PCR Amplification and Sequencing of Single Copy DNA Molecules

Abstract

DNA amplification via PCR (polymerase chain reaction) provides an unlimited supply of material for subsequent genetic analyses even in cases where only a single copy of the DNA molecule is present in the sample. There are several requirements for producing high quantities of uniformly amplified DNAs that are not altered, distorted or mutated by the process of the amplification, for example the choice of polymerase enzyme, cycling conditions and the starting copy number. In the case of single copy amplifications, high numbers of thermal cycles are needed to produce sufficient quantities of amplicons for follow-up analysis, which requires highly stable thermal polymerases and high fidelity in the polymerase to minimize misincorporations. In this paper, we examine the in-vitro amplification of single copy DNA molecules with subsequent sequence analysis of the PCR generated products from a single copy DNA molecule. Two different polymerases were investigated, including platinum Pfx polymerase and AmpliTaq® polymerase using hot-start PCR strategies. Amplification of a 500 bp segment of λ-DNA (48 kbp) with quantification using capillary gel electrophoresis and laser-induced fluorescence indicated that Pfx gave a higher yield of amplicons compared to AmpliTaq using similar cycling conditions with fewer false termination or side products, including primer-dimers. Following PCR amplification of single copy DNAs, the amplicons were sequenced using standard Sanger, dideoxy chain termination methods. Analysis of the sequencing data for single copy amplification products indicated that a read length of 424 bases could be achieved, with a read accuracy of 99.3%, similar to the results obtained for a starting copy number of 100.