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 (>3000 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.

Figure 1

Flow chart of the avidin-based protocol for capturing recombination hot spots sequences.

Figure 2

Principle of the capture protocol. The hybridization of oligos 1 and 2 creates an EcoRI restriction site and a 5’ end overhang that is complementary to the sequence in the recombination hotspot 5’-CCN CCN TNN CCN C-3’. The annealed capture oligonucleotides are immobilized on streptavidin-magnetic coated beads through the biotin-conjugated 5’ end in oligo 1.

Figure 3

Capture and release of a fluorophore-conjugated oligonucleotide. Oligonuclotide oligo-3-FAM is labeled with fluorescein and contains the hotspot sequence for recombination. The numbers denote the sequential washes of streptavidin beads before and after treatment with EcoRI. Fraction 9 depicts the fluorescence in beads after the final wash (N=3; the image represents a representative example).

Figure 4

Enrichment of a DNA containing the hotspot motif from a mixture of short, double-stranded DNA. (A) Synthetic dsDNA oligonucleotides: 100-mer containing the hotspot sequence for recombination; 50-mer not containing the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). (C) As described for “A” but now the 50-mer contains the hotspot sequence. (D) As described for “B.” Abbreviation: M, marker.

Figure 5

Capture of long dsDNA with oligo-Blue. (A) EcoRI fragment of pBluescript II sk (+), containing a 13-mer with a degree of degeneration similar to the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). Abbreviation: M, marker.

Figure 6

Enrichment of DNA for fragments containing the hotspot sequence in human cell lines. (A) Select fragments without (lanes 1 – 3) and with (lanes 4 – 6) the hotspot sequence were amplified by nested PCR before enrichment by capture protocol, using EcoRI-digested DNA from MCF-7 cells. (B) As described for “A” but after enrichment by using the capture protocol. (C) As described for “A” but using DNA from HEK293 cells. (D) As described for “B” but using DNA from HEK293 cells. (E) As described for “A” but using DNA from IMR90 fibroblasts. (F) As described for “B” but using DNA from IMR90 fibroblasts. Abbreviation: M, marker.