Determination of the identities of single nucleotide polymorphisms, point mutations and characteristic nucleotides in dna

A method for identifying a target nucleotide in a nucleic acid molecule, the nucleic acid molecule having a 3′ sequence and a 5′ sequence, wherein the target nucleotide is located between the 3′ sequence and the 5′ sequence of the nucleic acid molecule, comprising the steps of:

• (a) mixing an oligonucleotide extension primer with the nucleic acid molecule in a plurality of reaction containers, wherein the oligonucleotide extension primer comprises a 3′ hydroxy terminus residue complementary to the 3′ sequence in the nucleic acid molecule directly adjacent to the target nucleotide on the 3′ side;
• (b) allowing the 3′ hydroxy terminus of the oligonucleotide extension primer to hybridize with the 3′ sequence in the nucleic acid molecule;
• (c) providing a distinct chain-extending Bridging Nucleotide to each of the reaction containers, such that the distinct Bridging Nucleotide in each container is complementary to a different possible nucleotidyl residue at the target nucleotide site;
• (d) conducting a template dependent extension of the oligonucleotide extension primer by adding a polymerase reaction mixture to each of the reaction containers to give an extended primer;
• and
• (e) measuring the incorporation of nucleotides into the extended primer in each of the reaction containers in order to identify the target nucleotide.