Simultaneous amplification of 5' and 3' cDNA ends based on template-switching effect and inverse PCR.
A new approach for simultaneously amplifying the 5' and 3' ends of a desired cDNA is described. The method combines the template-switching effect with inverse PCR, which generates the flanking 5' and 3' regions of a certain cDNA with very low or no background. It requires only minimal amounts of total RNA for the synthesis of first-strand cDNA, while the same cDNA can be used to amplify flanking sequences of any cDNA species present in the sample. This method is reliable and easy to perform, which is very useful for isolating cDNA species of rare transcripts.
Huang
Jun-Chao
JC
The University of Hong Kong, Hong Kong, PR China.
Chen
Feng
F
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D048030:RNA, Algal / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D013698:Templates, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D048030:RNA, Algal; D018749:RNA, Plant; D019914:Taq Polymerase