Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences.
Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3' or 5' end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3' or 5' T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3' and 5' ends of numerous cDNAs from a single cDNA synthesis reaction.
Bower
Neil I
NI
Scottish Oceans Institute, School of Biology, University of St Andrews, St Andrews, Fife KY168LB, UK. nib@st-andrews.ac.uk
Johnston
Ian A
IA
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D020317:Salmo salar / Q000235:genetics; D013698:Templates, Genetic; D051981:Uracil-DNA Glycosidase; D015027:Zebrafish / Q000235:genetics
D018076:DNA, Complementary; D003856:Deoxyuracil Nucleotides; D051981:Uracil-DNA Glycosidase