PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.
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.
Dai
Zhong-Min
ZM
Institute of Cell Biology and Genetics, 317, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Zhu
Xiao-Jing
XJ
Chen
Qi
Q
Yang
Wei-Jun
WJ
D001483:Base Sequence; D018076:DNA, Complementary / Q000138:chemical synthesis; D004277:DNA, Single-Stranded / Q000737:chemistry*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013698:Templates, Genetic
D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D012194:RNA-Directed DNA Polymerase