Recent developments in RACE-PCR for the full-length cDNA identification

Abstract

Novel strategies have been developed for the identification of full-length cDNA using bioinformatics tools and multiplexed PCR methods. Usually, sequences are either incomplete or have missing UTR-sequences. Researchers still use the Rapid Amplification of cDNA Ends technique to obtain the full-length cDNA sequences. An oligo(dT) anchor-primer with a hairpin structure is designed for amplification of 3′ cDNA ends. Arbitrary degenerate and sequence-specific reverse primers were also developed for the amplification of 5′ cDNA ends, and tail-PCR is performed until the 5′ sequence of multi-assembled fragment reaches the exon-1 region which can be identified by aligning these fragments to reference genome database. Inhibitory and functional adapters are specially designed; adapter with phosphate can attach to full-length mRNAs with cap structure. In prokaryotes, a technique is reported to capture primary transcripts based on capping the 5′ triphosphorylated RNA. For the identification of full-length cDNA, specific-primers need to be designed for further processes.