Branch capture reactions: displacers derived from asymmetric PCR.
Branch capture reactions (BCR) contain three DNA species: (i) a recipient restriction fragment terminating in an overhang, (ii) a displacer strand containing two adjacent sequences, with one complementary to the overhang and to contiguous nucleotides within the recipient duplex and (iii) a linker which is complementary to the second displacer sequence. Branched complexes containing all three species may be captured by ligation of the linker to the recipient overhang. The use of 5-MedC in the displacer facilitates BCR. High temperature ligation with a thermostable enzyme increased specificity for ligation to the correct recipient in a complex mixture of restriction fragments. Displacer synthesis by PCR permitted separate reactions of formation of stable displacement complexes and of high-temperature ligation. Ethylene glycol-containing buffer permitted PCR with 5-MedCTP or high G + C products using thermostable polymerases. BCR may be used to modify the ends of one recipient DNA duplex in a population of duplex DNA fragments. Modification of the recipient could be used to facilitate detection, affinity chromatography or cloning. By using PCR to obtain a BCR displacer, the sequence non-homologous to the recipient duplex may be expanded to include the sequence of a selectable marker, thus facilitating chromosome walking.
Wong
D M
DM
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029.
Weinstock
P H
PH
Wetmur
J G
JG
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000737:chemistry; D004795:Enzyme Stability; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D013696:Temperature
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D003841:Deoxycytidine; D004247:DNA; C016569:5-methyldeoxycytidine; D004259:DNA-Directed DNA Polymerase