Double stranded scission of DNA directed through sequence-specific R-loop formation.
R-loop formation with short (100 nt) RNAs provides a highly flexible and stringent method to achieve sequence-specific separation of target DNA at any given sequence. After stabilization of R-loops with glyoxal and removal of the RNA through RNase treatment the remaining single-stranded DNA bubble provides a highly favorable substrate for attenuated micrococcal nuclease. We investigated this method for sequence-specific scission of double-stranded DNA and achieved quantitative scission of 3-5 kb plasmids. The applicability to larger size DNA is demonstrated through specific excision of the intervening segment between two R-loops from a P1 plasmid of approximately 120 kb.
Landgraf
R
R
Department of Biological Chemistry, School of Medicine, University of California-Los Angeles 90024-1570, USA.
Chen
C H
CH
Sigman
D S
DS
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D006037:Glyoxal; D066298:In Vitro Techniques; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012313:RNA / Q000737:chemistry*; D013329:Structure-Activity Relationship
D004277:DNA, Single-Stranded; D006037:Glyoxal; D012313:RNA; D004247:DNA; D008836:Micrococcal Nuclease