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 -
