Randomized Terminal Linker—dependent PCR：A Versatile and Sensitive Method for Detection of DNA Damage
摘要
Objective To design and develop a novel, sensitive and versatile method for in vivo foot printing and studies of DNA damage, such as DNA adducts and strand breaks. Methods Starting with mammalian genomic DNA, single-stranded products were made by repeated primer extension, these products were ligated to a double-stranded linker having a randomized 3′ overhang, and used for PCR.DNA breaks in p53 gene produced by restriction endonuclease AfaI were detected by using this new method followed by Southern hybridization with DIG-labeled probe. Results This randomized terminal linker-dependent PCR (RDPCR) method could generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR), and it was more rapid, convenient and accurate than the terminal transferase-dependent PCR (TDPCR). Conclusion DNA strand breakage can be detected sensitively in the gene level by RDPCR. Any lesion that blocks primer extension should be detectable.