases of such information are known in the art and include, for example, the Multiplex-Ready Marker Database available from Molecular Plant Breeding CRC, Victoria, Australia; the Plant Simple Sequence Repeat Database available from Clemson University Genomics Institute, Clemson, SC, USA; or the cotton SSR database available from Cotton Functional Genomics Center, University of California, Davis, CA, USA.
  • primer(s) or sets of primers is/are designed, produced or obtained that is/are capable of amplifying said amplification products.
  • primers may be, for example, produced using a method described herein or obtained from a commercial source.
  • primers or sets of primers are selected that produce amplification products having a sufficient difference in length to permit their resolution by a means that fractionates nucleic acid according to its length. For example, primers capable of producing amplification products having at least 5 nucleotide residues difference in length are selected. Preferably, primers capable of producing amplification products having at least 6 nucleotide residues difference in length are selected. Preferably, primers capable of producing amplification products having at least 7 nucleotide residues difference in length are selected. Preferably, primers capable of producing amplification products having at least 8 nucleotide residues difference in length are selected.
  • primers capable of producing amplification products having at least 9 nucleotide residues difference in length are selected.
  • primers capable of producing amplification products having at least 10 nucleotide residues difference in length are selected.
  • primers capable of producing amplification products having at least 15 nucleotide residues difference in length are selected.
  • primers capable of producing amplification products having at least 20 nucleotide residues difference in length are selected.
  • primers capable of producing amplification products having at least 25 nucleotide residues difference in length are selected.
  • primers capable of producing amplification products having at least 50 nucleotide residues difference in length are selected.
  • the primer(s) or set(s) of primers is(are) selected using a computer-based algorithm or nucleic acids to be amplified is(are) selected using a computer-based algorithm.
  • a computer-based algorithm is used that analyses the predicted length of each of the amplification products that are proposed to be amplified. Said algorithm then selects those amplification products that are of sufficiently different lengths to permit their resolution by a means that fractionates nucleic acid according to its length. Based on the selected amplification products, primers capable of amplifying said amplification products are selected, produced or obtained.
  • the algorithm selects one or more sets of first primers suitable for amplifying a plurality of amplification products.
  • the algorithm preferably groups primers suitable for producing amplification products of sufficiently different lengths to permit their resolution by a means that fractionates nucleic acid according to its length to produce a set of primers, wherein the primers are grouped to reduce or minimize the number of sets of first primers required to amplify the plurality of nucleic acids.
  • the algorithm selects one or more nucleic acids to be amplified or detected in a reaction from a plurality of nucleic acids.
  • the algorithm preferably groups nucleic acids of sufficiently different lengths to permit their resolution by a means that fractionates nucleic acid according to its length, wherein the nucleic acids are grouped to reduce or minimize the number of groups required to amplify the plurality of nucleic acids.
  • Suitable algorithms will be apparent to the skilled artisan and/or are commercially available.
  • the BINNER software available from Molecular Plant Breeding CRC, Victoria, Australia is a software program that implements a suitable algorithm.
  • the algorithm selects suitable primers or nucleic acids from a database of allele length data for nucleic acids.
  • suitable databases will be apparent to the skilled artisan and include a database discussed supra.
  • a suitable database is produced, e.g., using suitable software, and accessed by the algorithm.
  • a database may be stored or saved on a computer-readable medium, such as, for example, a diskette, a CD-ROM or a computer.
  • the algorithm selects, using this data, those primers capable of producing amplification products that are of sufficiently different lengths to permit their resolution by a means that fractionates nucleic acid according to its length.
  • the algorithm selects nucleic acids or amplification products that are of sufficiently different lengths to permit their resolution by a means that fractionates nucleic acid according to its length.
  • the algorithm preferably determines one or more groups of nucleic acids that are of sufficiently different lengths to permit their resolution by a means that fractionates nucleic acid according to its length or nucleic acids capable of producing amplification products comprising same.
  • the number of groups is minimized or reduced.
  • the algorithm is stored in a computer-readable medium, for example, a computer-readable diskette, a computer-readable CD-ROM or memory of a computer or web-based database or programme.
  • Computer-based medium shall also be taken to include a web-based means, such as, for example, a web-site or access to a web-site.
  • the computer-based algorithm may be in the form of a computer program or a computer code.
  • the present invention clearly contemplates such a computer-based algorithm, whether in the form of a computer program or a computer code or stored on a computer-based medium.
  • the computer-based algorithm is provided in any form as a component of a kit.
  • first primer(s) or set(s) of first primers shall be taken to apply mutatis mutandis to methods for selecting amplification products that may be amplified in a multiplex reaction and/or detected in a single reaction.
  • a specific the primer is produced and/or synthesized.
  • Methods for producing/synthesizing a primer of the present invention are known in the art. For example, oligonucleotide synthesis is described, in Gait (Ed) (In: Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, 1984).
  • a probe or primer may be obtained by biological synthesis (eg. by digestion of a nucleic acid with a restriction endonuclease) or by chemical synthesis. For short sequences (up to about 100 nucleotides) chemical synthesis is preferable.
  • a nucleotide comprising deoxynucleotides is produced using standard solid-phase phosphoramidite chemistry. Essentially, this method uses protected nucleoside phosphoramidites to produce a short oligonucleotide (i.e., up to about 80 nucleotides). Typically, an initial 5'-protected nucleoside is attached to a polymer resin by its 3'-hydroxy group. The 5' hydroxyl group is then de-protected and the subsequent nucleoside-3'-phophoramidite in the sequence is coupled to the de-protected group.
  • An internucleotide bond is then formed by oxidizing the linked nucleosides to form a phosphotriester.
  • an oligonucleotide of desired length and sequence is obtained. Suitable methods of oligonucleotide synthesis are described, for example, in Caruthers, M. H., et al., "Methods in Enzymology," Vol. 154, pp. 287-314 (1988).
  • oligonucleotide synthesis include, for example, phosphotriester and phosphodiester methods (Narang, et al. Meth. Enzymol 68: 90, 1979) and synthesis on a support (Beaucage, et al Tetrahedron Letters 22: 1859-1862, 1981), and others described in "Synthesis and Applications of DNA and RNA," S. A. Narang, editor, Academic Press, New York, 1987, and the references contained therein.
  • a plurality of primers are produced using standard techniques, each primer comprising a portion of a desired primer and a region that allows for annealing to another primer.
  • the primers are then used in an overlap extension method that comprises allowing the primers to anneal and synthesizing copies of a complete primer using a polymerase.
  • an overlap extension method comprises allowing the primers to anneal and synthesizing copies of a complete primer using a polymerase.
  • a primer of the invention may also include one or more nucleic acid analogs.
  • a primer comprises a phosphate ester analog and/or a pentose sugar analog.
  • a primer of the invention comprises polynucleotide in which the phosphate ester and/or sugar phosphate ester linkages are replaced with other types of linkages, such as N-(2-aminoethyl)-glycine amides and other amides (see, e.g., Nielsen et al, Science 254: 1497-1500, 1991; WO 92/20702; and USSN 5,719,262); morpholinos (see, for example, USSN 5,698,685); carbamates (for example, as described in Stirchak & Summerton, J Org.
  • Phosphate ester analogs include, but are not limited to, (i) C 1 -C 4 alkylphosphonate, e.g.
  • a probe or primer of the invention comprises one or more LNA and/or PNA residues.
  • Probes or primers comprising one or more LNA or PNA residues have been previously shown to anneal to nucleic acid template at a higher temperature than a probe or primer that comprises substantially the same sequence but does not comprise the LNA or PNA residues.
  • incorporation of LNA into a probe or primer has been shown to result in increased signal produced in reactions in which the level of the probe or primer is limiting (Latorra et al., MoI. Cell Probes 17: 253-259, 2003).
  • At least one primer of the invention comprises or is conjugated to a label.
  • label refers to any moiety which can be attached to a primer of the invention and: (i) provides a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the second label, e.g.
  • FRET Fluorescent Resonance Energy Transfer
  • stabilize annealing e.g., duplex formation
  • provide a member of a binding complex or affinity set e.g., affinity, antibody/antigen, ionic complexation, hapten/ligand, e.g. biotin/avidin.
  • Labelling of a primer is accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods.
  • Labels include, but are not limited to, light-emitting or light-absorbing compounds which generate or quench a detectable fluorescent, chemiluminescent, or bioluminescent signal (for example, as described in Kricka, L. in Nonisotopic DNA Probe Techniques (1992), Academic Press, San Diego, pp. 3-28).
  • Fluorescent reporter dyes useful for labelling biomolecules include, but are not limited to, fluoresceins (see, for example USSN 5,188,934; 6,008,379; or USSN 6,020,481), rhodamines (as described, for example, in USSN 5,366,860; USSN 5,847,162; USSN 5,936,087; or USSN 6,051,719), benzophenoxazines (for example, as described in USSN U.S. Pat. No.
  • exemplary fluorescein dyes include, but are not limited to, 6-carboxyfluorescein; 2',4',1,4,-tetrachlorofluorescein; and 2',4',5',7 I ,l,4-hexachlorofluorescein.
  • Labels also include, but are not limited to, semiconductor nanocrystals, or quantum dots (as described, for example in USSN 5,990,479 or USSN 6,207,392). Suitable methods for linking a label to a primer (or labelling a primer) are also describe din the references supra.
  • the probe or primer is produced with a fluorescent nucleotide analog to facilitate detection.
  • a fluorescent nucleotide analog for example, coupling allylamine-dUTP to the succinimidyl-ester derivatives of a fluorescent dye or a hapten (such as biotin or digoxigenin) enables preparation of many common fluorescent nucleotides.
  • a fluorescent dye or a hapten such as biotin or digoxigenin
  • Other fluorescent nucleotide analogs are also known in the art and described, for example, Jameson, Methods Enzymol. 278:363-390, 1997 or USSN 6,268,132.
  • Such nucleotide analogs are incorporated into nucleic acids, e.g., DNA and/or RNA, or oligonucleotides, via either enzymatic or chemical synthesis (e.g., a method described supra).
  • a primer of the invention is labelled with a fluorescent dye, such as, for example, 6-carboxyfluorescein (FAM), VIC, NED or PET.
  • FAM 6-carboxyfluorescein
  • VIC VIC
  • NED NED
  • PET fluorescent dye
  • a simple two-step process is used. In the first step, an amine-modif ⁇ ed nucleotide, 5-(3-aminoallyl)-dUTP, is incorporated into DNA using conventional enzymatic labeling methods. This step ensures relatively uniform labeling of the probe with primary amine groups. In the second step, the amine- modified DNA is chemically labeled using an amine-reactive fluorescent dye.
  • Various commercial kits for labelling a primer are known in the art and available from, for example, Molecular Probes (Invitrogen detection Technology) (Eugene, OR, USA) or Applied Biosystems (Foster City, CA, USA).
  • a first primer or group thereof and/or a second primer or group thereof is synthesized.
  • a first primer of the invention is produced by coupling an oligonucleotide comprising a tag region to an oligonucleotide comprising a sequence enabling specific annealing to a nucleic acid template.
  • an oligonucleotide comprising a tag region is linked to another oligonucleotide using a RNA ligase, such as, for example T4 RNA ligase (as available from New England Biolabs).
  • RNA ligase catalyzes ligation of a 5' phosphoryl-terminated nucleic acid donor to a 3' hydroxyl-terminated nucleic acid acceptor through the formation of a 3'-5' phosphodiester bond, with hydrolysis of ATP to AMP and PPj.
  • Suitable methods for the ligation of DNA and/or RNA molecules using a RNA ligase are known in the art and/or described in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) and Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
  • the present invention additionally provides a first and/or second primer of the invention, for example, as produced using a method known in the art and/or described herein.
  • a second primer of the invention is capable of annealing to an amplicon produced using a first primer.
  • kits comprising one or more first primers and/or one or more second primers.
  • the kit optionally comprises reagents suitable for amplification of a nucleic acid using the method of the invention (e.g., a buffer and/or one or more deoxynucleotides and/or a polymerase).
  • reagents suitable for amplification of a nucleic acid using the method of the invention e.g., a buffer and/or one or more deoxynucleotides and/or a polymerase.
  • the kit is packaged with instructions for use.
  • the method of the present invention is based on the amplification of a template nucleic acid using multiple rounds of PCR in a single reaction vessel. Accordingly, this single reaction vessel contains all of the components required for the performance of the multiple PCRs.
  • Reagents required for a PCR include for example, one or more primers (described herein), a suitable polymerase, deoxynucleotides and/or ribonucleotides, a buffer. Suitable reagents are described for example, in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995).
  • a suitable polymerase for use in the method of the invention include, a DNA polymerase, a RNA polymerase, a reverse transcriptase, a T7 polymerase, a SP6 polymerase, a T3 polymerase, SequenaseTM, a Klenow fragment, a Taq polymerase, a Taq polymerase derivative, a Taq polymerase variant, a Pfu polymerase, a Pfx polymerase, an AmpliTaqTM FS polymerase, a thermostable DNA polymerase with minimal or no 3 '-5' exonuclease activity, or an enzymatically active variant or fragment of any of the above polymerases.
  • a polymerase used in the method of the invention is a thermostable polymerase.
  • a mixture of two or more polymerases is used.
  • the mixture of a Pfx or Pfu polymerase and a Taq polymerase has been previously shown to be useful for amplifying templates comprising a high GC content or for amplifying a large template.
  • Suitable commercial sources for a polymerase useful for the performance of the invention will be apparent to the skilled artisan and include, for example, Stratagene (La Jolla, CA, USA), Promega (Madison, WI, USA), Invitrogen (Carlsbad, CA, USA), Applied Biosystems (Foster City, CA, USA) and New England Biolabs (Beverly, MA, USA).
  • the amplification reaction used is a PCR or a variant thereof.
  • a suitable variant of a PCR includes, for example, one-armed (or single- primer PCR), reverse-transcriptase mediated PCR (RT-PCR), nested PCR, touch-up and loop incorporated primers (TULIP) PCR, touch-down PCR, competitive PCR, rapid competitive PCR (RC-PCR) or multiplex PCR.
  • the amplification reaction is a PCR or a multiplex PCR.
  • the amplification reaction is a PCR or a multiplex PCR.
  • PCR Methods of PCR are known in the art and described, for example, in Dieffenbach (ed) and Dveksler (ed) ⁇ In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995).
  • two non-complementary nucleic acid primers comprising at least about 8, more preferably, at least about 15 or 20 nucleotides are annealed to different strands of a template nucleic acid, and amplicons of the template are amplified enzymatically using a polymerase, preferably, a thermostable DNA polymerase.
  • an initial reaction is performed wherein one or more nucleic acid templates are amplified using one or more sets of first primers.
  • Each primer of a set of primers anneals to a different strand of the template nucleic acid such that the target region is defined by the sites of annealing.
  • the second set of primers is unable to anneal to target nucleic acid.
  • the intervening region of nucleic acid is then replicated enzymatically, for example, using a polymerase. By cycling the temperature to enable denaturation of template nucleic acid, annealing of the primer/s and polymerase mediated replication of nucleic acid, the nucleic acid template is amplified.
  • the annealing temperature is reduced and the second set of primers are able to anneal to amplicons produced using the first set of primers.
  • the nucleic acid template is amplified.
  • first primers used in the reaction comprise a tag sequence it will be apparent to the skilled artisan that each primer in the first set of primers need not comprise the same tag region. Rather, the primer that anneals to the sense strand of the nucleic acid template comprises one tag and the primer that anneals to the antisense strand comprises a distinct tag.
  • the present inventors have produced a primer capable of annealing to the sense strand of a nucleic acid template that comprises tag region and a primer capable of annealing to the complement thereof. Accordingly, a set of different second primers is used to amplify the amplicons produced by the first primers.
  • the present invention uses a form of amplification (e.g., PCR) known as exhaustive PCR.
  • PCR a form of amplification
  • Exhaustive PCR is thus named as a primer or set of primers used in the reaction is exhausted (or incorporated into amplicons) such that a detectable level of amplification initiated by that primer or set of primers no longer detectably occurs.
  • the inventors have reduced the likelihood of non-specific annealing of the first primer at the reduced annealing temperature used for the second primer. Furthermore, the use of exhaustive PCR also reduces the likelihood of the first and second primer or sets of primers interacting and producing "primer-dimers".
  • a first primer is used at a concentration of between about 7.5nM and about 25OnM, more preferably between about 2OnM and about 20OnM, even more preferably between about 5OnM and about 15OnM and still more preferably between about 75nM and about 10OnM.
  • a first primer is used at about 23nM or about 3OnM of primer or about 4OnM of primer or about 8OnM of primer.
  • the second amplification reaction (i.e., a reaction using a second primer or set thereof) is additionally performed under exhaustive conditions. Accordingly, a PCR is performed under conditions sufficient to amplify the amplicon produced by the first primers said conditions comprising an annealing temperature suitable for annealing of a second primer or set of second primers and a sufficient number of amplification cycles for the second primer(s) to be substantially incorporated into the amplification product(s) and little or no residual unincorporated second primer.
  • the level or amount of amplified nucleic acid produced in the amplification reaction is controlled by the amount of the first primer or set thereof and/or the second primer or set thereof.
  • a second primer is used at a concentration between about 7.5nM and about 40OnM (e.g., about 23nM or about 3OnM or about 4OnM or about 8OnM), even more preferably about 75 nM to about 250 nM and still more preferably about 75 nM to about 200 nM.
  • a second primer is present at a concentration of about 75nM or 10OnM, 20OnM, 30OnM or 40OnM..
  • a limiting amount of a primer is about 23nM of primer or about 3OnM of primer or about 4OnM of primer or about 8OnM of primer.
  • a second primer is preferably used at a concentration of between about 50 and 10OnM and more preferably, about 75nM.
  • the concentration may be increased, however, this is not necessarily required.
  • the present inventors have amplified two template nucleic acids using a second primer concentration between about 50 and 10OnM and more preferably, about 75nM.
  • a preferred concentration of second primer is at least about 9OnM or 10OnM.
  • concentration of 10OnM of second primer is sufficient to amplify three template nucleic acids using the method of the invention.
  • RNA is reverse transcribed using a reverse transcriptase (such as, for example, Moloney Murine Leukemia Virus) to produce cDNA.
  • a reverse transcriptase such as, for example, Moloney Murine Leukemia Virus
  • the reverse transcription of the RNA is primed using, for example, a random primer (e.g., a hexa-nucleotide random primer) or oligo-dT (that binds to a poly-adenylation signal in mRNA).
  • a locus-specific primer is used to prime the reverse transcription (e.g., a first primer of the invention).
  • a sample is heated to ensure production of single stranded nucleic acid and then cooled to enable annealing of the primer.
  • the sample is then incubated under conditions sufficient for reverse-transcription of the nucleic acid adjacent to an annealed primer by a reverse transcriptase.
  • the cDNA is used as a template nucleic acid for a PCR reaction, e.g., as described supra.
  • single primer PCR uses only one primer to amplify nucleic acid.
  • this technique comprises annealing a first primer to template nucleic acid at sufficiently low stringency to ensure that it anneals to multiple sites in the template.
  • nucleic acid products are produced in those cases wherein the primer has annealed sufficiently closely to enable amplification.
  • the amplified nucleic acid is amplified further.
  • Such a method is useful for, for example, cloning nucleic acid homologs or determining a molecular marker that is diagnostic or characteristic of a disease or trait.
  • the PCR performed using the second primer of the invention is a single primer PCR.
  • each of the first primers of the invention comprise the same tag at their 5'-termmus and are used in a PCR amplification.
  • a single second primer is then used to amplify the amplicons produced with the first primer.
  • a nested PCR is performed. Nested PCR is described in detail in, for example, Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995). Essentially a nested PCR reaction involves the use of two sets of primers that are specific to a sequence of interest. The first set of primers is used to amplify the nucleic acid template to a desired level. The second set of primers is designed to anneal to a region of the nucleic acid between the sites of annealing of the first primers and further amplify the nucleic acid template.
  • Such a method is useful for, for example, amplifying a nucleic acid template using a small amount of template nucleic acid (e.g., nucleic acid from a single cell).
  • a nested PCR reaction is preferably performed using a second set of primers that comprise a tag region to permit annealing of a further set of primers (i.e. to enable performance of the method of the invention).
  • a nested PCR reaction is performed with all primers in a single closed tube.
  • the initial PCR is performed in one tube and the second and third PCRs (i.e. the method of the invention) are performed in a separate closed tube.
  • the method of the invention is performed in a multiplex format.
  • a multiplex reaction is used to amplify a plurality of distinct nucleic acids of interest in a single reaction.
  • an initial amplification reaction is performed using a plurality of sets of first primers, each set capable of amplifying a specific nucleic acid template.
  • a second primer or set of second primers is then used to amplify the amplicons produced using the first primers in what is effectively, a "singleplex" reaction (i.e., a single second primer or set of second primers is used to amplify a plurality of different amplicons).
  • each of the first primers that anneals to a sense stand comprises a tag region and each of the primers that anneals to the antisense strand comprises another tag region.
  • Each of the nucleic acid templates is then amplified with the sets of first primers. Following amplification with the first primers, the annealing temperature is reduced and the amplification products further amplified using the second primer/s.
  • This method enables the simultaneous amplification of all nucleic acids amplified in the initial stage of amplification.
  • tag regions are used for the set of first primers.
  • a subset of amplification products is amplified using a specific set of second primers.
  • all primers comprise the same tag, thereby enabling amplification with a single second primer.
  • the present inventors have found that the method of the invention requires little optimisation to successfully amplify a plurality of nucleic acids of interest in a single multiplex reaction.
  • the amount of each of the first sets of primers required for exhaustive PCR varies between primers or sets of primer.
  • the amount of each set of primers is preferably selected to ensure that each nucleic acid template is amplified to a desired level.
  • Methods for determining a suitable amount of each primer or set thereof will be apparent to the skilled artisan and include, for example, performing an amplification reaction with each primer set individually to determine a suitable amount of primer (i.e., empirically).
  • a multiplex PCR of the invention comprises an additional amplification step prior to amplification using a locus-specific primer.
  • this optional step comprises performing an amplification reaction under conditions suitable for annealing of the first and second sets of primers.
  • amplification is performed for a limited number of cycles (for example, 1 to 5 amplification cycles).
  • Such an optional amplification is useful for, for example, performing a multiplex reaction in which plurality of locus specific primers have different Tms.
  • a multiplex reaction is performed with two or three or four or five or six or more primers comprising a locus specific region.
  • the amplicon/s produced using the method of the invention is/are separated using gel electrophoresis.
  • the separated amplicon/s is/are then detected using a detectable marker that selectively binds nucleic acid, such as, for example, ethidium bromide, 4'-6-diamidino-2-phenylinodole (DAPI), methylene blue or SYBR® green I or II (available from Sigma Aldrich).
  • DAPI 4'-6-diamidino-2-phenylinodole
  • SYBR® green I or II available from Sigma Aldrich.
  • Suitable methods for detection of a nucleic acid using gel electrophoresis are known in the art and described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) and Sambrook et al (ui: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New
  • the nucleic acid is separated using one dimensional agarose, agaorse-acrylamide or polyacrylamide gel electrophoresis. Such separation techniques separate nucleic acids on the basis of molecular weight.
  • an amplicon is separated using two dimensional electrophoresis and detected using a detectable marker (e.g., as described supra).
  • a detectable marker e.g., as described supra.
  • Two dimensional agarose gel electrophoresis is adapted from the procedure by Bell and Byers Anal. Biochem.
  • the first dimension gel is run at low voltage in low percentage agarose to separate DNA molecules in proportion to their mass.
  • the second dimension is run at high voltage in a gel of higher agarose concentration in the presence of ethidium bromide so that the mobility of a non-linear molecule is drastically influenced by its shape.
  • an amplification product is characterized or isolated using capillary electrophoresis.
  • Capillary electrophoresis is reviewed in, for example, Heller, Electrophoresis 22:629-43, 2001; Dovichi et al, Methods MoI Biol 167:225-39, 2001; Mitchelson, Methods MoI Biol 162:3-26, 2001; or Dolnik, J Biochem Biophys Methods 47:103-19, 1999.
  • Capillary electrophoresis uses high voltage to separate molecules according to their size and charge. A voltage gradient is produced in a column (i.e. a capillary) and this gradient drives molecules of different sizes and charges through the tube at different rates.
  • an amplification product is identified and/or isolated using chromatography.
  • ion pair-reversed phase HPLC has been shown to be useful for isolating a PCR product (Shaw-Bruha and Lamb, Biotechniques. 28:794-7, 2000.
  • the present invention additionally contemplates using a primer that comprises a detectable marker to facilitate detection of an amplicon.
  • a primer of the invention is labelled with a detectable marker using a method known in the art and/or described herein and used in the method of the invention.
  • a labelled second primer is particularly useful in the performance of a multiplex reaction of the invention as it facilitates the rapid and inexpensive labelling of all nucleic acid amplified with said primer (i.e., all amplicons in a single multiplex reaction are labelled with one primer).
  • the use of a plurality of tag sequences and their corresponding labelled second primers facilitates labelling of subsets of nucleic acids with different labels.
  • Amplified nucleic acid is then readily detected by detecting the label.
  • the detection technique may comprise, for example, the use of a photographic film.
  • the nucleic acid is detected, for example, by exposing the gel to light of a suitable wavelength to excite the label and detecting the fluorescence produced therefrom.
  • the present invention particularly contemplates an automated or semi-automated method for detection of a nucleic acid of the invention.
  • the present inventors have used polyacrylamide gel electrophoresis to separate nucleic acids and an automated system to detect each amplified nucleic acid.
  • Such automated systems are available from, for example, Applied Biosystems.
  • the amplified nucleic acid is detected using, for example, mass spectrometry (e.g., MALDI-TOF).
  • mass spectrometry e.g., MALDI-TOF
  • a sample comprising nucleic acid amplified using the method of the invention is incorporated into a matrix, such as for example 3-hydroxypropionic acid, ⁇ -cyano-4-hydroxycinnamic acid, 3,5 dimethoxy-4- hydroxycinnamic acid (Sinapinic acid) or 2,5 dihydroxybenzoic acid (Gentisic acid).
  • a matrix such as for example 3-hydroxypropionic acid, ⁇ -cyano-4-hydroxycinnamic acid, 3,5 dimethoxy-4- hydroxycinnamic acid (Sinapinic acid) or 2,5 dihydroxybenzoic acid (Gentisic acid).
  • the sample and matrix are then spotted onto a metal plate and subjected to irradiation by a laser, promoting the formation of molecular ions.
  • the mass of the produced molecular ion is analyzed by its time of flight (TOF), essentially as described by Yates, J Mass Spectrom. 33, 1-19, 1998 and references cited therein.
  • a time of flight instrument measures the mass to charge ratio (m/z) ratio of an ion by determining the time required for it to traverse the length of a flight tube.
  • a TOF mass analyzer includes an ion mirror at one end of the flight tube that reflects said ion back through the flight tube to a detector. Accordingly, an ion mirror serves to increase the length of a flight tube, increasing the accuracy of this form of analysis.
  • the present invention additionally contemplates the use of a high-throughput method to detect a nucleic acid amplified using a method of the present invention.
  • a nucleic acid microarray is used to detect a nucleic acid amplified using the method of the invention.
  • a detection reaction is performed to detect a nucleic acid produced by the method of the invention.
  • the detection reaction is performed to detect the entire amplified nucleic acid, or alternatively, a detection reaction is performed to detect a portion of the amplified nucleic acid.
  • the method of the invention is useful for amplifying a nucleic acid comprising a genetic marker such as, for example a SSR or a SNP and subsequently detecting the genetic marker.
  • the present invention provides the means to rapidly, specifically and inexpensively amplify a template nucleic acid (or a plurality of nucleic acid templates) for use with such a detection reaction.
  • the nucleic acid is amplified using a primer labelled with a tag, such as, for example, biotin.
  • a primer labelled with a tag such as, for example, biotin.
  • a biotinylated primer facilitates isolation of amplified nucleic acid, e.g., using a streptavidin coated chip or bead.
  • the isolated amplified nucleic acid or nucleic acid therein is then detected using a method known in the art and/or described herein.
  • the nucleic acid is denatured to produce single-stranded DNA, and a genetic marker of interest therein detected using a hybridization reaction, e.g., a hybridization reaction described below.
  • the detection reaction is a hybridization reaction.
  • a suitable hybridization based assay will be apparent to the skilled artisan and/or described herein.
  • a fluorescently labelled locked nucleic acid (LNA) molecule or fluorescently labelled protein-nucleic acid (PNA) molecule is used to detect a nucleic acid (e.g., a specific nucleotide in a sample, e.g., a SNP) (as described in Simeonov and Nikiforov, Nucleic Acids Research, 30(17): 1-5, 2002).
  • LNA and PNA molecules bind, with high affinity, to nucleic acid, in particular, DNA.
  • Flurophores conjugated to the LNA or PNA probe fluoresce at a significantly greater level upon hybridization of the probe to nucleic acid template compared to a probe that has not hybridized to a target nucleic acid.
  • the level of increase of fluorescence is not enhanced to the same level when even a single nucleotide mismatch occurs. Accordingly, the degree of fluorescence detected in a sample is indicative of the presence of a mismatch between the LNA or PNA probe and the target nucleic acid, such as, in the presence of a SNP or SSR in a nucleic acid amplified using a method described supra.
  • LNA or PNA detection technology is amenable to a high-throughput detection of one or more markers immobilising an LNA or PNA probe to a solid support, as described in Oram et ah, Clin. Chem. 45: 1898- 1905, 1999.
  • Molecular BeaconsTM are useful for detecting specific nucleotides, eg., a SNP or a SSR in an amplified nucleic acid produced using the method of the invention (see, for example, Mhlang and Malmberg, Methods 25: 463-471, 2001).
  • Molecular BeaconsTM are single stranded nucleic acid molecules with a stem-and-loop structure.
  • the loop structure is complementary to the region surrounding the nucleic acid of interest.
  • the stem structure is formed by annealing two "arms" that are complementary to each other and that are on either side of the probe (loop).
  • a fluorescent moiety is bound to one arm and a quenching moiety, that suppresses any detectable fluorescence when the molecular beacon is not bound to a target sequence, is bound to the other arm.
  • a quenching moiety that suppresses any detectable fluorescence when the molecular beacon is not bound to a target sequence.
  • a nucleic acid of interest (e.g., SNP or SSR) in an amplified product can also be identified by hybridization to nucleic acid arrays, an example of which is described in WO 95/11995.
  • WO 95/11995 also describes subarrays that are optimized for detection of a variant form of a precharacterized polymorphism.
  • Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence.
  • the second group of probes is designed by the same principles, except that the probes exhibit complementarity to the second reference sequence.
  • a second group (or further groups) can be particularly useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (e.g., two or more mutations within 9 to 21 bases).
  • RNA-DNA duplex formed is a target for RNase H thereby cleaving the probe.
  • the cleaved probe is then detected using, for example, electrophoresis or MALDI-TOF.
  • a genetic marker within a nucleic acid amplified using the method of the invention is detected using an amplification reaction.
  • a suitable amplification reaction will be apparent to the skilled artisan.
  • a ligase chain reaction (described in EU 320,308 and US 4,883,750) uses at least two oligonucleotides that bind to a nucleic acid template in such a way that they are adjacent.
  • a ligase enzyme is then used to link the oligonucleotides.
  • the ligated oligonucleotides then become a target for further oligonucleotides.
  • the ligated fragments are then detected, for example, using electrophoresis, or MALDI-TOF.
  • one or more of the probes is labelled with a detectable marker, thereby facilitating rapid detection.
  • a probe or primer that hybridizes to the nucleic acid at the site of the genetic marker such that it is only capable of being linked to the second probe or primer in the presence of a specific marker (e.g., a specific allele) the presence or absence of a marker is detected.
  • a specific marker e.g., a specific allele
  • a nucleic acid e.g., a SNP
  • SSCP single stranded conformational polymorphism
  • SSCP analysis relies upon the formation of secondary structures in nucleic acids and the sequence dependent nature of these secondary structures, hi one form of this analysis an amplification method, such as, for example, a method described supra, is used to amplify a nucleic acid that comprises a SNP.
  • the amplified nucleic acids are then denatured, cooled and analyzed using, for example, non-denaturing polyarcrylamide gel electrophoresis, mass spectrometry, or liquid chromatography (eg. HPLC or dHPLC).
  • Regions that comprise different sequences form different secondary structures, and as a consequence migrate at different rates through, for example, a gel and/or a charged field.
  • a detectable marker may be incorporated into a probe/primer useful in SSCP analysis to facilitate rapid marker detection.
  • Allele specific PCR (as described, for example, In Liu et al, Genome Research, 7: 389- 398, 1997) is also useful for determining the presence of one or other allele of a SNP.
  • An oligonucleotide is designed, in which the most 3' base of the oligonucleotide anneals to a specific form of the SNP of interest (i.e., allele).
  • PCR products are then detected using, for example, gel or capillary electrophoresis or mass spectrometry.
  • a nucleic acid comprising an allele to be tested is amplified using the method of the present invention.
  • the amplified nucleic acid is then used in a reaction with one or more allele specific primers to determine the presence or absence of an amplification product of interest.
  • a first primer of the invention is an allele specific primer. Accordingly, only in the presence of a specific allele will the first primer anneal to the template nucleic acid sufficiently to produce an amplification product. Accordingly, detection of an amplification product is indicative of indicative of a specific allele.
  • Primer extension methods are also useful for the detection of a SNP or SSR or a nucleic acid of interest.
  • An oligonucleotide is used that anneals to the region of a nucleic acid adjacent to the nucleic acid of interest. This oligonucleotide is then used in a primer extension protocol with a polymerase and a free nucleotide diphosphate that corresponds to either or any of the possible bases that occur at the nucleic acid of interest.
  • the nucleotide- diphosphate is labelled with a detectable marker (e.g. a flurophore).
  • a detectable marker e.g. a flurophore
  • unbound labelled nucleotide diphosphates are removed, e.g. using size exclusion chromatography or electrophoresis, or hydrolized, using for example, alkaline phosphatase, and the incorporation of the labelled nucleotide into the oligonucleotide is detected, indicating the base that is present at the site of the nucleic acid of interest.
  • the presence of a genetic marker in a nucleic acid amplified using the method of the invention is detected using pyrosequencing, such as, for example, as described in Uhlmann et at, Electrophoresis, 23: 4072 -4079, 2002.
  • pyrosequencing such as, for example, as described in Uhlmann et at, Electrophoresis, 23: 4072 -4079, 2002.
  • this method is a form of real-time sequencing that uses a primer that anneals to a site adjacent or close to the site of a genetic marker of interest. Following annealing of the primer and template in the presence of a DNA polymerase each of four modified deoxynucleotide triphosphates is added separately according to a predetermined dispensation order.
  • PPi inorganic pyrophosphate
  • a primer of the present invention comprises an additional region (e.g., a second tag region) that comprises a binding site for a polymerase, preferably, an PvNA polymerase.
  • a primer comprises a T7 or a T3 polymerase binding site.
  • a polymerase binding site permits detection and/or identification of the genetic marker in amplified nucleic acid using a method known in the art, such as, for example, RNaseCut (Krebs et at, Nucleic Acids Research, 31: e37, 2003).
  • the RNaseCut method comprises amplifying nucleic acid comprising a genetic marker of interest, e.g., a SNP, using a primer of the present invention additionally comprising an PvNA polymerase binding site.
  • the amplified nucleic acid is then transcribed using the RNA polymerase, and the resulting RNA digested with a RNase capable of cleaving a particular RNA sequence, such as, for example, guanosine- specific ribonuclease Tl.
  • the resulting RNA fragments are then analysed using mass spectrometry to determine, for example, a change in the number of RNA fragments produced compared to a control sample.
  • the present invention is useful for amplification and detection of a genetic marker, the invention has clear application in determining relationships between one or more individuals, isolates of an organism, cultivars of an organism, species or genera. Furthermore, the present invention is useful for identifying an individual, isolate of an organism, cultivar of an organism, species or genus. Accordingly, the present invention additionally provides a method comprising performing a method described herein to detect one or more polymorphic nucleic acid/s in an individual, isolate of an organism, cultivar of an organism, species or genus wherein the polymorphic nucleic acid detected identifies and/or characterizes the individual, isolate of an organism, cultivar of an organism, species or genus.
  • the detection of polymorphic nucleic acid facilitates differentiation between related organisms. For example, should a population of inbred or highly related organisms be used for a genetic mapping experiment, the method of the invention is useful for determining those organisms that, for example, comprise a phenotype of interest, and only comprise one region of nucleic acid different to a related organism.
  • the method of the invention is useful for a form of genetic mapping, such as, for example bulked segregant analysis (BSA).
  • BSA bulked segregant analysis
  • this form of analysis uses nucleic acid from a plurality of organisms (preferably, plants) that only differ in one trait (e.g., as a result of mutation or introgression).
  • Nucleic acid from organisms with one phenotype is pooled, as is nucleic acid from organisms with the other phenotype.
  • a region of nucleic acid in which the two pools of nucleic acid differ is determined.
  • Such a method is particularly useful for, for example, mapping of a gene responsible for a monogenic trait or a quantitative trait. Suitable methods for BSA are described, for example, in Wang and Paterson Theor. Appl Genet. 55:355-361, 1994 and Mackay and Caligari Crop Science 40:626- 630, 2000.
  • the present invention contemplates performing a multiplex reaction to identify or characterize an individual, isolate of an organism, cultivar of an organism, species or genus, for example, the detection of a plurality of genetic markers such as, for example, SNP or SSR.
  • genetic marker shall be taken to mean a nucleic acid that is polymorphic between, for example, two or more individuals (e.g., related individuals), isolates of an organism, cultivars of an organism, species or genera.
  • a genetic marker is additionally linked to or associated with a trait of interest.
  • linked to shall be taken to mean that there is sufficient proximity between a genetic marker (e.g., a polymorphic nucleic acid) and a nucleic acid that causes a trait of interest to permit said linked nucleic acid to be predictive of said trait.
  • a genetic marker e.g., a polymorphic nucleic acid
  • the term "associated with” shall be taken to mean that the presence of a specific genetic marker is significantly correlated with a trait of interest in an organism or a population of organisms.
  • the presence of the genetic marker is significantly correlated with the presence of the trait of interest in a population of unrelated organisms.
  • a suitable amplification and/or detection reaction is described supra and is to be taken to apply mutatis mutandis to the present embodiment of the invention.
  • the present embodiment of the invention is used to amplify a plurality of nucleic acids, each comprising a SNP.
  • the allele present at the site of each SNP is determined using a method described herein.
  • the method of the present invention has broad reaching application in any assay that detects one or more genetic markers. Accordingly, the present invention is useful for, for example, marker assisted breeding programs (e.g., animal husbandry), gene mapping, identification of specific strains, isolates or cultivars of plants, identification of organisms likely to have a trait of interest and diagnosis of a disease or disorder.
  • marker assisted breeding programs e.g., animal husbandry
  • Genetic markers are used for a variety of purposes in association with plants. For example, one or more genetic markers is (are) used to identify a specific plant variety.
  • a plant that is protected by an intellectual property right is characterised to determine one or more genetic markers that are specific to said plant. This then enables simple and rapid characterisation of similar plants to determine whether or not an intellectual property right has been infringed.
  • a plant or plant matter e.g., a foodstuff comprising plant matter
  • a genetically modified organism is useful for, for example, determining whether or not a food product must be labelled to state that it contains a genetically modified product, or alternatively, for the detection of a specific genetically modified organism that is the subject of an intellectual property right.
  • AATTTATCCTAGTTTGCGCGCTA (SEQ ID NO: 18); (iii) CrylAb gene (Bt) TGCTATGCGGGAGCTGCG (SEQ ID NO: 19); and
  • a set of first primers for use in the present invention is produced.
  • the second primers are then used to amplify amplicons produced using the previously described primers, thereby enabling detection of a plant or plant matter derived from a genetically modified organism.
  • Additional suitable primers for the detection of transgenic plants, such as, for example, maize of soybean are known in the art and described, for example, in Germini et at, J. Agric.food Chem., 52: 3275-3280, 2004.
  • the present invention is used to determine a plant that is likely to comprise a phenotype of interest.
  • a phenotype is, for example, resistance to a pest, enhanced growth or biomass production or enhanced fruit production, amongst others.
  • Moczulski and Salmanowicz J Appl. Genetics 44: 459-471, 2003, describe an assay for determining wheat with an enhanced bread-making phenotype (based on the presence of HMW glutenin alleles).
  • the following primers are produced with a tag sequence located at their 5' end: (i) ACGTTCCCCTACAGGTACTA (SEQ ID NO: 23); and
  • TATCACTGGCTAGCCGACAA SEQ ID NO: 24
  • CCATCGAAATGGCTAAGCGG SEQ ID NO: 25
  • GTCCAGAAGTTGGGAAGTGC SEQ ID NO: 26
  • GAAACCTGCTGCGGACAAG SEQ ID NO: 32
  • GTTGGCCGGTCGGCTGCCATG SEQ ID NO: 33
  • primers are then used to amplify nucleic acid derived from a wheat plant in a single reaction.
  • Using second primers corresponding to a region of the respective tag sequences each of the amplification products are further amplified.
  • wheat strains positive for amplification products produced by the primer pairs a wheat with good bread making quality is selected.
  • the present invention is useful for marker-assisted selection of a plant that is resistant to a pathogen.
  • a set of primers comprising the following nucleotide sequences are produced linked to a tag sequence: (i) TCTGGTGAGGCAAACCTTCTGG (SEQ ID NO: 35); and TCTGGTGAGGGAGGTGTGATGACG (SEQ ID NO: 36).
  • a plant preferably a wheat plant, is identified that is resistant to powdery mildew.
  • the instant assay may be used in a multiplex reaction to determine a plant that is resistant to a plurality of pathogens.
  • a method that facilitates the rapid analysis of a plurality of genetic markers is useful for gene mapping experiments.
  • Such a method enables, for example, the differentiation between, for example, two individuals that differ in only one nucleic acid (for example, a region of nucleic acid introduced by crossing). Accordingly, the method of the invention enables rapid and inexpensive marker based genetic mapping.
  • genetic markers are detected to differentiate between specific cultivars of plant, to determine genetic relationships between specific cultivars of a plant and to select plants with desirable characteristics for further analysis.
  • the present inventors have clearly demonstrated the applicability of the instant invention to the differentiation between various strains of wheat and/or barley. Accordingly, the method of the invention is clearly useful for, for example, identifying a strain of wheat or barley of commercial value or of interest. Such a method is useful for, for example, ensuring the purity of a crop or determining the presence of a contaminating strain of wheat or barley in a sample.
  • one embodiment of the present invention provides a process of characterising or identifying one or more strains of wheat or barley said method comprising: (i) providing in a reaction vessel reagents suitable for performing PCR comprising: (a) an amount of a first primer or set of first primers sufficient to permit amplification of a nucleic acid template without substantial residual unincorporated primer, wherein each first primer comprises a locus-specific and a tag sequence; and (b) a second primer or set of second primers; (ii) performing PCR under conditions sufficient to amplify the nucleic acid template thereby producing an amplification product, wherein said conditions comprise an annealing temperature suitable for annealing of the first primer or set of first primers but not detectably of the second primer or set of second primers and a sufficient number of amplification cycles for the primer(s) to be substantially incorporated into the amplification product and little or no residual unincorporated primer; (iii) performing PCR under conditions sufficient to amplify the amplification product at
  • this embodiment of the invention is useful for amplifying nucleic acids that are polymorphic between individuals (including related individuals), it shall be taken to apply mutatis mutandis to a method for differentiating between two or more related individuals.
  • the present invention provides a process of characterising or identifying one or more individuals, strains or species of the genus Prunus said method comprising: (i) providing in a reaction vessel reagents suitable for performing PCR comprising:
  • each first primer comprises a locus-specific sequence and a tag sequence
  • a second primer or set of second primers performing PCR under conditions sufficient to amplify the nucleic acid template thereby producing an amplification product, wherein said conditions comprise an annealing temperature suitable for annealing of the first primer or set of first primers but not detectably of the second primer or set of second primers and a sufficient number of amplification cycles for the primer(s) to be substantially incorporated into the amplification product and little or no residual unincorporated primer;
  • the invention is useful for differentiating between related organisms or individuals. Accordingly, this embodiment shall be taken to apply mutatis mutandis to a method for differentiating between two or more related organisms or individuals. Genetic markers in humans and animals
  • the method of the present invention for detecting one or more genetic markers is also useful for, for example, marker assisted breeding of animals and/or to select for those animals with one or more desired traits.
  • the assay is used to screen animals for enhanced commercial properties, such as, for example, food quality for human consumption.
  • Such an assay is performed to detect one or more markers that is (are) associated with increased marbling in beef.
  • Marbled beef is of commercial importance as consumers in several countries pay a premium price for beef with a high level of marbling. Recently, several markers have been reported that are associated with an increased level of marbling.
  • Ciobanu et al, J. Anim. Set 82: 2829-2839, 2004 and Chang et al, Vet. J. 165: 157- 163, 2003 describe markers useful for determining an increased pork quality from a pig.
  • the marker described by Ciobanu et al occurs in the calpastatin gene, while the marker described by Chang et al, is a dinucleotide repeat (CT) in the desmin gene.
  • CT dinucleotide repeat
  • a plurality of markers in the melanocortin-4 receptor that are associated with increased meat quality in swine are disclosed in USSN 20040261138.
  • markers associated with increased meat quality in pigs include, for example, the microsatellite markers SW413, SW1482, SW439, S0005 and SW904 (described in USSN 20040101842).
  • each marker described herein may be detected individually using the method of the present invention.
  • a plurality of the markers may be detected in a single assay (i.e., a multiplex assay) using a method described herein.
  • Such methods are also applicable to, for example, selecting enhanced race horses (e.g., with enhanced speed and/or endurance), selecting sheep that produce superior wool, or selecting a mammal (e.g., a cow) that produces superior quality milk.
  • the method of the invention is useful for "typing" an animal or human. Such a process is used for, for example, maternity and/or paternity testing.
  • an assay involves detecting a plurality of markers (e.g., SSR) that are highly polymorphic in a population in the test organism and in one or more of the suspected parents. By comparing the nucleic acid detected in the samples, it is possible to determine the nature of the relationship (if any) between the test sample and the suspect parent.
  • markers e.g., SSR
  • Such a typing assay important for human testing is regularly used to determine the lineage of an animal, such as, for example, a horse, a cow or a bull.
  • Bowling et ah Animal Genetics, 28: 247-252, 1997 describe a parallel test that detects 11 dinucleotide microsatellite markers and 15 blood group or protein markers to determine the parentage of a horse with 99.99 percent accuracy.
  • the method of the present invention is useful for detecting the microsatellite markers described by Bowling et al. either in parallel or in a multiplex reaction.
  • the microsatellite markers detected were AHT4, AHT5, HMSl, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTGlO and VHL20.
  • the present invention is useful for the detection of genetic differences, it is particularly useful for the diagnosis of a disease or disorder or the presence of one or more infectious agents in a sample.
  • the method of the invention is useful for detecting a genetic change that is associated with a disease or disorder in a human or a non-human animal.
  • Exemplary common genetic diseases or disorders in humans include, for example, cystic fibrosis, sickle cell anemia, ⁇ -thalasemia, albinism, Huntington's disease, Down's Syndrome or muscular dystrophy.
  • Exemplary common diseases in sheep include, for example, dermatosparaxis, erythrocyte glutathione deficiency, globoid cell leukodystrophy, glycogen storage disease, anury, cataract, glomerulonephritis, and lethal grey.
  • Examples of common genetic diseases in goats include, for example, gynecomastia and anotia-microtia complex.
  • Exemplary genetic diseases in horses include, for example, hyperkalemic periodic paralysis (HYPP), combined immune deficiency syndrome (CID), overo-lethal white syndrome and epitheliogenesis.
  • HYPP hyperkalemic periodic paralysis
  • CID combined immune deficiency syndrome
  • epitheliogenesis epitheliogenesis
  • a screen may be developed using the method of the invention to screen for any or all of these disorders in a specific organism.
  • a screen to amplify nucleic acid comprising a mutation associated with cystic fibrosis may be performed using the following primers:
  • TTGTACCAGCTCACTACCTA SEQ ID NO: 40
  • GCAGAGTACCTGAAACAGGA SEQ ID NO: 41
  • CAAAAGTACCTGTTGCTCCA (SEQ ID NO: 54).
  • each primer conjugated to a tag sequence the primers are then used in a multiplex PCR according to the method of the present invention.
  • all amplified nucleic acid is further amplified with a set of tag specific primers.
  • the specific mutations are detected, for example, using DGGE essentially as described in Fanen et al, Genomics, 13, 770-776, 1992.
  • a diagnostic method of the invention is performed to detect the presence of a respiratory tract infection using one or more of the primer sets described by Tempelton et al, J. CHn. Microbiol, 42: 1564-1569, 2004 with a tag sequence of the invention.
  • a second reaction with a second primer of the invention is performed. Using such a reaction the following infections are detected: influenza A virus, influenza B virus, RSV, PIVl, and PIV3 infections.
  • the method is used for the detection of an infection in a non-human animal.
  • 2004 are used to detect bovine arboviruses using a RT-PCR assay of the invention.
  • RNA isolated from a bovine source is reverse transcribed and amplified using the primers described by Ohashi et al, supra tagged with a tag region of the invention. Following substantial incorporation of each of the primers a second reaction is performed with a second primer of the invention. By determining the molecular weight of the amplified nucleic acid/s the presence and identity of a bovine arbovirus is determined.
  • the present invention is also useful for detecting an infection in a plant.
  • the present invention is useful for detecting a fungal infection in a plant, e.g., an infection by Rhynchosporium secalis.
  • Additional plant infections that may be detected by the method of the present invention include, for example, Tilletia indica Mitra or T. wa ⁇ keri, both of which cause karnal bunt in wheat.
  • Suitable primers for the detection of such a microorganism include, for example, a pair of primers comprising a tag sequence as described herein according to any embodiment and a locus specific sequence comprising a nucleotide sequence such as, for example,
  • GCAGAATTCAGTGAATCATCAAG SEQ ID NO: 5343
  • GCAACACTCAAAATCCAACAAT SEQ ID NO: 5345
  • the nucleic acid that is amplified is within a biological sample.
  • the biological sample has been previously isolated from a subject and, as a consequence, the method is performed ex vivo.
  • suitable biological samples will be apparent to the skilled artisan and include, for example, a body fluid (e.g., blood, saliva or sputum) or a pollen sample, a soil sample or a leaf sample.
  • the method of the invention may comprise comparing the nucleic acid amplified and detected to nucleic acid amplified and detected using a variety of infectious organisms, to thereby detect or determine the organism causing the infection.
  • These comparative samples may be amplified at the same time as the test sample, or alternatively, may be previously amplified and, for example, stored in a library of amplification products.
  • the present invention additionally contemplates a multiplex method for diagnosing a disease or disorder or detecting an infectious organism. Suitable multiplex methods are known in the art and/or described herein.
  • PCR primers for markers were synthesized with a generic 19-bp nucleotide sequence at their 5' end (Primer sequences are set forth in Tables 6, 7, 12 and 16, and in the Sequence Listing). Specifically, the forward primer (as labelled in Tables 6, 7, 12 and 17) for each marker was synthesised with the 19-bp nucleotide sequence: 5 '- CACGACGTTGTAAAACGAC -3 ' (SEQ ID NO: 13).
  • the reverse primer (as labelled in Tables 6, 7, 12 and 17) for each marker was synthesised with the 19-bp nucleotide sequence: 5'- GTACATTAAGTTCCCATTAC -3' (SEQ ID NO: 14).
  • Primer aliquots for each marker were prepared by mixing equimolar amounts of appropriate forward and reverse primer in Ix TE (1 mM EDTA, 10 mM Tris-HCL, pH 8.0), and are hereafter referred to as locus-specific primers.
  • TagF and tagR were also synthesised and comprise the sequences: 5' ACGACGTTGTAAAA 3' (SEQ ID NO: 55); or 5' CATTAAGTTCCCATTA 3' (SEQ ID NO: 56).
  • the tagF primer was dye-labelled at its 5' end with VIC®, FAMTM, NEDTM or PETTM (Applied Biosystems).
  • the optimal concentration of locus-specific primer required for marker amplification was determined empirically. Typically, PCR was performed for each marker using 20, 30, 40 or 80 nM of locus-specific primer. PCR products were electrophoresed on a GelScan2000 instrument as described herein, and the optimal primer concentration was determined by visual inspection of the yield of target amplicon and PCR specificity. The optimal concentration of locus-specific primer was defined as the primer concentration producing a strongly amplified target fragment(s) with a high level of PCR specificity. In instances where it was desirable to adjust the PCR specificity observed for a marker, additional locus-specific primer concentrations were tested. Locus-specific primer concentrations as low as 7.5 nM, and as high as 240 nM, were used to achieve the desired level of PCR specificity.
  • locus-specific primers for several markers were added to each reaction at the optimal concentration determined using singleplex amplification.
  • PCR was performed for a total of 55 cycles with the profile: 60 s at 92°C, 90 s at 50°C, 60 s at 72°C for five cycles. The next 20 cycles were with 30 s at 92°C, 90 s at 63°C, and 60 s at 72°C, followed by 40 cycles with 15 s at 92°C, 60 s at 54°C, and 60 s at 72°C, and a final extension step of 10 min at 72°C.
  • Electrophoresis and visualization of SSR alleles was performed on a GelScan2000 (Corbett Research), or an ABI3700 instrument or an ABI3730 instrument (Applied Biosystems).
  • the PCR products were mixed with an equal volume of gel loading buffer (98% formamide, 10 mM EDTA, and 0.5% basic fuchsin as tracking dye), heated for 3 min at 95 °C, chilled quickly on ice and separated on a 4% sequencing gel (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001)).
  • gel loading buffer 98% formamide, 10 mM EDTA, and 0.5% basic fuchsin as tracking dye
  • PCR products for each set of markers were mixed together in a ratio of 1:2:2:5 for VTC:FAM:NED:PET, desalted by ethanol precipitation (Sambrook et al (hi: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001)), and resuspended in an appropriate volume of sterile water to give a final dilution factor of 1:500.
  • the diluted PCR products were separated by capillary electrophoresis on the ABI3700 using LIZ-labeled GeneScan500 as an internal size standard (Applied Biosystems).
  • PCR products for each set of markers were mixed together at a ratio of 3:4:2:4 for VIC:FAM:NED:PET, desalted by vacuum filtration on an AcroPrep 384-well filter plate with a 1OK Omega membrane (PALL Life Sciences) essentially according to the manufactures instructions, and resuspended in an appropriate volume of sterile water to give a final dilution factor of 1:500.
  • the diluted PCR products were separated by capillary electrophoresis on the ABI3730 using LIZ- labeled GeneScan500 as an internal size standard (Applied Biosystems).
  • PCR fragment sizes were determined using GeneMapper v3.7 analysis software (Applied Biosystems). The pooling of PCR products with different dye-labels at the described ratios for each ABI instrument was to account for differences in the relative fluorescence of each fluorophore dye, and the detection sensitivity of each instrument. 1.2 DNA quality and concentration does not affect amplification
  • the effect of the concentration of DNA used in the multiplex assay of the invention on PCR specificity and amplification yield was determined by comparing the SSR profiles of 64 barley and wheat markers amplified using 10, 30, 50, 70, 100, 150 and 250 ng of genomic DNA purified from leaf material using a salt-based extraction method (Rogowsky et al, Theoretical and Applied Genetics 82, 537-554 1991). Each PCR was performed in triplicate.
  • SSRs markers shown in Figure Ia are representative of the effect that genomic DNA concentration had on the amplification yield and PCR specificity. DNA was loaded onto the gel represented in Figure 1 a in the following order:
  • genomic DNA was extracted from the barley variety Sloop.
  • SSRs shown in Figure Ib are representative of the effect that genomic DNA quality had on the amplification yield and PCR specificity.
  • PCR products were separated by gel electrophoresis using a 4% denaturing polyacrylamide gel and detected by fluorescence using a GelScan2000 instrument.
  • the results of these experiments demonstrate that the concentration and quality of genomic DNA had no observable effect on PCR specificity and amplification yield.
  • the tolerance of the PCR assay of the invention to a broad range of DNA template concentrations indicated that quantification of genomic DNA samples is not necessary.
  • the tolerance of the assay to high concentrations of genomic DNA indicated that the assay was not susceptible to PCR over-cycling effects. Such over- cycling effects often result in assay failure due to the conversion of PCR products into random high molecular sized fragments (Bell and DeMarini, Nucl. Acids Res. 19, 5079, 1991).
  • the compatibility of the assay with low-quality DNA samples prepared by sodium hydroxide extraction is also useful for facilitating high throughput genotyping in marker-assisted breeding.
  • This experiment aimed to determine how PCR specificity and amplification yield was affected by the concentration of locus-specific primer used in the assay.
  • DNA fingerprints of 32 wheat SSRs amplified using 40, 20, 10, 5, 3.3, 2.5, 2 or 1.6 nM of locus-specific primer were compared.
  • Each PCR was performed in triplicate using genomic DNA from the genetically diverse wheat varieties Olympic, Gabo and Chinese Spring. PCR products were separated on a 4% denaturing polyacrylamide gel and detected by fluorescence using a GelScan2000 instrument (see Materials and Methods).
  • the amplification products shown in Figure 2 are representative of the different types of effects that the concentration of locus-specific primer had on amplification yield and PCR specificity.
  • Table 3 Source of nucleic acid used in PCR am lification and location in Fi ure 2.
  • LSPconc is the concentration of locus-specific primer (mM) used in the multiplex- ready PCR assay
  • the concentration of locus-specific primer affects both the PCR specificity and yield of amplification product(s) for each marker.
  • the marker gwm642 amplified two microsatellite loci when the concentration of locus-specific primer in the multiplex-ready assay was ⁇ 5 nM.
  • the concentration of locus-specific primer in the multiplex-ready assay was ⁇ 5 nM.
  • the concentration of locus-specific primer in the multiplex-ready assay was ⁇ 5 nM.
  • locus-specific primer concentrations i.e., >10 nM
  • the microsatellite locus producing the smallest SSR fragment was preferentially amplified.
  • a locus-specific primer concentration of 40 nM achieved relatively uniform amplification of the two microsatellite loci amplified by the marker gwml74.
  • PCR assay of the invention is capable of coamplifying up to six wheat markers without assay optimization.
  • 32 marker panels consisting of twelve 6- plex and twenty 4-plex reactions were tested. Each PCR was performed in duplicate across six genetically diverse wheat varieties. PCR products were separated on a 4% denaturing polyacrylamide gel and detected by fluorescence using a GelScan2000 instrument.
  • the multiplex marker panels shown in Figure 3 are representative of the PCR specificity and amplification yield achieved.
  • the DNA was loaded onto the gel depicted in Figure 3 in the order shown in Table 4.
  • Table 4 Source of nucleic acid used in PCR amplification and location in Figure 3. Lane Wheat Variety
  • Table 5 Markers amplified in multiplex reactions and the location in Figure 3 of the electro horesed amplicons roduced
  • Marker names correspond to primer sets set forth in Table 6.
  • 1 LSPopt is the optimal concentration of the locus specific primer for marker amplification in nM.
  • SSR markers amplified from six genetically diverse wheat varieties using the assay of the invention and conventional PCR assays were compared.
  • the markers were amplified using the primers set forth in Table 6.
  • Microsatellite marker amplification by conventional PCR was performed in a 6 ⁇ l reaction mixture containing 0.2 mM dNTP, Ix PCR buffer containing 1.5 mM MgCl 2 (Qiagen), 200 nmol each of dye-labelled forward and unlabelled reverse primer, 50 ng genomic DNA, and 0.15 U DNA polymerase (Qiagen).
  • Conventional PCR amplification was performed for a total of 48 cycles with the touchdown profile: 30 s at 94 0 C, 30 s at 60°C, and 30 s at 72°C.
  • PCR profile was completed with a final extension step of 10 min at 72 0 C. PCR products were separated on a 4% denaturing polyacrylamide gel and detected by fluorescence using a GelScan2000 instrument.
  • the wheat varieties used in this experiment from left to right in Figure 4 were: Rialto, Spark, Halberd, Cranbrook, Sunco and Tasman.
  • the level of nucleic acid amplified for each marker is approximately equal Using the semi-automated ABI3700 instrument and Genotyper version 3.7 analysis software (Applied Biosystems), the average fluorescence peak height for each of 366 markers was calculated from the fluorescence peak heights observed for eight genetically diverse barley (or wheat) cultivars. Additionally, using the semi-automated ABI3730 instrument and Genemapper version 3.7 analysis software (Applied Biosystems), the average fluorescence peak height for each of 1070 markers was calculated from the fluorescence peak heights observed for eight genetically diverse barley (or wheat) cultivars.
  • One hundred and four primer sets for amplifying a characterised marker from Prunus spp. were produced essentially as described in Example 1.1.1.
  • the nucleotide sequence of the locus specific region of the primers used is set forth in Table 7.
  • Forward primers were synthesized with a 19-bp tag sequence and the reverse primers synthesized with a 20-bp tag sequence.
  • the optimum concentration of each locus-specific primer was determined using singleplex reactions, essentially as described in Section 1.1.2, above, and are listed in Table 7.
  • the template nucleic acid used in the reactions was derived from apricot and cherry varieties (P.armeniaca and P. avium, respectively).
  • Conditions for the PCR reactions were essentially as described in Section 1.1.3, above.
  • Tag primers were also assessed for their ability to anneal to and initiate replication of nucleic acid from the apricot and cherry genomes. PCR reactions were performed with primer concentrations of 75nM, 20OnM, 30OnM or 40OnM. No amplification was detected with these primers in the absence of locus-specific primers, indicating that the tag primers do not anneal to and initiate amplification of nucleic acid in the apricot and cherry genomes.
  • Table 7 Nucleotide sequence of locus specific regions for amplifying template nucleic acid from Prunus sp.
  • BPPCT002 SSR TCGACAGCTTGATCTTGACC CAATGCCTACGGAGATAAAAGAC 100
  • BPPCT009 SSR ATTCGGGTCGAACTCCCT ACGAGCACTAGAGTAACCCTCTC 100
  • BPPCT015 SSR ATGGAAGGGAAGAGAAATCG GTCATCTCAGTCAACTTTTCCG 140
  • BPPCT022 SSR TTGCGTCTCGCAGGTTATA CTACCCCTGCCACAAGCT 100
  • BPPCT024 SSR GAGGAATGTGCCTCTTCTGG CTCCCGTACGCGTTTACC 100
  • BPPCT026 SSR ATACCTTTGCCACTTGCG TGAGTTGGAAGAAAACGTAACA 140
  • BPPCT031 SSR CTGGGGAGAAGAAGTGGC GCTTTCATGCCACCTCTCTA 140
  • BPPCT032 SSR TTAAGCCACAACATCCATGAT AATGGTCTAAGGAGCACACG 100
  • BPPCT033 SSR GTAGCCGGAGCCGTGTAT CTAGAACCCTATAAACACATGGC 100
  • BPPCT034 SSR CTACCTGAAATAAGCAGAGCCAT CAATGGAGAATGGGGTGC 100
  • BPPCT040 SSR ATGAGGACGTGTCTGAATGG AGCCAAACCCCTCTTATACG 100
  • BPPCT042 SSR AACCCTACTGGTTCCTCAGC GACCAGTCCTTTAGTTGGAGC 140
  • PS12A02 SSR GCCACCAATGGTTCTTCC AGCACCAGATGCACCTGA 120
  • the average fluorescence peak height for each of 64 markers was calculated from the fluorescence peak heights observed for six apricot and six cherry varieties.
  • the multiplex marker panels shown in Figure 8 are representative of the PCR specificity and amplification yield achieved.
  • the DNA was loaded onto the gel depicted in Figure 8 in the order shown in Table 8.
  • Table 8 Source of nucleic acid used in PCR amplification and location of the electro horesed am licon in Fi ure 8.
  • the 10-plex marker panels shown in Figure 9 are representative of the PCR specificity and amplification yield achieved.
  • the DNA was loaded onto the gel depicted in Figure 9 in the order shown in Table 10.
  • Table 10 Source of nucleic acid used in PCR amplification and location of the electrophoresed am licon in Fi ure 9.
  • Table 11 Markers depicted in multiplex reactions and the location in Figure 9 of the electrophoresed amplicons roduced
  • Marker names correspond to primer sets set forth in Table 7.
  • primer sets for amplifying characterised markers from domesticated cattle (Bos taurus) and sheep (Ovis aries) were produced essentially as described in Example 1.1.1.
  • the nucleotide sequence of the locus specific region of the primers used is set forth in Table 12. Forward primers were synthesised with a 19-bp tag sequence and the reverse primers synthesised with a 20-bp sequence as described in Example 1.1.1.
  • each locus specific primer was determined using singleplex reactions, essentially as described in Section 1.1.2, above, and is listed in Table 12. However, the template nucleic acid used in the reactions was derived from cattle or sheep. Conditions for the PCR reactions were essentially as described in Section 1.1.3, above.
  • Tag primers were also assessed for their ability to anneal to and initiate replication of nucleic acid from cattle and sheep genomes. PCR reactions were performed with primer concentrations of 75nM, 20OnM, 30OnM or 40OnM. No amplification was detected with these primers in the absence of locus-specific primers, indicating that the tag primers do not anneal to and initiate amplification of nucleic acid in cattle and sheep genomes.
  • Table 12 Nucleotide sequence of locus specific regions for amplifying template nucleic acid from cattle and sheep.
  • HMH1R(MBO84) SSR GAAAGCTGGAGCAAACATCC AACTGCCACCACTGTCAGG 15
  • CSSM31 SSR CCAAGTTTAGTACTTGTAAGTAGA GACTCTCTAGCACTTTATCTGTGT 20
  • ADCY2(MB085) SSR AAAGTGACACAACAGCTTCTCC ACAAGTGAGTGCGTAACAAAGG 15
  • LSPconc is the concentration (in nM) of locus-specific primer used in the multiplex-ready PCR assay
  • Table 13 Source of nucleic acid used in PCR amplification and location of the electrophoresed amplicon in Fi ure 10.
  • LSPconc is the concentration (in nM) of locus-specific primer used in multiplex-ready PCR
  • Table 14 Source and amount of nucleic acid used in PCR amplification and location of
  • the 3-plex marker panels shown in Figure 12 are representative of the PCR specificity and amplification yield achieved.
  • the DNA was loaded onto the gel depicted in Figure 12 in the order shown in Table 15.
  • Table 15 Source of nucleic acid used in PCR amplification and location of the electrophoresed am licon in Fi ure 12.
  • Table 16 Markers depicted in multiplex reactions and the location in Figure 12 of the electro horesed am licons roduced
  • Marker names correspond to primer sets set forth in Table 12.
  • Example 1.1.1 Twenty four primer sets for amplifying characterised microsatellite markers from the fungal pathogen Rhynchosporium secalis (causal agent of scald disease in barley) were produced essentially as described in Example 1.1.1.
  • the nucleotide sequence of the locus specific region of the primers used is set forth in Table 17. Forward primers were synthesised with a 19-bp tag sequence and the reverse primers synthesised with a 20-bp sequence as described in Example 1.1.1.
  • Section 1.1.2 above, and are listed in Table 16.
  • the template nucleic acid used in the reactions was a heterogenous mixture of genomic DNA from barley and the fungal pathogen.
  • Genomic DNA was isolated from barley leaf tissue infected with the fungal pathogen using a salt-based extraction method (Rogowsky et al, supra). Each PCR was performed in triplicate. Conditions for the PCR reactions were essentially as described in Section 1.1.3, above. Successful amplification of 88% (21/24) of the markers tested showed that the assay of the present invention is suitable for in planta detection of fungal pathogen.
  • Tag primers were also assessed for their ability to anneal to and initiate replication of nucleic acid from the heterogenous mixture of barley and R. secalis genomic DNA. PCR reactions were performed with primer concentrations of 75nM, 20OnM, 30OnM or 40OnM. No amplification was detected with these primers in the absence of locus- specific primers, indicating that the tag primers do not anneal to and initiate amplification of nucleic acid in barley or R. secalis genomes.
  • Table 17 Nucleotide sequence of locus specific regions for amplifying template nucleic acid from Rhynchosporium secalis.
  • SRS_TgAg5 SSR ACACACAGTTCATTCCAGTTTCAAG TGTGTGTGTGTGTGTGAGAGAGAG 200
  • SRS-TgAgI 9 SSR CTATCTGAAGCGAAGAAGGTAAGTG TGTGTGTGTGTGTGTGAGAGAGAG 60
  • SRS_TgAg28 SSR GTAATGGACTATGGACTGTGGAGAA TGTGTGTGTGTGTGTGAGAGAGAG 200
  • SRS_TgAg29 SSR GTCAGGTATGTAGGCAAGGTAGTCG TGTGTGTGTGTGTGAGAGAGAG 60
  • SRS_TgAg31 SSR TAGAGAGAAGGGTTTACAAAGACGA TGTGTGTGTGTGTGAGAGAGAG 60
  • SRS_TgAg32 SSR TAGAGGAATAGACACCGTCAACAA TGTGTGTGTGTGTGAGAGAGAG 200
  • SRS_TgAg35 SSR TGGTCTGTTCATTTATTGACTCGTA TGTGTGTGTGTGTGTGAGAGAGAG 200
  • LSPconc is the concentration (in nM) of locus-specific primer used in the multiplex-ready PCR assay
  • BHSfNER is. a software program that determines the most cost-efficient way to separate a set of markers, e.g., electrophoretically.
  • the program uses a few simple rules to construct marker panels for the markers of interest.
  • Each marker panel consists of amplification products that can be resolved, e.g., by electrophoresis; i.e. marker panels are constructed so that each amplification product has a unique length.
  • the mathematical algorithm essentially calculates from all possible marker combinations the minimum number of panels in which the markers of interest can be placed. This minimizes the cost of marker separation, since the number of marker panels determines the total number of capillaries that are required for electrophoretic separation. This process is also useful for determining which markers are to be amplified in a single multiplex amplification reaction.
  • the BINNER software supports individual user accounts to permit users to upload and manage marker data. Following login, a list of markers is shown for which allele length data is available, such as, for example, allele length data for barley and wheat markers that are published in the Multiplex-Ready Marker Database. This allele length data was generated using eight genetically diverse varieties. The varieties used for barley were Alexis, Chebec, Clipper, Harrington, Haruna Nijo, Sahara 3771, Sloop and WI3408; and for wheat were Barunga, VPM Cook, Chinese Spring, Gabo, WI7984 (a synthetic hexaploid), NorinlO, Olympic and Opata85.
  • marker allele length data is uploaded to BINNER and used to develop marker panels for, for example, specific germplasm or a specific species or a specific genus.
  • suitable marker allele length data are saved as a Microsoft® Excel® file and uploaded to the BINNER software.
  • Marker panels for deployment or separation on an automated DNA fragment analyser are then constructed automatically by BINNER for a set of user-defined markers using either default or imported allele length data.
  • BINNER To determine suitable marker panels, the minimum amount of distance (in base-pairs) that BINNER must leave between markers when building a marker panel is selected. It is recommended that a minimum padding of 10-bp is used for marker polymorphism screening unless the allele length range for each marker is known for the germplasm of interest. For genetic mapping, a minimum padding of 5-bp is recommended.
  • Each panel of markers created by BESENER contains a list of markers that have correct spatial separation to avoid allele overlap when separated on an automated DNA fragment analyser. These panels are then used to select appropriate primers or set of primers for use in a multiplex amplification reaction to produce amplification products that can be resolved, e.g., using electrophoresis.

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Abstract

La présente invention concerne un procédé qui amplifie les acides nucléiques à l’aide d’une analyse PCR. L’invention se rapporte à un procédé destiné à amplifier un acide nucléique dans un tube fermé unique, par exemple. Le procédé se compose de plusieurs étapes : la première amplification comprend une quantité d’amorce à un locus spécifique marqué ou une extension de ladite amorce en quantité appropriée pour effectuer un PCR détaillé ; lors de la seconde amplification, le produit de la première amplification est amplifié à l’aide d’amorceurs marqués ayant une température de fusion inférieure à celle des amorceurs marqués avec un locus marqué et annelés selon laséquence marquée incorporée dans le produit de ladite première amplification à une température d’ annelage inférieure à celle qui est utilisée lors de la première amplification. La présente invention a également trait au multiplexage de l’amplification. Le présent procédé concerne aussi des procédés destinés à caractériser ou identifier des individus ou établir le diagnostique d’une maladie ou d’un trouble.
PCT/AU2006/000318 2005-03-11 2006-03-10 Procede destine a amplifier les acides nucleiques Ceased WO2006094360A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2911144A1 (fr) * 2007-01-10 2008-07-11 Inst Francais Des Boissons De Methode d'obtention d'un profil genetique specifique d'une variete d'orge a l'aide de couples d'amorces.
WO2009052547A1 (fr) 2007-10-22 2009-04-30 Monoquant Pty Ltd Procédé d'amplification d'adn
WO2009068211A1 (fr) * 2007-11-30 2009-06-04 Olympus Life Science Research Europa Gmbh Procédé pour déterminer la présence ou l'absence de plusieurs séquences cibles dans un échantillon biologique
EP1947196A4 (fr) * 2005-11-08 2009-09-09 Olympus Corp Procédé d'amplification de plusieurs séquences d'acides nucléiques en vue de leur différenciation
WO2010046441A1 (fr) * 2008-10-23 2010-04-29 Qiagen Gmbh Quantification d’arn utilisant une normalisation interne
WO2011059508A2 (fr) 2009-11-16 2011-05-19 Genomics Usa, Inc. Méthodes s'appliquant à la pcr et au typage hla au moyen de sang brut
EP2559774A1 (fr) * 2011-08-17 2013-02-20 Roche Diagniostics GmbH Procédé amélioré pour l'amplification d'acides nucléiques cible au moyen d'une approche à plusieurs amorces
CN103451286A (zh) * 2013-08-28 2013-12-18 宁夏农林科学院农作物研究所 用于检测小麦抗旱性的引物及其应用
CN105274198A (zh) * 2015-05-26 2016-01-27 江苏省农业科学院 一种基于转录组测序开发鸟巢蕨est-ssr引物的方法
WO2016184902A1 (fr) 2015-05-18 2016-11-24 Saga Diagnostics Ab Détection d'acides nucléiques et variants
CN106957914A (zh) * 2017-04-08 2017-07-18 中国农业科学院郑州果树研究所 樱桃品种的鉴定方法
US10337066B2 (en) 2009-11-16 2019-07-02 Genomics Usa, Inc. Methods for PCR and HLA typing using unpurified samples
CN110172525A (zh) * 2019-06-26 2019-08-27 广西壮族自治区林业科学研究院 林木差异表达基因ssr引物组及多态性ssr标记开发方法
CN114686579A (zh) * 2020-12-28 2022-07-01 广东菲鹏生物有限公司 用于核酸样本扩增的组合物、试剂盒、方法及系统
CN116064927A (zh) * 2022-07-06 2023-05-05 北京博晖创新生物技术集团股份有限公司 一种用于呼吸道病原体检测的引物探针组合物及应用

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EP1947196A4 (fr) * 2005-11-08 2009-09-09 Olympus Corp Procédé d'amplification de plusieurs séquences d'acides nucléiques en vue de leur différenciation
EP1944380A1 (fr) * 2007-01-10 2008-07-16 Institut Français des Boissons, de la Brasserie et de la Malterie Méthode d'obtention d'un profil génétique spécifique d'une variété d'orge à l'aide de couples d'amorces
FR2911144A1 (fr) * 2007-01-10 2008-07-11 Inst Francais Des Boissons De Methode d'obtention d'un profil genetique specifique d'une variete d'orge a l'aide de couples d'amorces.
WO2009052547A1 (fr) 2007-10-22 2009-04-30 Monoquant Pty Ltd Procédé d'amplification d'adn
EP2193208A4 (fr) * 2007-10-22 2011-01-26 Monoquant Pty Ltd Procédé d'amplification d'adn
WO2009068211A1 (fr) * 2007-11-30 2009-06-04 Olympus Life Science Research Europa Gmbh Procédé pour déterminer la présence ou l'absence de plusieurs séquences cibles dans un échantillon biologique
WO2010046441A1 (fr) * 2008-10-23 2010-04-29 Qiagen Gmbh Quantification d’arn utilisant une normalisation interne
EP3342880A1 (fr) * 2009-11-16 2018-07-04 Genomics USA, Inc. Méthodes s'appliquant à la rt-pcr et au typage hla au moyen de sang brut
WO2011059508A2 (fr) 2009-11-16 2011-05-19 Genomics Usa, Inc. Méthodes s'appliquant à la pcr et au typage hla au moyen de sang brut
CN102834525A (zh) * 2009-11-16 2012-12-19 美国基因组学有限公司 使用未经加工的血液的pcr和hla分型的方法
US10337066B2 (en) 2009-11-16 2019-07-02 Genomics Usa, Inc. Methods for PCR and HLA typing using unpurified samples
EP2501827A4 (fr) * 2009-11-16 2013-05-15 Genomics Usa Inc Méthodes s'appliquant à la pcr et au typage hla au moyen de sang brut
US20130045894A1 (en) * 2011-08-17 2013-02-21 Bruno Frey Method for Amplification of Target Nucleic Acids Using a Multi-Primer Approach
EP2559774A1 (fr) * 2011-08-17 2013-02-20 Roche Diagniostics GmbH Procédé amélioré pour l'amplification d'acides nucléiques cible au moyen d'une approche à plusieurs amorces
CN103451286A (zh) * 2013-08-28 2013-12-18 宁夏农林科学院农作物研究所 用于检测小麦抗旱性的引物及其应用
WO2016184902A1 (fr) 2015-05-18 2016-11-24 Saga Diagnostics Ab Détection d'acides nucléiques et variants
US11066707B2 (en) 2015-05-18 2021-07-20 Saga Diagnostics Ab Detection of target nucleic acid variants
CN105274198A (zh) * 2015-05-26 2016-01-27 江苏省农业科学院 一种基于转录组测序开发鸟巢蕨est-ssr引物的方法
CN106957914A (zh) * 2017-04-08 2017-07-18 中国农业科学院郑州果树研究所 樱桃品种的鉴定方法
CN106957914B (zh) * 2017-04-08 2020-12-01 中国农业科学院郑州果树研究所 樱桃品种的鉴定方法
CN110172525A (zh) * 2019-06-26 2019-08-27 广西壮族自治区林业科学研究院 林木差异表达基因ssr引物组及多态性ssr标记开发方法
CN114686579A (zh) * 2020-12-28 2022-07-01 广东菲鹏生物有限公司 用于核酸样本扩增的组合物、试剂盒、方法及系统
CN116064927A (zh) * 2022-07-06 2023-05-05 北京博晖创新生物技术集团股份有限公司 一种用于呼吸道病原体检测的引物探针组合物及应用

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