One-Pot Isolation of a Desired Human Genome Fragment by Using a Biotinylated pcPNA/S1 Nuclease Combination

Abstract

Scission of the human genome at predetermined sites and isolation of a particular fragment are of great interest for the analysis of lesion/modification sites, in proteomics, and for gene therapy. However, methods for human genome scission and specific fragment isolation are limited. Here, we report a novel one-pot method for the site-specific scission of DNA by using a biotinylated pcPNA/S1 nuclease combination and isolation of a desired fragment by streptavidin-coated magnetic beads. The proof of concept was initially demonstrated for the clipping of plasmid DNA and isolation of the required fragment. Our method was then successfully applied for the isolation of a fragment from the cell-derived human genome.

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

“…We have been developing artificial restriction DNA cutting (ARCUT) systems for siteselective scission of human genomic DNA. In this approach, double-duplex invasion of a desired target site by a pair of pcPNAs cre-ates a single-stranded region in each strand that can be cleaved by Ce(IV)/EDTA complex (Komiyama et al, 2008b) or by S1 nuclease (Li et al, 2014;Rajendran et al, 2018;Shigi et al, 2015). The Ce(IV)/EDTA complex can interfere with other biomolecules, such as enzymes, but nuclease S1-based approach is biocompatible.…”

“…After cleavage, the pcPNA strands remain bound to the termini of each scission fragment. A fragment of interest can therefore be affinity purified if the complementary pcPNA is conjugated to biotin (Rajendran et al, 2018). We have successfully used this approach to isolate BFP gene fragments from plasmid or from human genomic DNA.…”

“…In this method, a pair of pcPNAs guide the scission site and Ce(IV)/EDTA complex cleaves the DNA strands. To make our method more compatible with biological conditions, we have modified our ARCUT method by replacing the metal complex with S1 nuclease (Li, Muneoka, Shigi, Sumaoka, & Komiyama, 2014;Rajendran, Shigi, Sumaoka, & Komiyama, 2018;Shigi et al, 2015). A companion article details protocols for site-specific scission of plasmid and genomic DNA using the S1-based ARCUT method (Rajendran, Shigi, Sumaoka, & Komiyama, 2019).…”

Artificial Restriction DNA Cutter Using Nuclease S1 for Site‐Selective Scission of Genomic DNA

“…This method is also very sensitive for a single‐base mutation in the recognition site (Li et al., ; Miyajima, Ishizuka, Yamamoto, Sumaoka, & Komiyama, ). To make our system more convenient for biochemists, we have replaced the metal complex with ssDNA‐cleaving enzyme S1 nuclease for the DNA scission (Li et al., ; Rajendran et al., ; Shigi et al., ). S1 nuclease is readily available, and it is active under a range of biological conditions, making it compatible with other molecular applications.…”

“…In this method, the pcPNAs base pair to the substrate DNA at the target site and guide the Ce(IV)/EDTA complex to cleave the phosphodiester bonds therein. To make our system more user‐friendly—especially for non‐chemists—we have replaced the Ce(IV)/EDTA complex with S1 nuclease (Li, Muneoka, Shigi, Sumaoka, & Komiyama, ; Rajendran, Shigi, Sumaoka, & Komiyama, ; Shigi et al., ). Whereas naturally occurring restriction enzymes recognize only a few base pairs (e.g., 4‐ to 8‐bp recognition sites for type II restriction enzymes), our pcPNAs guided restriction system recognizes 20 bp and exhibits exceptionally high sequence specificity.…”