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

Anthony G Uren; Harald Mikkers; Jaap Kool; Louise van der Weyden; Anders H Lund; Catherine H Wilson; Richard Rance; Jos Jonkers; Maarten van Lohuizen; Anton Berns; David J Adams

doi:10.1038/nprot.2009.64

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

Anthony G Uren, Harald Mikkers, Jaap Kool, Louise van der Weyden, Anders H Lund, Catherine H Wilson, Richard Rance, Jos Jonkers, Maarten van Lohuizen, Anton Berns, David J Adams

117 Citations (Scopus)

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.

Original language	English
Journal	Nature Protocols (Print Edition)
Volume	4
Issue number	5
Pages (from-to)	789-98
Number of pages	9
ISSN	1754-2189
DOIs	https://doi.org/10.1038/nprot.2009.64
Publication status	Published - 2009

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title = "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 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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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.

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DO - 10.1038/nprot.2009.64

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JO - Nature Protocols

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

Abstract

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