42 - Rapid Amplification of Complementary DNA Ends for Generation of Full-Length Complementary DNAs: Thermal RACE

Abstract

This chapter discusses about the rapid amplification of complementary DNA (cDNA) ends for the generation of full length cDNAs. The generation of a full-length cDNA represents the most challenging part of a cloning project, particularly with respect to obtaining the 5′ end of the cDNA. cDNAs are derived from the reverse transcription of messenger RNA (mRNA). Thermal rapid amplification of cDNA ends (RACE) is a PCR technique through which previously unobtained 3′ and 5′ ends of a cDNA can be amplified starting with the knowledge of a small stretch of sequence from within an internal region of the cDNA. This technique provides an alternative to constructing and screening conventional libraries in order to obtain the remainder of the sequence for a partially cloned cDNA. Partial cDNAs are generated using PCR to amplify copies of the region between a single point in mRNA transcript and its 3′ or 5′ end. This chapter also presents the analysis of amplification products during execution of RACE protocol. The production of specific partial cDNAs by the RACE protocol is assessed using Southern blot hybridization analysis.