Identification of differentially expressed 5'-end mRNA variants by an improved RACE technique (PEETA).

AUTOR(ES)

Flouriot, G

RESUMO

A rapid and efficient procedure is described for mapping and cloning the 5'-ends of mRNAs, including those generated from a unique gene by alternative splicing and promoter usage. This method involves reverse transcription of the targeted mRNAs from a long, highly labeled specific primer, resolution of the extension products on a DNA sequencing gel, elution and poly(dC) tailing of the single-stranded cDNAs of interest, amplification of these cDNAs by PCR using an oligo(dG) adapter-primer and a gene-specific primer and finally DNA sequencing of the subcloned PCR fragments. The overall method is called PEETA (primer extension, electrophoresis, elution, tailing and amplification).

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