Modification of Rapid Amplication of cDNA Ends for the Extension of cDNA libraries.

Abstract

Rapid Amplification of cDNA Ends (RACE) is a technique used in molecular biology to obtain full length sequence of an RNA transcript found within cell. RACE results in the production of a copy of the RNA sequence of interest, which is converted through reverse transcription to cDNA, followed by PCR amplification of the cDNA copies.

A cDNA capture method was devised to construct full-length cDNA libraries. In this method,alibrary of cDNA fragments generated from substractive hybridiazation studies (full-length cDNA library RAD),allowed the respective full-length cDNA cognates to be amplified by PCR and then cloned.

Five clones from subtractive hydridization studies,identified as containing a high proportion of differentially expressedgenes (coded for hypothetical proteins), were chosen as templates for the preparation of full length cDNA. Primers were designed using Genefisher software (Bioinformatics Bielefeld) .Typically, 30-35 cycles of PCR were employed. As a target forthe capture reaction; double-stranded cDNA was amplified by PCRusing the modification of SMART II cDNA library construction kit (Clontech)with the same RNA sample that had served as the tester samplefor the respective RAD reaction

This modified RACE technique resulted in the production of a copy of RNA sequence of interest, produced through reversed transcriptase followed by a successful PCR amplification of cDNA copies. The cDNA libraries were successfully cloned and sequenced, leading to a successful extension of two unknown sequences by 200 base pairs.