The DNA sequence specificity of hedamycin damage determined by ligation-mediated PCR and linear amplification.
The DNA sequence specificity of hedamycin damage was compared using two techniques: Ligation-mediated PCR (LMPCR) and Linear Amplification (LA). The electrophoretic mobility of LA products were made comparable with LMPCR products by using a primer with a 27 bp non-binding 5'-extension. By direct comparison of the damage intensity (as represented by each of the methods), considerable bias was found in the LMPCR system with respect to LA--resulting in the under representation of lesions with T as the base 3' to the damage site. In view of this observation some caution should be exercised in the interpretation of data for DNA damage/repair specificity derived by LMPCR. In addition the extended primer LA method used in these experiments, could be applied to generate a dideoxy sequencing ladder for analysis of LMPCR products. This would negate the need to prepare Maxam-Gilbert chemical sequencing fragments for amplification through LMPCR.
Cairns
M J
MJ
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia. v.murray@unsw.edu.au
Murray
V
V
D000477:Alkylating Agents / Q000494:pharmacology*; D000880:Anthraquinones / Q000494:pharmacology*; D001483:Base Sequence; D004249:DNA Damage; D011088:DNA Ligases; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D006150:Guanine Nucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction
D000477:Alkylating Agents; D000880:Anthraquinones; D006150:Guanine Nucleotides; C001328:hedamycin; D011088:DNA Ligases