Abstract
Having knowledge of the entire 3′ sequence of a cDNA is often important because the non-coding terminal region can contain signals that regulate the stability or subcellular localization of the mRNA. Also, some messages use alternative genomic sites for cleavage and polyadenylation that can alter the above properties, or change the encoded protein. Full-length cDNAs can be obtained from complex mixtures of cellular mRNA using rapid amplification of cDNA ends (RACE) PCR as long as part of the mRNA sequence is known; adding non-specific tags to the ends of the cDNA allows the regions between the known parts of the sequence and the ends to be amplified. In 3′ RACE, the poly(A) tail functions as a non-specific tag at the 3′ end of the mRNA. cDNA ends can be obtained in 1–3 days using this protocol.
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References
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Acknowledgements
The authors thank Yue Zhang and the publisher for permission to use and adapt material from “RACE all the way to the end” (ref. 6). This work was supported by awards NIHDDK 64166 and NIHGM71520 to M.A.F., and NIHGM071475 and a Scientist Development Grant from the American Heart Association to G.D. (0430096N).
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Scotto–Lavino, E., Du, G. & Frohman, M. 3′ End cDNA amplification using classic RACE. Nat Protoc 1, 2742–2745 (2006). https://doi.org/10.1038/nprot.2006.481
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DOI: https://doi.org/10.1038/nprot.2006.481
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