A gel-free SNP genotyping method: bioluminometric assay coupled with modified primer extension reactions (BAMPER) directly from double-stranded PCR products

Human Mutation
Guohua ZhouHideki Kambara

Abstract

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 compone...Continue Reading

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Citations

Jun 20, 2006·TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·K ShirasawaT Nishio
Sep 6, 2005·Nucleic Acids Research·Guo-Hua ZhouHideki Kambara
Nov 8, 2015·Applied and Environmental Microbiology·Lana J McMillanJulie A Maupin-Furlow
Jun 13, 2015·Clinical Chemistry and Laboratory Medicine : CCLM·Wenqian SongWeijian Yu
Nov 2, 2007·Chemical Reviews·Karel Kleparník, Petr Bocek

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