Rapid generation of subclones for DNA sequencing using the reverse cloning procedure.
A fast and reliable procedure for generating subclones necessary for sequencing long stretches of DNA has been developed. The reverse cloning procedure involves cloning a fragment of DNA into a single-stranded plasmid or phage vector containing a polycloning region; synthesizing variable lengths of double-stranded DNA using a "Universal Primer"; isolating the double-stranded DNA; and force cloning the double-stranded DNA fragments into a complementary vector with the polycloning region in the reverse orientation. The resulting clones can be sequenced, using the same Universal Primer and T7 DNA polymerase, to provide overlapping DNA sequences. The reverse cloning procedure can be used to construct deletion mutations.
Liu
Z J
ZJ
Dept. of Genetics & Cell Biology, University of Minnesota, St. Paul 55108-1095.
Hackett
P B
PB
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids; D013604:T-Phages / Q000235:genetics
D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase