Affinity Isolation of Defined Genomic Fragments Cleaved by Nuclease S1-based Artificial Restriction DNA Cutter.

Arivazhagan Rajendran, Narumi Shigi, Jun Sumaoka, Makoto Komiyama

Journal: Current protocols in nucleic acid chemistry 2019;76(1):e76

PMID: 30753751

Abstract

The human genome is highly susceptible to various modifications, lesions, and damage. To analyze lesions and proteins bound to a defined region of the human genome, the genome should be fragmented at desired sites and the region of interest should be isolated. The few available methods for isolating a desired region of the human genome have serious drawbacks and can only be applied to specific sequences or require tedious experimental procedures. We have recently developed a novel method to isolate a desired fragment of the genome released by site-specific scission of DNA using a pair of pseudo-complementary peptide nucleic acids (pcPNAs) and S1 nuclease. When conjugated to biotin, one of the pcPNAs can be used to affinity purify the cleavage product. Here we report a detailed protocol to isolate defined kilobase-length DNA fragments that can be applied to plasmid or genomic DNA and is not limited by sequence. © 2019 by John Wiley & Sons, Inc.

Address: Institute of Advanced Energy, Kyoto University, Kyoto, Japan.; Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan.; Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.; Department of Applied Chemistry, School of Engineering, Tokyo University of Technology, Tokyo, Japan.; College of Food Science and Engineering, Ocean University of China, Qingdao, China.

DOI: 10.1002/cpnc.76

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https://doi.org/10.1002/cpnc.76

MeSH Terms: Cells, Cultured, Chromatography, Affinity, DNA Cleavage, DNA, Single-Stranded, Electrophoresis, Agar Gel, Genome, Human, Humans, Hydrolysis, Peptide Nucleic Acids, Plasmids