A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.
A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.
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DOI：
10.1038/nprot.2009.64
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发表时间：
2009
期刊：
影响因子：
14.8
通讯作者：
Adams, David J.
中科院分区：
文献类型：
--
作者：
Uren, Anthony G.;Mikkers, Harald;Kool, Jaap;van der Weyden, Louise;Lund, Anders H.;Wilson, Catherine H.;Rance, Richard;Jonkers, Jos;van Lohuizen, Maarten;Berns, Anton;Adams, David J.
插入诱变剂如病毒和转座子是在小鼠中进行前向遗传筛选以发现癌症基因的有用工具。这些筛选在使用数百只小鼠时是最有效的，然而直到最近，在这种规模上进行筛选的主要限制是低成本的插入位点分离和测序。在这里，我们提出了一种高通量分离插入位点的方法，使用高效的splinkerter - pcr方法结合毛细管或454测序。该方案包括对DNA分离、DNA消化、连接子或splinkerter连接、一级和二级PCR扩增和测序过程的描述。该方法耗时约1周，使我们能够从小鼠肿瘤中分离出数十万个插入位点，并且与其他方法不同，该方法专门针对小鼠白血病病毒（MuLV）产生的插入位点的分离进行了优化，并且可以很容易地在96孔板格式中进行插入位点的高效多重分离。
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 a major limitation to performing screens on this scale has been the cost effective isolation and sequencing of insertion sites. 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 tumours and, unlike other methods, has been specifically optimised for the isolation of insertion sites generated with the murine leukaemia virus (MuLV), and can easily be performed in 96 well plate format for the efficient multiplex isolation of insertion sites.
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影响因子：
14.9
作者：
Parameswaran P;Jalili R;Tao L;Shokralla S;Gharizadeh B;Ronaghi M;Fire AZ
通讯作者：
Fire AZ
人类癌基因组中体细胞突变的模式。
影响因子：
64.8
作者：
Greenman, Christopher;Stephens, Philip;Smith, Raffaella;Dalgliesh, Gillian L.;Hunter, Christopher;Bignell, Graham;Davies, Helen;Teague, Jon;Butler, Adam;Edkins, Sarah;O'Meara, Sarah;Vastrik, Imre;Schmidt, Esther E.;Avis, Tim;Barthorpe, Syd;Bhamra, Gurpreet;Buck, Gemma;Choudhury, Bhudipa;Clements, Jody;Cole, Jennifer;Dicks, Ed;Forbes, Simon;Gray, Kris;Halliday, Kelly;Harrison, Rachel;Hills, Katy;Hinton, Jon;Jenkinson, Andy;Jones, David;Menzies, Andy;Mironenko, Tatiana;Perry, Janet;Raine, Keiran;Richardson, Dave;Shepherd, Rebecca;Small, Alexandra;Tofts, Calli;Varian, Jennifer;Webb, Tony;West, Sofie;Widaa, Sara;Yates, Andy;Cahill, Daniel P.;Louis, David N.;Goldstraw, Peter;Nicholson, Andrew G.;Brasseur, Francis;Looijenga, Leendert;Weber, Barbara L.;Chiew, Yoke-Eng;deFazio, Anna;Greaves, Mel F.;Green, Anthony R.;Campbell, Peter;Birney, Ewan;Easton, Douglas F.;Chenevix-Trench, Georgia;Tan, Min-Han;Khoo, Sok Kean;Teh, Bin Tean;Yuen, Siu Tsan;Leung, Suet Yi;Wooster, Richard;Futreal, P. Andrew;Stratton, Michael R.
通讯作者：
Stratton, Michael R.
影响因子：
4.3
作者：
Ambrosi A;Cattoglio C;Di Serio C
通讯作者：
Di Serio C
影响因子：
7
作者：
Wang, Gary P.;Ciuffi, Angela;Bushman, Frederic D.
通讯作者：
Bushman, Frederic D.
影响因子：
5.8
作者：
Kong, Jun;Zhu, Fei;Adams, David J.
通讯作者：
Adams, David J.