We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted either to the poly(A)+ region of the message or to an enzymatically synthesized d( A) tail. Pairwise combinations of specific and general primers allow for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products can be cloned, sequenced directly by genomic sequencing, or labeled for sequencing by amplifying with a radioactive primer. We illustrate the power of this approach by deriving the cDNA sequences for the skeletal muscle a-tropomyosins of European common frog (Rana temporaria) and zebrafish (Brachydaniorerio) using only 300 ng of a total poly(A) + preparation. In these examples, we gained initial entry into the tropomyosin messages by using heterologous primers (to conserved regions) derived from the rat skeletal muscle a-tropomyosin sequence. The frog and zebrafish sequences are used in an analysis of tropomyosin evolution across the vertebrate phylogenetic spectrum. The results underscore the conservative nature of the tropomyosin molecule and support the notion of a constrained heptapeptide unit as the fundamental structural motif of tropomyosin.
direct sequencing, tropomyosin, zebrafish (Brachydanio rerio)/, European common frog (Rana temporaria), sequence evolution
Ohara, Osamu; Dorit, Robert L.; and Gilbert, Walter, "One-Sided Polymerase Chain Reaction: The Amplification of cDNA" (1989). Biological Sciences: Faculty Publications, Smith College, Northampton, MA.