Universal fast walking appiled to cDNA

Abstract

The elucidation of cDNA sequence remains problematic in cases such as genes possessing long coding regions, low expression levels, or poor library coverage. The recently described Universal Fast Walk (UFW) procedure offers a means of determining DNA sequence adjacent to characterised regions. To date, however, the approach has been applied only to genomic DNA. We demonstrate the first successful application of the UFW procedure to the elucidation of cDNA sequence, a previously unknown region of the large tammar wallaby ATRX gene in the theoretically more challenging 3′ direction. To do this, we modified the previously published method by including an initial linear amplification and a final, fully nested PCR. We also exchanged buffers between preparative enzyme reactions to ensure optimal conditions for successive steps. These additional steps ensured a product not observed in their absence. UFW, therefore, represents a powerful alternative mechanism for the cloning and sequencing of cDNA, harnessing the exquisite sensitivity and specificity of fully nested PCR in challenging cloning scenarios where conventional 5′ or 3′ RACE may fail.