Tzertzinis George, Tabor Stanley, Nichols Nicole M
Curr Protoc Mol Biol. 2008 Oct;Chapter 3:Unit3.7. doi: 10.1002/0471142727.mb0307s84.
Reverse transcriptases (RTs) are multifunctional enzymes, but are mainly used as RNA-directed DNA polymerases in first-strand cDNA synthesis. Specifically, oligodeoxynucleotides are used as primers for extension on RNA templates. The DNA synthesized from an RNA template is referred to as complementary DNA (cDNA) and is often used as a template for PCR or converted to dsDNA for cloning. This unit describes appropriate reaction conditions for RTs from Moloney murine leukemia virus (MMLV) and avian myeloblastosis virus (AMV), along with applications such as synthesizing cDNA, 3' fill-in reactions, and labeling the 3' terminus of DNA fragments with 5' protruding ends, and DNA sequencing.
逆转录酶(RTs)是多功能酶,但主要用作第一链cDNA合成中以RNA为模板的DNA聚合酶。具体而言,寡脱氧核苷酸用作在RNA模板上延伸的引物。从RNA模板合成的DNA称为互补DNA(cDNA),常用于PCR模板或转化为双链DNA用于克隆。本单元介绍了莫洛尼鼠白血病病毒(MMLV)和禽成髓细胞瘤病毒(AMV)逆转录酶的合适反应条件,以及诸如合成cDNA、3' 填充反应、用5' 突出末端标记DNA片段的3' 末端和DNA测序等应用。