Abstract: The present invention provides a method of identifying a base at a target position in a sample nucleic acid sequence, said method comprising: subjecting a primer hybridised to said sample nucleic acid immediately adjacent to the target position, to a polymerase primer extension reaction in the presence of a nucleotide, whereby the nucleotide will only become incorporated if it is complementary to the base in the target position, and determining whether or not said nucleotide is incorporated by detecting whether Ppi is released, the identity of the target base being determined from the identity of any nucleotide incorporated, wherein, where said nucleotide comprises an adenine base, an ?-thio triphosphate analogue of said nucleotide is used, and the Rp isomer of said analogue and/or the degradation products of said analogue are eliminated from the polymerase reaction step.

Abstract: The present invention provides a method of identifying a base at a target position in a single-stranded sample DNA sequence wherein an extension primer, which hybridizes to the sample DNA immediately adjacent to the target position, is provided and the sample DNA and extension primer are subjected to a polymerization reaction in the presence of a deoxynucleotide or dideoxynucleotide, whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate if it is complementary to the base in the target position. Release of pyrophosphate is detected enzymatically and pyrophosphate detection enzyme(s) are included in the polymerization step.