A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.
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 of thousands of insertion sites from mouse tumors and, unlike other methods, has been specifically optimized for the murine leukemia virus (MuLV), and can easily be performed in a 96-well plate format for the efficient multiplex isolation of insertion sites.
Uren
Anthony G
AG
Division of Molecular Genetics, Cancer Genomics Centre, Netherlands Cancer Institute, Plesmanlaan, Amsterdam, The Netherlands.
Mikkers
Harald
H
Kool
Jaap
J
van der Weyden
Louise
L
Lund
Anders H
AH
Wilson
Catherine H
CH
Rance
Richard
R
Jonkers
Jos
J
van Lohuizen
Maarten
M
Berns
Anton
A
Adams
David J
DJ
D000818:Animals; D004587:Electrophoresis, Agar Gel; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000502:physiology*; D051379:Mice; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D016662:Virus Integration