Optimal conditions for supercoil DNA sequencing with the Escherichia coli DNA polymerase I large fragment.
Sequencing ladders produced from supercoiled DNA templates with the Escherichia coli DNA polymerase Klenow fragment are often unreadable because of a high background and misincorporated nucleotides. This study showed that contaminating RNA molecules can interfere with template:primer hybridization. Procedures are provided for the purification of template DNA and stringent conditions for primer-template hybridization that overcome these problems.
Lim
H M
HM
University of Delaware, Newark 19716.
Pène
J J
JJ
D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D010957:Plasmids; D013698:Templates, Genetic
D004278:DNA, Superhelical; D004256:DNA Polymerase I