Rapid Amplification of cDNA Ends (RACE)

Abstract

AbstractRapid amplification of cDNA ends (RACE) or “one-sided” polymerase chain reaction (PCR) or “anchored” PCR is a polymerase chain reaction-based technique that results in the cloning of full-length cDNA sequences in case only a partial cDNA sequence is available. The 5′ RACE system is a set of prequalified reagents utilized for the synthesis of first-strand cDNA, purification of first-strand products, homopolymeric tailing, and preparation of target cDNA for subsequent amplification by PCR. The next step is the amplification of tailed cDNA by PCR with a mixture of three primers: a nested gene-specific primer (GSP2), which anneals 3′ to GSP1, a combination of a complementary homopolymer-containing anchor primer and a corresponding adapter primer, which permit amplification from the homopolymeric tail. Outline of the methodology of the individual steps of 5′ RACE is isolation of RNA, design of 5′ RACE primers, design of 5′ RACE primers, first-strand cDNA synthesis from total RNA, removal of RNA template by RNase mix, purification of first-strand product, homopolymeric tailing of cDNA, amplification of target cDNA, cloning 5′ RACE amplification products. Detailed protocol with illustrations has been discussed along with a troubleshooting guide.KeywordsRACEAnchored PCRNested gene-specific primerHomopolymeric tailing of cDNA