PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples

J Biotechnol. 2007 Feb 20;128(3):435-43. doi: 10.1016/j.jbiotec.2006.10.018. Epub 2006 Nov 16.

Abstract

In the present study, we analyzed the kinetics of the PCR-suppression effect (PS-effect) and firstly demonstrate that lower annealing temperature enhances PS-effect. Furthermore, we controlled the average size of complex PCR products over a wide range, and simultaneously amplified targets from 0.25 to 10kb by regulating the degree of suppression in a single-primer PCR. In addition, we describe an improved template-switching full-length cDNA synthesis method that greatly reduces truncated cDNA. This study provides a general guide for the design of PS-effect related PCR and is useful for representative or long-transcript enriched cDNA library construction, especially when only a small amount of total RNA is available.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA, Complementary / chemical synthesis
  • DNA, Single-Stranded / chemistry*
  • Kinetics
  • Models, Biological
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • RNA-Directed DNA Polymerase / metabolism
  • Templates, Genetic

Substances

  • DNA, Complementary
  • DNA, Single-Stranded
  • RNA-Directed DNA Polymerase