Determination of genotype of an amplification product at multiple allelic sites

A method for determining which allelic sites are present in a sample of DNA, the method comprising:

• amplifying the sample of DNA in the presence of:
• i) a polymerase having 5′ nuclease activity;
• ii) a forward primer and a reverse primer that hybridize to the sample of DNA, and iii) four oligonucleotide probes that differ from each other by at least one nucleotide base, such that the first and second oligonucleotide probes compete to hybridize to a first allelic site and the third and fourth oligonucleotide probes compete to hybridize to a second allelic site;
• wherein all of the oligonucleotide probes comprise a different fluorescer, and a quencher that quenches the fluorescence of the fluorescer;
• and
• determining the presence or absence of the first and second allelic sites by detecting a fluorescence spectrum of the amplification reaction resulting from 5′ nuclease digestion of the oligonucleotide probes that hybridized to the sample DNA.

A kit for identifying in a single 5′ nuclease assay which alleles at multiple different allelic sites are present in a sample of DNA, the kit comprising:

• a first set of oligonucleotide probes comprising first and second oligonucleotide probes for detecting which alleles are present at a first allelic site by single 5′ nuclease assay, the first and second oligonucleotide probes being designed to distinguish between at least two alleles at the first allelic site, which differ from each other by at least one base position, wherein the first set of oligonucleotide probes is digestible by a 5′ to 3′ nuclease, and does not self-hybridize;
• a second set of oligonucleotide probes comprising third and fourth oligonucleotide probes for detecting which alleles are present at a second, different allelic site by the single 5′ nuclease assay, the third and fourth oligonucleotide probes being designed to distinguish between at least two alleles at the second allelic site, which differ from each other by at least one base position, wherein the second set of oligonucleotide probes is digestible by a 5′ to 3′ nuclease, and does not self-hybridize;
• and
• a first primer that is capable of hybridizing to a sequence in the first allelic site 3′ relative to where the probes of the first set hybridize to the first allelic site and a second primer that is capable of hybridizing to a sequence in the second allelic site 3′ relative to where the probes of the second set hybridize to the second allelic site;
• wherein all of the oligonucleotide probes comprise a different fluorescer and all of the oligonucleotide probes comprise a quencher that quenches the fluorescence of the fluorescer such that the first and second sets of oligonucleotide probes can be used to determine which alleles are present at the first and second allelic sites in the single 5′ nuclease assay.

A kit for identifying in a single 5′ nuclease assay which alleles are present in a sample of DNA, the kit comprising:

• first and second oligonucleotide probes for detecting which alleles are present at a first allelic site, wherein the first and second oligonucleotide probes:
• i) are digestible by a 5′ to 3′ nuclease;
• ii) do not self hybridize;
• and iii) differ from each other by at least one nucleotide base;
• third and fourth oligonucleotide probes for detecting which alleles are present at a second allelic site, wherein the third and fourth oligonucleotide probes:
• i) are digestible by a 5′ to 3′ nuclease;
• ii) do not self hybridize;
• and iii) differ from each other by at least one nucleotide base;
• a first primer that is capable of hybridizing to a sequence of the first allelic site 3′ relative to where the first or second probes hybridize to the first allelic site;
• and
• a second primer that is capable of hybridizing to a sequence of the second allelic site 3′ relative to where the third or fourth probes hybridize to the second allelic site;
• wherein all of the oligonucleotide probes comprise a different fluorescer and all of the oligonucleotide probes comprise a quencher that quenches the fluorescence of the fluorescer such that the probes can be used to determine which alleles are present at the first and second allelic sites in the single 5′ nuclease assay.