A gel-free SNP genotyping method: bioluminometric assay coupled with modified primer extension reactions (BAMPER) directly from double-stranded PCR products.
Inexpensive, high-throughput genotyping methods are needed for analyzing human genetic variations. We have successfully applied the regular bioluminometric assay coupled with modified primer extension reactions (BAMPER) method to single-nucleotide polymorphism (SNP) typing as well as the allele frequency determination for various SNPs. This method includes the production of single-strand target DNA from a genome and a primer extension reaction coupled with inorganic pyrophosphate (PPi) detection by a bioluminometric assay. It is an efficient way to get accurate allele frequencies for various SNPs, while single-strand DNA preparation is labor intensive. The procedure can be simplified in the typing of SNPs. We demonstrate that a modified BAMPER method in which we need not prepare a single-strand DNA can be carried out in one tube. A PCR product is directly used as a template for SNP typing in the new BAMPER method. Generally, tremendous amounts of PPi are produced in a PCR process, as well as many residual dNTPs, and residual PCR primers remain in the PCR products, which cause a large background signal in a bioluminometric assay. Here, shrimp alkaline phosphatase (SAP) and E. coli exonuclease I were used to degrade these components prior to BAMPER detection. The specific primer extension reactions in BAMPER were carried out under thermocycle conditions. The primers were extended to produce large amounts of PPi only when their bases at 3'-termini were complementary to the target. The extension products, PPis, were converted to ATP to be analyzed using the luciferin-luciferase detection system. We successfully demonstrated that PCR products can be directly genotyped by BAMPER in one tube for SNPs with various GC contents. As all reactions can be carried out in a single tube, the method will be useful for realizing a fully automated genotyping system.