Rapid amplification of 5′ complementary DNA ends (5′ RACE)

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    This method is used to extend partial cDNA clones by amplifying the 5′ sequences of the corresponding mRNAs1,2,3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target—in the case of 5′ RACE, to a homopolymeric tail added (via terminal transferase) to the 3′ termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5′ sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.

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    References

    1. 1

      Frohman, M.A., Dush, M.K. & Martin, G.R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002 (1988).

    2. 2

      Schaefer, B.C. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal. Biochem. 227, 255–273 (1995).

    3. 3

      Zhang, Y. & Frohman, M.A. Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs. Methods Mol. Biol. 69, 61–87 (1997).

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    Rapid amplification of 5′ complementary DNA ends (5′ RACE). Nat Methods 2, 629–630 (2005) doi:10.1038/nmeth0805-629

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