Amplification of cDNA Ends Using PCR Suppression Effect and Step-Out PCR

In Chapter 10 , we describe two methods of complementary DNA (cDNA) synthesis and amplification. In this chapter, we will refer to these two methods of cDNA synthesis and amplification as “method A” (the technique based on classical double-stranded cDNA synthesis and subsequent adapter ligation, implemented in the Marathon kit from Clontech) and “method B” (based on the template-switching effect, implemented in the SMART cDNA synthesis kit from Clontech). These methods not only allow for amplification of representative cDNA populations from microscopic tissue samples but also provide an excellent starting point for amplification of unknown flanks of a known cDNA fragment. There are many techniques designed for this purpose (1 –10 ); among gene hunters, they go under the generic name RACE (rapid amplification of cDNA ends). It must be remembered, though, that this name formally belongs to a particular technique introduced by Frohman et al. (8 ).