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5′ end cDNA amplification using classic RACE

Nature Protocols volume 1, pages 2555–2562 (2006)Cite this article

Abstract

The 5′ ends of transcripts provide important information about transcription initiation sites and the approximate locations of local cis-acting enhancer elements; it is therefore important to establish the 5′ ends with some precision. RACE (rapid amplification of cDNA ends) PCR is useful for quickly obtaining full length cDNAs for mRNAs for which only part of the sequence is known and to identify alternative 5′ or 3′ ends of fully sequenced genes. The method consists of using PCR to amplify, from complex mixtures of cellular mRNA, the regions between the known parts of the sequence and non-specific tags appended to the ends of the cDNA. Whereas the poly(A) tail serves to provide such a tag at the 3′ end of the mRNA, an artificial one needs to be generated at the 5′ end, and various approaches have been described to address this step. The classical scheme for 5′ RACE described here is simple, suffices in many instances in which RACE is needed and can be performed in 1–3 days.

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Figure 1: A schematic representation of the setting in which Classic RACE is used.
Figure 2: A schematic representation of Classic RACE.
Figure 3: The advantage of new RACE over classic RACE.
Figure 4: Schematic representation of cap-switching RACE.

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Acknowledgements

The author thanks Yue Zhang and the publisher for permission to use and adapt material from 'RACE all the way to the end' (ref. 35). 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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Authors and Affiliations

  1. Graduate Program in Molecular & Cellular Pharmacology, Stony Brook University, Stony Brook, 11794, New York, U.S.A.

    Elizabeth Scotto–Lavino & Michael A Frohman

  2. Department of Pharmacological Sciences & Center for Developmental Genetics, Stony Brook University, Stony Brook, 11794, New York, U.S.A.

    Elizabeth Scotto–Lavino, Guangwei Du & Michael A Frohman

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  1. Elizabeth Scotto–Lavino
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Correspondence to Michael A Frohman.

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Scotto–Lavino, E., Du, G. & Frohman, M. 5′ end cDNA amplification using classic RACE. Nat Protoc 1, 2555–2562 (2006). https://doi.org/10.1038/nprot.2006.480

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