Randomized terminal linker-dependent PCR: a versatile and sensitive method for detection of DNA damage.
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).


CONCLUSIONS
DNA strand breakage can be detected sensitively in the gene level by RDPCR. Any lesion that blocks primer extension should be detectable.
Zhang
Zhi-Wei
ZW
Department of Environmental Health, School of Public Health, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China. zzwwcums@263.net
Heng
Zheng-Chang
ZC
D000818:Animals; D018736:DNA Adducts; D004249:DNA Damage; D017931:DNA Primers; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D008322:Mammals; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D018736:DNA Adducts; D017931:DNA Primers