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• Volpicella, M
• Leoni, C
• Costanza, A
• Fanizza, I
• Placido, A
• Ceci, L
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• Volpicella, M
• Leoni, C
• Costanza, A
• Fanizza, I
• Placido, A
• Ceci, L
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• Volpicella, M
• Leoni, C
• Costanza, A
• Fanizza, I
• Placido, A
• Ceci, L

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Biology 2012, 1(3), 495-507; doi:10.3390/biology1030495

Review

Genome Walking by Next Generation Sequencing Approaches

Mariateresa Volpicella ^1,† , Claudia Leoni ^1,† , Alessandra Costanza ¹ , Immacolata Fanizza ¹ , Antonio Placido ¹  and Luigi R. Ceci ^2,*

¹ Department of Biosciences, Biotechnology and Pharmacological Sciences, University of Bari, Via Amendola 165/A, 70126 Bari, Italy ² Institute of Biomembranes and Bioenergetics, Italian National Research Council (CNR), Via Amendola 165/A, 70126 Bari, Italy ^† These authors contributed equally to this work.

* Author to whom correspondence should be addressed.

Received: 1 August 2012; in revised form: 31 August 2012 / Accepted: 25 September 2012 / Published: 1 October 2012

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Biology)

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Abstract: Genome Walking (GW) comprises a number of PCR-based methods for the identification of nucleotide sequences flanking known regions. The different methods have been used for several purposes: from de novo sequencing, useful for the identification of unknown regions, to the characterization of insertion sites for viruses and transposons. In the latter cases Genome Walking methods have been recently boosted by coupling to Next Generation Sequencing technologies. This review will focus on the development of several protocols for the application of Next Generation Sequencing (NGS) technologies to GW, which have been developed in the course of analysis of insertional libraries. These analyses find broad application in protocols for functional genomics and gene therapy. Thanks to the application of NGS technologies, the original vision of GW as a procedure for walking along an unknown genome is now changing into the possibility of observing the parallel marching of hundreds of thousands of primers across the borders of inserted DNA molecules in host genomes.

Keywords: genome walking; insertional mutagenesis; next generation sequencing; functional genomics; gene therapy; transposon tagging; 454-pyrosequencing; Illumina; SOLiD; Ion Torrent

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