Enrichment of meiotic recombination hotspot sequences by avidin capture technology

Abstract

About 40% of the hotspots for meiotic recombination contain the degenerate consensus sequence 5’-CCNCCNTNNCCNC-3’. Here we present a novel protocol for enriching hotspot sequences from digested genomic DNA by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. The captured hotspots can be released by simple digestion with restriction enzymes for subsequent characterization by second generation sequencing or PCR. The capture protocol specifically enriches hotspot sequences, judged by using fluorophore-conjugated synthetic oligonucleotides and synthetic double-stranded oligonucleotides in combination with PCR. The capture protocol enriches single stranded DNA, denatured double-stranded DNA, and large fragments (>3,000 bp) of digested plasmid DNA with good efficacy. No false positive and false negatives were detected when enriching digested DNA from human cell cultures and primary human cells. The protocol can probably be adapted to enriching sequences other than the hotspot sequence by altering the sequence in the capture oligonucleotide. We intend to apply this protocol in studies assessing effects of micronutrient status on meiotic recombination events in human sperm.

Efficient sequence‐specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins

“…; St. John & Quinn ; Camara Teixeira et al . ; Gagan & Van Allen ); however, in vitro enChIP technology is a potentially useful alternative method that has potential advantages over existing DNA hybridization methods. For example, in vitro enChIP can isolate target double‐stranded DNA (dsDNA) without the need for the denaturing step required in DNA hybridization approaches.…”

Efficient sequence-specific isolation of DNA fragments and chromatin byin vitroenChIP technology using recombinant CRISPR ribonucleoproteins

“…Currently, a DNA hybridization using biotinylated DNA probes combined with the biotin-avidin purification system is used for a number of the aforementioned applications (Diamandis & Christopoulos, 1991;Ito et al, 1992;Mangiapan et al, 1996;St. John & Quinn, 2008;Camara Teixeira et al, 2013;Gagan & Van Allen, 2015); however, in vitro enChIP technology is a potentially useful alternative method.…”