A simplified pyrosequencing protocol based on linear-after-the-exponential (LATE)-PCR using whole blood as the starting material directly

Abstract

Pyrosequencing has been one of the most commonly used methods for genotyping; however, generally it needs single-stranded DNA (ssDNA) preparation from PCR amplicons as well as purified genomic DNA extraction from whole blood. To simplify the process of a pyrosequencing protocol, we proposed an improved linear-after-the-exponential (LATE)-PCR by employing whole blood as the starting material. A successful LATE-PCR was achieved by using a common Taq DNA polymerase in high pH buffer (HpH-buffer). As amplicons from LATE-PCR contain a large amount of ssDNA, pyrosequencing can be performed on the amplicons directly. Since DNA extraction and ssDNA preparation are omitted, the labor, cost and cross-contamination risk is decreased compared to conventional pyrosequencing-based genotyping protocols. The results for typing three polymorphisms related to personalized medicine of fluorouracil indicate that the proposed whole-blood LATE-PCR can be well coupled with pyrosequencing, thus becoming a potential tool in personalized medicine.