Whole-Genome Amplification by Improved Primer Extension Preamplification PCR (I-PEP-PCR)

Abstract

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). Primer extension preamplification PCR (PEP-PCR), in contrast to degenerate oligonucleotide primed PCR (DOP-PCR), uses totally degenerate 15-mer PCR primers. An additional difference is that in PEP-PCR, the number of potential priming sites is orders of magnitude larger. The effectiveness of PEP-PCR has been increased by several alterations. The improved PEP (I-PEP) PCR approach, described in this protocol, uses a DNA polymerase cocktail that includes Taq DNA polymerase (to carry out the primer extension as in a traditional PCR) and a proofreading DNA polymerase (to provide 3′-to-5′-exonuclease activity, excising misincorporated nucleotides that slow the progression of Taq DNA polymerase). The result is far more efficient WGA, with increased fidelity due to the removal of the misincorporated nucleotides. Similar to DOP-PCR, PEP-PCR generates a smear of DNA fragments that are visible on an agarose gel.