Branch migration mediated DNA labeling and cloning.
The sequence-dependent attachment (capture) of an oligodeoxynucleotide duplex containing a single-stranded tail can be mediated by branch migration into the end of a DNA molecule. Substitution of bromodeoxycytidine (BrdC) for deoxycytidine (dC) increased DNA-DNA hybrid stability. BrdC-containing oligodeoxynucleotides displaced dC-containing strands from duplexes with blunt ends or 3'-overhangs. In the later case the rate of displacement was of the same order of magnitude as DNA reassociation. A BrdC-containing displacer oligodeoxynucleotide was used for transient sequence-specific invasion at a particular PstI site. The product was captured by use of T4 DNA ligase and a linker oligodeoxynucleotide. The capture rate was more than 300 times the rate observed for an unrelated PstI site. This high degree of specificity required BrdC substitution. In addition, deliberate incorporation of an incorrect nucleotide into a displacer strand demonstrated that branch migration was terminated at a mismatch. A branched, BrdC-containing ligated product of a capture reaction was cloned and sequenced. The specific capture reaction may be used to label a particular DNA fragment prior to electrophoresis, to mark the specific fragment for affinity chromatography, or to facilitate cloning by introducing a new overhanging sequence compatible with a restriction endonuclease site in a cloning vector.
Quartin
R S
RS
Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029.
Plewinska
M
M
Wetmur
J G
JG
D001345:Autoradiography; D001483:Base Sequence; D001972:Bromodeoxycytidine; D003001:Cloning, Molecular; D004247:DNA; D003841:Deoxycytidine / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008025:Ligases; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D010759:Phosphorus Isotopes; D015183:Restriction Mapping
D009841:Oligonucleotides; D010759:Phosphorus Isotopes; D003841:Deoxycytidine; D001972:Bromodeoxycytidine; D004247:DNA; D008025:Ligases