A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.

ABSTRACT: Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA isolation, DNA digestion, linker or splinkerette ligation, primary and secondary PCR amplification, and sequencing. This method, which takes about 1 week to perform, has allowed us to isolate hundreds

SUBMITTER: Uren AG 

PROVIDER: S-EPMC3627465 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature