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The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

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Abstract

Insertional mutagenesis is a productive strategy for the exploration of genetic regulation of important biological and pathological processes, such as tumorigenesis. Successful implementation of this strategy depends heavily on an efficient approach to the identification of insertion sites present in the host genome. Here, we have introduced an easy and efficient protocol, called Adenosine-ended Primer Extension Polymerase Chain Reaction (APE-PCR), which represents several advantages, including the Addition technique we previously developed, primer extension approach coupled with biotin-streptavidin based purification, introduction of nano-scale magnetic particles, and digestion of DNA with a combination of enzymes. We have demonstrated that APE-PCR is able to amplify more and larger specific proviral insertion site (PIS)-derived fragments, with a lower non-specific background produced, fewer steps and less DNA samples required, flexibility in choice of restriction enzymes applied, at a lower cost. Replacement of regular magnetic beads with nano-scale ones in the protocol can further increase its power. Moreover, even with small amount of sample DNA, PISs can be recovered and analyzed. Thus, based on the results provided from this study, we believe that APE-PCR represents an efficient method in mapping of PISs and likely, the insertion sites of other types of DNA elements as well.

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Abbreviations

APE-PCR:

adenosine-ended primer extension polymerase chain reaction

LAM-PCR:

linear amplificationmediated polymerase chain reaction

PCR:

polymerase chain reaction

PIS:

proviral insertion site

References

  • Altschul S.F., Gish W., Miller W., Myers E.W. & Lipman D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403–410.

    CAS  PubMed  Google Scholar 

  • Alvarez-Erviti L., Seow Y., Yin H., Betts C., Lakhal S. & Wood M.J. 2011. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29: 341–345.

    Article  CAS  PubMed  Google Scholar 

  • Brady T., Roth S.L., Malani N., Wang G.P., Berry C.C., Leboulch P., Hacein-Bey-Abina S., Cavazzana-Calvo M., Papapetrou E.P., Sadelain M., Savilahti H. & Bushman F.D. 2011. A method to sequence and quantify DNA integration for monitoring outcome in gene therapy. Nucleic Acids Res. 39: e72.

    Article  CAS  PubMed  Google Scholar 

  • Carlson C.M. & Largaespada D.A. 2005. Insertional mutagenesis in mice: new perspectives and tools. Nat. Genet. 6: 568–580.

    CAS  Google Scholar 

  • Collier L.S., Carlson C.M., Ravimohan S., Dupuy A.J. & Largaespada D.A. 2005. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436: 272–276.

    Article  CAS  PubMed  Google Scholar 

  • Davis M.E., Zuckerman J.E., Choi C.H., Seligson D., Tolcher A., Alabi C.A., Yen Y., Heidel J.D. & Ribas A. 2010. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464: 1067–1070.

    Article  CAS  PubMed  Google Scholar 

  • Devon R.S., Porteous D.J. & Brookes A.J. 1995. Splinkerettes-improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res. 23: 1644–1645.

    Article  CAS  PubMed  Google Scholar 

  • Dubertret B., Skourides P., Norris D.J., Noireaux V., Brivanlou A.H. & Libchaber A. 2002. In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298: 1759–1762.

    Article  CAS  PubMed  Google Scholar 

  • Gabriel R., Eckenberg R., Paruzynski A., Bartholomae C.C., Nowrouzi A., Arens A., Howe S.J., et al. 2009. Comprehensive genomic access to vector integration in clinical gene therapy. Nat Med. 15: 1431–1436.

    Article  CAS  PubMed  Google Scholar 

  • Harkey M.A., Kaul R., Jacobs M.A., Kurre P., Bovee D., Levy R. & Blau C.A. 2007. Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization forclonal analysis. Stem Cells Dev. 16: 381–392.

    Article  CAS  PubMed  Google Scholar 

  • Jenkins N.A., Copeland N.G., Taylor B.A., Bedigian H.G. & Lee B.K. 1982. Ecotropic murine leukemia virus DNA content of normal and lymphomatous tissues of BXH-2 recombinant inbred mice. J. Virol. 42: 379–388.

    CAS  PubMed  Google Scholar 

  • Kool J. & Berns A. 2009. High-throughput insertional mutagenesis screens in mice to identify oncogenic networks. Nat. Rev. Cancer. 9: 389–399.

    Article  CAS  PubMed  Google Scholar 

  • Lander E.S., Linton L.M., Birren B., Nusbaum C., Zody M.C., Baldwin J., Devon K., et al. 2001. Initial sequencing and analysis of the human genome. Nature. 409: 860–921.

    Article  CAS  PubMed  Google Scholar 

  • Largaespada D.A. 2000. Genetic heterogeneity in acute myeloid leukemia: maximizing information flow from MuLV mutagenesis studies. Leukemia 14: 1174–1184.

    Article  CAS  PubMed  Google Scholar 

  • Leoni C., Volpicella M., De Leo F., Gallerani R. & Ceci L.R. 2011. Genome walking in eukaryotes. FEBS J. 278: 3953–3977.

    Article  CAS  PubMed  Google Scholar 

  • Medintz I.L., Uyeda H.T., Goldman E.R. & Mattoussi H. 2005. Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 4: 435–446.

    Article  CAS  PubMed  Google Scholar 

  • Mikkers H., Allen J., Knipscheer P., Romeijn L., Hart A., Vink E. & Berns A. 2002. High-throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat. Genet. 32: 153–159.

    Article  CAS  PubMed  Google Scholar 

  • Mikkers H. & Berns A. 2003. Retroviral insertional mutagenesis: tagging cancer pathways. Adv. Cancer. Res. 88: 53–99.

    Article  CAS  PubMed  Google Scholar 

  • Pérez-Mancera P.A., Rust A.G., van der Weyden L., Kristiansen G., Li A., Sarver A.L., Silverstein K.A., Grützmann R., et al. 2012. The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma. Nature 486: 266–270.

    PubMed  Google Scholar 

  • Schmidt M., Schwarzwaelder K., Bartholomae C., Zaoui K., Ball C., Pilz I., Braun S., Glimm H. & von Kalle C. 2007. High-resolution insertion-site analysis by linear amplificationmediated PCR (LAM-PCR). Nat Methods 4: 1051–1057.

    Article  CAS  PubMed  Google Scholar 

  • Skarnes W.C., von Melchner H., Wurst W., Hicks G., Nord A.S., Cox T., Young S.G., Ruiz P., Soriano P., Tessier-Lavigne M., Conklin B.R., Stanford W.L. & Rossant J. 2004. A public gene trap resource for mouse functional genomics. Nat. Genet. 36: 543–544.

    Article  CAS  PubMed  Google Scholar 

  • Sugahara K.N., Teesalu T., Karmali P.P., Kotamraju V.R., Agemy L., Greenwald D.R. & Ruoslahti E. 2010. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science 328: 1031–1035.

    Article  CAS  PubMed  Google Scholar 

  • Uren A.G., Mikkers H., Kool J., van der Weyden L., Lund A.H., Wilson C.H., Rance R., Jonkers J., van Lohuizen M., Berns A. & Adams D.J. 2009. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nat. Methods4: 789–798.

    CAS  Google Scholar 

  • Vassiliou G.S., Cooper J.L., Rad R., Li J., Rice S., Uren A., Rad L., Ellis P., Andrews R., Banerjee R., Grove C., Wang W., Liu P., Wright P., Arends M. & Bradley A. 2011. Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice. Nat. Genet. 43: 470–475.

    Article  CAS  PubMed  Google Scholar 

  • Venter J.C., Adams M.D., Myers E.W., Li P.W., Mural R.J., Sutton G.G., Smith H.O., et al. 2001. The sequence of the human genome. Science. 291: 1304–1351.

    Article  CAS  PubMed  Google Scholar 

  • Volpicella M., Leoni C., Fanizza I., Rius S., Gallerani R. & Ceci L.R. 2012. Genome walking by Klenow polymerase. Anal. Biochem. 430: 200–202.

    Article  CAS  PubMed  Google Scholar 

  • Yin B. 2011. Isolation of genomic insertion sites of proviruses using Splinkerette-PCR-Based procedures. Methods Mol. Biol. 687: 25–42.

    Article  CAS  PubMed  Google Scholar 

  • Yin B., Delwel R., Valk P.J., Wallace M.R., Loh M.L., Shannon K.M. & Largaespada D.A. 2009. A retroviral mutagenesis screen reveals strong cooperation between Bcl11a overexpression and loss of the Nf1 tumor suppressor gene. Blood 113: 1075–1085.

    Article  CAS  PubMed  Google Scholar 

  • Yin B., Kogan S.C., Dickins R.A., Lowe S.W. & Largaespada D.A. 2006. Trp53 loss during in vitro selection contributes to acquired Ara-C resistance in acute myeloid leukemia. Exp. Hematol. 34: 631–641.

    Article  CAS  PubMed  Google Scholar 

  • Yin B. & Largaespada D.A. 2007. PCR-based procedures to isolate insertion sites of DNA elements. Biotechniques 43: 79–84.

    Article  CAS  PubMed  Google Scholar 

  • Yin B., Morgan K., Hasz D.E., Mao Z. & Largaespada D.A. 2006. Nf1 gene inactivation in acute myeloid leukemia cells confers cytarabine resistance through MAPK and mTOR pathways. Leukemia 20: 151–154.

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Bin Yin.

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Xu, Z., Li, Y., Mao, Z.J. et al. The development of APE-PCR for the cloning of genomic insertion sites of DNA elements. Biologia 68, 766–772 (2013). https://doi.org/10.2478/s11756-013-0214-2

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  • DOI: https://doi.org/10.2478/s11756-013-0214-2

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