BROAD RANGE PCR AMPLIFICATION TECHNIQUES

摘要

Disclosed herein are methods and kits for determining whether a nucleic acid sequence includes a particular allele of a polymorphic sequence. One such method includes the steps of: (a) contacting a nucleic acid sequence, in the same or a separate reaction, with a first pair of PCR primers and a second pair of PCR primers under conditions that allow hybridization of the PCR primers to the nucleic acid sequence, the first pair of PCR primers hybridizing to opposite strands of the nucleic acid sequence and bordering the position of the polymorphic sequence, and the second pair of PCR primers hybridizing to opposite strands of the nucleic acid sequence and bordering the position of the polymorphic sequence, the PCR primers being characterized as follows: (i) one of the first pair of PCR primers (a) being complementary at its 3'-terminal nucleotide to a first allele of the polymorphic sequence (allele A), (b) being non-complementary at its 3'-terminal nucleotide to a second allele of the polymorphic sequence (allele B), and (c) being non-complementary to the nucleic acid sequence at a single non-complementary nucleotide in its 3'-terminal nucleotides 2-6; and (ii) one of the second pair of PCR primers (a) being complementary at its 3'-terminal nucleotide to the first allele of the polymorphic sequence (allele A), (b) being non-complementary at its 3'-terminal nucleotide to the second allele of the polymorphic sequence (allele B), and (c) being non-complementary to the nucleic acid sequence at one (and, preferably, two) or more nucleotides in its 3'-terminal nucleotides 2-6; (b) carrying out the amplification reactions; and (c) detecting an amplification product as an indication of the presence, in the nucleic acid sequence, of the first allele of the polymorphic sequence (allele A). In a preferred embodiment, this method is used to identify single nucleotide polymorphisms, for example, for genomic mapping purposes.