RACE using only a gene-specific primer |
| |
Authors: | Masanori Hirano |
| |
Institution: | (1) HuBit Genomix, 2-19 Hayabusa-cho, Chiyoda-ku, 102-0092 Tokyo, Japan |
| |
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. |
| |
Keywords: | RACE single gene-specific primer terminal inverted repeat template-switching model of Taq DNA polymerase |
本文献已被 PubMed SpringerLink 等数据库收录! |
|