Abstract
'New RACE' (rapid amplification of cDNA ends) PCR is a method for obtaining full-length cDNA for mRNA for which only part of the sequence is known. Starting with cellular mRNA, PCR is used to amplify regions between the known parts of the sequence and nonspecific tags at the ends of the cDNA. In 'new RACE', an anchor is ligated to the 5′ end of the mRNA before reverse transcription, resulting in the selective production of full-length 5′ cDNA ends. Although 'new RACE' can also be used to amplify 3′ ends, only the protocol for obtaining 5′ ends is presented here. This protocol can be completed in 1–3 days.
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References
Liu, X. & Gorovsky, M.A. Mapping the 5′ and 3′ ends of tetrahymena-thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE). Nucleic Acids Res. 21, 4954–4960 (1993).
Fromont-Racine, M., Bertrand, E., Pictet, R. & Grange, T. A highly sensitive method for mapping the 5′ termini of mRNAs. Nucleic Acids Res. 21, 1683–1684 (1993).
Scotto-Lavino, E., Du, G. & Frohman, M.A. 5′ end cDNA amplification using Classic RACE. Nat. Protocols 1, 2555–2562 (2006).
Volloch, V., Schweitzer, B., Zhang, X. & Rits, S. Identification of negative-strand complements to cytochrome oxidase subunit III RNA in Trypanosoma brucei. Proc. Natl. Acad. Sci. USA 88, 10671–10675 (1991).
Mandl, C.W., Heinz, F.X., Puchhammer-Stockl, E. & Kunz, C. Sequencing the termini of capped viral RNA by 5′-3′ ligation and PCR. Biotechniques 10, 484–486 (1991).
Tessier, D.C., Brousseau, R. & Vernet, T. Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase. Anal. Biochem. 158, 171–178 (1986).
Frohman, M.A., Dickinson, M.E., Hogan, B.L.M. & Martin, G.R. Localization of two new and related homeobox-containing genes to chromosomes 1 and 5, near the phenotypically similar mutant loci dominant hemimelia (Dh) and hemimelic extra-toes (Hx). Mouse Genome 91, 323–325 (1993).
Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K. & Mattick, J.S. Touchdown PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19, 4008 (1991).
Frohman, M.A. On beyond classic RACE (rapid amplification of cDNA ends). PCR Methods Appl. 4, S40–S58 (1994).
Scotto-Lavino, E., Du, G. & Frohman, M.A. 3′ end cDNA amplification using classic RACE. Nat. Protocols 1, 2742–2745 (2006).
Zhang, Y. in Generation of cDNA Libraries (ed. Ying, S.-Y.) 13–24 (Humana Press, Totowa, New Jersey, 2003).
Acknowledgements
We thank Y. Zhang and Humana Press for permission to use and adapt material from a prior review (ref. 11). Supported by the National Institutes of Health (NIHDDK 64166 and NIHGM71520 to M.A.F., and NIHGM071475 to G.D.) and the American Heart Association (Scientist Development Grant (0430096N to G.D.).
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Scotto-Lavino, E., Du, G. & Frohman, M. Amplification of 5′ end cDNA with 'new RACE'. Nat Protoc 1, 3056–3061 (2006). https://doi.org/10.1038/nprot.2006.479
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DOI: https://doi.org/10.1038/nprot.2006.479
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