Simultaneous amplification of 5′ and 3′ cDNA ends based on template-switching effect and inverse PCR

Abstract

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.