Recovery of upstream cDNA sequences by a PCR-based biotin-capture method

The world-wide, large-scale sequencing efforts have generated an abundance of partial cDNA sequences, ie., expressed sequence tags (ESTs) accessible i n the public databases. To enable functional characterization of these part ial cDNA sequences, general and robust methods for recovery of upstream ful l-coding cDNA sequences are needed. Here, a novel biotin- and PCR-assisted capture method was used directly on poly(A)(+) RNA for the purpose of gener ating a full-coding sequence of a gene with only partially known sequence a nd for which a full-length clone of the gene was not found in existing cDNA libraries. The presented method involves linear extension by reverse trans ciptase from a biotinylated primer annealing in a region with known sequenc e. After capture of the generated single-stranded cDNA onto paramagnetic be ads, unspecifically annealing primers, i.e., arbitrary primers, were used t o generate cDNA fragments that could be amplified by PCR and thereafter dir ectly sequenced without subcloning. By using the presented strategy, which is to be seen as a complement to rapid amplification of cDNA ends (RACE)-re lated methods, we were able to recover full-coding sequence versions of two potential splice variants of the target gene. The general applicability of the novel method for recovery and sequencing of cDNA sequences is discusse d.