One-sided polymerase chain reaction: the amplification of cDNA.

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