Rapid Amplification of cDNA Ends (RACE)

– use of PCR to amplify cDNA ends

– type of anchored PCR

– PCR with 1 gene-specific primer & 1 non-specific primer

– uses of RACE

– identify missing cDNA ends

– 5' ends often missing

– self-priming or incomplete first strand

(secondary structure)

– 3' ends sometimes missing

– if hexanucleotide primer used

– amplify rare cDNAs – using PCR

– isolate different forms of mRNA from same gene

– e.g. different intron processing

– using gene-specific probe

5' RACE – more common technique

– use gene-specific primer to prime 1^st strand cDNA

– homopolymer tail with polyA or polyC

– start PCR using 2 primers

– 1 primer is 2^nd gene-specific primer + RE site

– inside 1^st primer

– nested primers control for false positives

– 2 false positives unlikely on same DNA

– other primer is polyT or polyI/G + RE site

– specific for homopolymer tail

– PCR amplifies region between primers

– 5' end of mRNA

– cloned using RE sites in primers

– note – homopolymers poor PCR primers

– polyT has very low melting point

– needs lower annealing temperature

– may hybridize nonspecifically

– polyG has very high melting point

– but can be lowered using inosine in primer

– inosine is precursor to A & G

– binds to C with 2 H-bonds

3' RACE – less common than 5'

– 1^st cDNA strand synthesized

– using polyT primer + RE site

– 2^nd strand primed by gene-specific primer

– amplified using: – nested internal primer + RE site

– & polyT primer + RE site