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

TY - JOUR

T1 - A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

AU - Uren, Anthony G

AU - Mikkers, Harald

AU - Kool, Jaap

AU - van der Weyden, Louise

AU - Lund, Anders H

AU - Wilson, Catherine H

AU - Rance, Richard

AU - Jonkers, Jos

AU - van Lohuizen, Maarten

AU - Berns, Anton

AU - Adams, David J

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

U2 - 10.1038/nprot.2009.64

DO - 10.1038/nprot.2009.64

M3 - Journal article

C2 - 19528954

SN - 1754-2189

VL - 4

SP - 789

EP - 798

JO - Nature Protocols (Print Edition)

JF - Nature Protocols (Print Edition)

IS - 5

ER -