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

This method is used to extend partial cDNA clones by amplifying the 5′ sequences of the corresponding mRNAs^1,2,3. 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.