RACE using only a gene-specific primer: application of a template-switching model

Abstract

This article describes a simple method for accurate rapid amplification of complementary deoxyribonucleic acid (cDNA) ends (RACE), the distinctive feature being that only a gene-specific primer is used, without an anchor or adapter primer. Under these conditions, Thermus aquaticus (Taq) polymerase synthesizes cDNA ends exactly, so that amplified products obtain a characteristic structure: a terminal inverted repeat composed of a gene-specific primer and occasionally several nucleotides from its 3' flanking sequence. These structures suggest a hypothetical mechanism of cDNA end synthesis in which Taq DNA polymerase synthesizes a sequence complementary to the gene-specific primer at the 3' end of the daughter strand by switching the template to the 5' terminal region through circularization of the DNA. As a result, the targeted cDNA will be efficiently amplified with only a single gene-specific primer. This technique, which provides highly specific amplification of the 5' and 3' ends of a cDNA, is especially useful for isolation of cDNA when the corresponding messenger ribonucleic acid is scarce.