Allele specific PCR for genotyping
First Claim
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1. A method of genotyping one or more loci in a DNA sample, comprising the steps of:
- combining a sample containing single stranded DNA or double stranded DNA, at least one primary primer specific for one locus on one strand of DNA in said sample, said primary primer having a first homologous portion which hybridizes to said one strand of DNA and a non-homologous portion which does not hybridize to said one strand of DNA, at least one secondary primer having a second homologous portion which comprises sequences identical to those of said non-homologous portion of said primary primer;
conducting polymerase chain reaction (PCR); and
identifying amplicons of said PCR which include said non-homologous portion, wherein said step of identifying allows the genotype of said one or more loci to be established.
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Abstract
Methodology for a two-level allele-specific extension (AS-PCR) reaction for use in ultra-high throughput genotyping is provided. A primary AS-PCR reaction is carried out with primers containing both sequence-specific and artificial, universal domains. The primers are designed to render the PCR products 1) allele specific and 2) amenable to amplification with secondary primers which also contain universal domains. The secondary primers are designed to maintain allele-specificity, and to provide a detectable label.
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12 Claims
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1. A method of genotyping one or more loci in a DNA sample, comprising the steps of:
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combining a sample containing single stranded DNA or double stranded DNA, at least one primary primer specific for one locus on one strand of DNA in said sample, said primary primer having a first homologous portion which hybridizes to said one strand of DNA and a non-homologous portion which does not hybridize to said one strand of DNA, at least one secondary primer having a second homologous portion which comprises sequences identical to those of said non-homologous portion of said primary primer;
conducting polymerase chain reaction (PCR); and
identifying amplicons of said PCR which include said non-homologous portion, wherein said step of identifying allows the genotype of said one or more loci to be established. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A primer set for genotyping one or more loci in a DNA sample, comprising,
at least one primary primer specific for one locus on one strand of DNA in said sample, said primary primer having a first homologous portion which hybridizes to said one strand of DNA and a non-homologous portion which does not hybridize to said one strand of DNA, and at least one secondary primer having a second homologous portion which comprises sequences identical to those of said non-homologous portion of said primary primer.
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10. A method of multiplex PCR for a plurality of loci in a DNA sample, comprising the steps of:
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carrying out a first round of PCR amplification with at least one primary primer specific for one locus on one strand of DNA in said sample, said primary primer having a first homologous portion which hybridizes to said one strand of DNA and a non-homologous portion which does not hybridize to said one strand of DNA, wherein said primary primers are present in an equal and limited amount, and carrying out a second round of amplification with at least one secondary primer having a second homologous portion which comprises sequences identical to those of said non-homologous portion of said primary primer;
wherein said secondary primers are present in a non-limiting amount. - View Dependent Claims (11)
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12. A kit, comprising
instructions for the design of primary primers having a first homologous portion which hybridizes to one strand of DNA and a non-homologous portion which does not hybridize to said strand of DNA, and secondary PCR primers having a second homologous portion which comprises sequences identical to those of said non-homologous portion of said primary primer.
Specification