Gene Specific-Primer Extension Preamplification (GS-PEP), a simple method to increase the copy number of a gene prior to PCR amplification

摘要

We have developed a simple method, designated as Gene Specific-Primer Extension Preamplification (GS-PEP), to increase the copy number of genomic DNA fragments for a gene of interest prior to PCR amplification. In this method, multiple cycles of primer-extension reaction are undertaken in a single tube using a mixture of primers derived from a defined strand of DNA sequences covering a gene. After 50 cycles of GS-PEP against 21pg of genomic DNA, which corresponds to approximately 3 copies of diploid human genome DNA, with a mixture of 6 primers for the p53 gene locus, all the 10 coding exons of the p53 gene were readily amplifiable by standard PCR at rates of 100%. In contrast, direct PCR against 21pg of genomic DNA was estimated to allow the amplification of only 3 exons. This procedure also enhanced the rate of amplification by PCR using genomic DNA extracted from formalin-fixed and paraffin-embedded tissues. Thus, GS-PEP would allow mutation analyses of various genes in cancer cells using small amounts and/or low qualities of DNA.