使用基于连接子介导的PCR方法分离前病毒的基因组插入位点。

Isolation of genomic insertion sites of proviruses using Splinkerette-PCR-based procedures.

作者信息

Yin Bin

机构信息

Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, Soochow University, Suzhou, China.

出版信息

Methods Mol Biol. 2011;687:25-42. doi: 10.1007/978-1-60761-944-4_3.

DOI:10.1007/978-1-60761-944-4_3

PMID:20967599

Abstract

The availability of whole genomic sequences provides a great framework for biologists to address a broad range of scientific questions. However, functions of most mammalian genes remain obscure. The forward genetics strategy of insertional mutagenesis uses DNA mutagens such as retroviruses and transposable elements; this strategy represents a powerful approach to functional genomics. A variety of methods to uncover insertion sites have been described. This chapter details SplinkTA-PCR and SplinkBlunt-PCR, modified from splinkerette-PCR, for mapping chromosomally the insertion sites of a murine leukemia virus that causes leukemia in the BXH-2 strain of mice. These protocols are easy to use, reliable, and efficient.

摘要

全基因组序列的可用性为生物学家解决广泛的科学问题提供了一个很好的框架。然而，大多数哺乳动物基因的功能仍然不清楚。插入诱变的正向遗传学策略使用逆转录病毒和转座元件等DNA诱变剂；这种策略是功能基因组学的一种强大方法。已经描述了多种揭示插入位点的方法。本章详细介绍了从连接子PCR改进而来的SplinkTA-PCR和SplinkBlunt-PCR，用于在染色体上定位一种在BXH-2小鼠品系中引起白血病的鼠白血病病毒的插入位点。这些方案易于使用、可靠且高效。