A reliable method for amplifying cDNA using the anchored-polymerase chain reaction (A-PCR).

A-PCR is a useful method for analyzing RNA transcripts (e.g., T-cell receptor mRNAs) where the sequence of the 5' portion of the molecule is not known. The procedure involves reverse transcription of mRNA using (dT)18 or a specific primer, homopolymer tailing of the first strand cDNA with dGTP and then by amplification by PCR using a non-specific anchor-oligo(dC) oligonucleotide on the 5' end and a specific primer on the 3' end (1, 2). Although the procedure appears straightforward, many investigators have found it difficult or impossible to amplify cDNA using the A-PCR method, largely because of technical difficulties. This report identifies the nature of these technical difficulties and provides a method for successful, reliable amplification of cDNA by A-PCR.