Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer.

Abstract

Maturation of eukaryotic mRNAs involves 3' end formation, which involves the addition of a poly(A) tail. In order to map the 3' end of a gene, the traditional method of choice is 3' rapid amplification of cDNA ends (3' RACE). Protocols for 3' RACE require the careful design and selection of nested primers within the 3' untranslated region (3' UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3' UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3' UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3' RACE. Expanded 3' RACE can detect unusual 3' UTRs, including gene fusions within the 3' UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.