Targeting in linear DNA duplexes with two complementary probe strands for hybrid stability.
A new in vitro hybridization reaction targets two short complementary RecA protein-coated DNA probes to homologous sequences at any position in a linear duplex DNA molecule. Stable hybrids are obtained after RecA protein removal when both complementary probe strands are present in a four-stranded hybrid, but not when one probe strand is present in a three-stranded hybrid. In four-stranded hybrids with one probe strand biotinylated and the other radiolabelled, the deproteinized hybrids can be isolated and detected by affinity capture on streptavidin-coated magnetic beads. RecA-mediated targeting of complementary biotinylated DNA probe strands allows the affinity capture of 48.5-kilobase duplex lambda genomic DNA. These reactions provide a means of isolating any desired duplex gene or chromosomal DNA fragment.
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Cell and Molecular Biology Laboratory, SRI International, Menlo Park, California 94025.
Zarling
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D010582:Bacteriophage lambda; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D015342:DNA Probes; D004279:DNA, Viral / Q000737:chemistry* / Q000302:isolation & purification; D007202:Indicators and Reagents; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D011938:Rec A Recombinases; D015183:Restriction Mapping; D013816:Thermodynamics
D015342:DNA Probes; D004279:DNA, Viral; D007202:Indicators and Reagents; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases