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.