Methods and compositions for genotyping

A method of performing a primer extension reaction, comprising:

• obtaining a sample comprising target nucleic acid from one or more individuals;
• obtaining an amplicon population having a sequence generated from the sample and a sequence generated from a tagged first strand amplification primer, by amplifying nucleic acids in the sample having a variant nucleotide that is a transversion flanked in the 5′ direction by an invariant nucleotide and flanked in the 3′ direction by an invariant nucleotide, wherein the tagged first strand primer is employed that comprises a 5′ tag substantially incapable of hybridizing to target nucleic acids in the sample, and wherein the 5′ tag contains the variant nucleotide with its flanking invariant nucleotides, and wherein a second strand amplification primer is employed;
• employing the amplicon population in a primer extension reaction wherein the identity of the variant nucleotide in the sequence generated from the sample is determined by hybridizing a first identification primer immediately adjacent to the variant nucleotide in the sequence generated from the sample;
• hybridizing a second identification primer immediately adjacent to the variant nucleotide in the sequence generated from the amplification primer;
• extending the first and the second identification primers in the presence of one or more nucleotides and a polymerizing agent;
• determining the identity of the variant nucleotide generated from the sample;
• and
• comparing extension product of the first identification primer and extension product of the second identification primer, thereby performing the primer extension reaction.

A method of performing primer extension utilizing at least two amplification primers comprising:

• obtaining a target nucleic acid comprising a variant nucleotide flanked by an invariant nucleotide;
• hybridizing to the target nucleic acid a first amplification primer having a 5′ tag comprising the variant nucleotide flanked by the invariant nucleotide, wherein the 5′ tag is substantially unable to hybridize to the target nucleic acid, and hybridizing a second amplification primer;
• and
• extending the amplification primers in the presence of at least one or more nucleotides and a polymerizing agent, thereby performing primer extension.

A method of monitoring the efficiency of incorporation of chain terminators into primers in a primer extension reaction, comprising:

• generating a population of amplicons from a mixed sample of target nucleic acid, wherein the population of amplicons comprises sequences at known ratios;
• performing primer extension reactions on the population of amplicons employing chain terminators and employing a population of primers specific for the sequences;
• detecting and measuring efficiency of incorporation of chain terminators into the population of primers at the known ratios, thereby monitoring the efficiency of incorporation of chain terminators into primers in a primer extension reaction.
• using the information generated on the efficiency of incorporation of chain terminators at known ratio to interpret observed efficiencies of incorporation of these chain terminators into primers targeted against polymorphisms of unknown ratio.

A method of performing a primer extension reaction, comprising:

• obtaining a sample comprising target nucleic acid from one or more individuals;
• obtaining an amplicon population having a sequence generated from the sample and a sequence generated from a tagged first strand amplification primer, by amplifying nucleic acids in the sample having a variant nucleotide, wherein the tagged first strand primer is employed that comprises a 5′ tag substantially incapable of hybridizing to target nucleic acids in the sample, and wherein the 5′ tag contains the variant nucleotide, and wherein a second strand amplification primer is employed;
• employing the amplicon population in a primer extension reaction wherein the identity of the variant nucleotide in the sequence generated from the sample is determined by hybridizing a first identification primer immediately adjacent to the variant nucleotide in the sequence generated from the sample;
• hybridizing a second identification primer immediately adjacent to the variant nucleotide in the sequence generated from the amplification primer;
• extending the first and the second identification primers in the presence of one or more nucleotides and a polymerizing agent;
• determining the identity of the variant nucleotide generated from the sample;
• and
• comparing extension product of the first identification primer and extension product of the second identification primer, thereby performing the primer extension reaction.