Determination of a genotype of an amplification product at multiple allelic sites

A method for genotyping a sample of DNA at at least two allelic sites by a 5' nuclease amplification reaction, the method comprising:

• performing a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5'.fwdarw.3' nuclease activity and at least one set of forward and reverse primers capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
• each set of allelic oligonucleotide probes is for detecting a different allelic site,
• each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence of the sample DNA to which the primer hybridizes 5' relative to a sequence to which the primer hybridizes, and
• at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
• digesting those allelic oligonucleotide probes which hybridize to the target sequence during the amplification by the nuclease activity of the polymerase;
• detecting a fluorescence spectrum of the amplification;
• calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum;
• and
• determining a presence or absence of the different allelic variants at the two or more different allelic sites based on the fluorescence contribution of each fluorescer to the fluorescence spectrum.

A method for genotyping a sample of DNA at at least two allelic sites by a 5' nuclease amplification reaction, the method comprising:

• performing a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5'.fwdarw.3' nuclease activity and at least one set of forward and reverse primers capable of hybridizing to the sample of DNA in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
• each set of allelic oligonucleotide probes is for detecting a different allelic site,
• each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and
• at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
• digesting those allelic oligonucleotide probes which hybridize to the sample DNA during the amplification by the nuclease activity of the polymerase;
• detecting a fluorescence spectrum of the amplification;
• calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum;
• and
• determining a genotype of the target sequence at the at least two different allelic sites based on the fluorescence contribution of the different fluorescers to the fluorescence spectrum.

A method for determining a fluorescence signature of a sample of DNA comprising:

• calculating fluorescence contributions of at least three fluorescers to a fluorescence spectrum taken of a nucleic acid amplification performed on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5'.fwdarw.3' nuclease activity and a primer capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and an internal standard and amplifying the at least two different allelic sites wherein:
• each set of allelic oligonucleotide probes is for detecting a different allelic site,
• each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and
• at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
• and
• normalizing the fluorescence contributions of each fluorescer relative to an internal standard, the normalized fluorescence contributions corresponding to a fluorescence signature for the DNA sample for the at least two different allelic sites.

A method for genotyping a sample of DNA at two or more different allelic sites comprising:

• calculating fluorescence contributions of at least three fluorescers to a fluorescence spectrum taken of a nucleic acid amplification performed on a target sequence having at least two different allelic sites using a nucleic acid polymerase having 5'.fwdarw.3' nuclease activity and a primer capable of hybridizing to the sample of DNA in the presence of two or more sets of allelic oligonucleotide probes and an internal standard and amplifying the at least two different allelic sites wherein:
• each set of allelic oligonucleotide probes is for detecting a different allelic site,
• each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes, and
• at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer;
• normalizing the fluorescence contributions of each fluorescer relative to an internal standard, the normalized fluorescence contributions corresponding to a fluorescence signature for the DNA sample for the at least two different allelic sites;
• and
• determining the genotype of the DNA sample at the at least two allelic sites by comparing the normalized fluorescence contributions of the DNA sample to normalized fluorescence contributions of control sequences having a known genotype at the at least two allelic sites.