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 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.
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Acknowledgements
A.G.U., J.K., M.v.L. and A.B. are funded by the NWO Genomics program and the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NWO). D.J.A. and his laboratory are funded by Cancer Research-UK and the Wellcome Trust. L.v.d.W. is funded by the Kay Kendall Leukemia Fund. We thank L. Collier for critically reading this manuscript.
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Supplementary information
Supplementary Table 1
10 bp sequences suitable for 'barcoding' splinkerette products for 454 sequencing. These sequences facilitate the computational 'demultiplexing' of pooled splinkerette-PCR products following 454 sequencing. (PDF 18 kb)
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Uren, A., Mikkers, H., Kool, J. et al. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nat Protoc 4, 789–798 (2009). https://doi.org/10.1038/nprot.2009.64
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DOI: https://doi.org/10.1038/nprot.2009.64
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