Evaluation of different amplification protocols for use in primer-extension preamplification.

Abstract

Different amplification protocols were evaluated for use with primer-extension preamplification (PEP). We hypothesized that a protocol known to improve amplification of long DNA fragments would improve efficacy of PEP. Eight DNA samples were preamplified by PCR using different protocols. Treatments consisted of the use of Taq DNA polymerase (T), Taq plus a second polymerase obtained from Pyrococcus furiosus (E) or Stoffel fragment (S) in PEP. After preamplification, six genetic markers were genotyped, and the number of scorable genotypes was recorded. A control reaction (C) consisted of amplification using genomic DNA as template. A second experiment was performed to evaluate preamplification efficiency using Taq DNA polymerase (5 units) and exponential dilutions of Pfu DNA polymerase. After preamplification, the same procedure was used to obtain a number of scorable genotypes. In the first experiment, treatment E was the most reliable approach for amplifying genomic DNA in PEP. Treatments T and S produced fewer scorable genotypes than treatments E or C. In the second experiment, low concentrations of Pfu DNA polymerase produced a similar percentage of scorable genotypes as higher concentrations. Low concentrations of Pfu DNA polymerase combined with Taq DNA polymerase is the most cost-effective procedure to maximize amplification of limited DNA samples in PEP.