Terminal transferase-dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts.
We report here a new, sensitive and versatile genomic sequencing method, which can be used for in vivo footprinting and studies of DNA adducts. Starting with mammalian genomic DNA, single-stranded products are made by repeated primer extension; these products are subjected to homopolymeric ribonucleotide tailing at the 3' termini with terminal deoxynucleotidyl transferase and then ligated to a double-stranded linker having a complementary 3' overhang, and used for PCR. This terminal transferase-dependent PCR (TDPCR) method can generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR). A UV photofootprint in the mouse Xist gene promoter can be easily detected using TDPCR. No special enzymes or chemical reagents are needed to convert DNA adducts into strand breaks. Any lesion that blocks primer extension should be detectable.
Komura
J
J
Biology Department, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Riggs
A D
AD
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000528:radiation effects*; D018736:DNA Adducts / Q000032:analysis*; D004249:DNA Damage; D018983:DNA Footprinting / Q000379:methods*; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D017931:DNA Primers; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics*; D014466:Ultraviolet Rays; D014960:X Chromosome
D018736:DNA Adducts; D017931:DNA Primers; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D014157:Transcription Factors; C077885:XIST non-coding RNA; D004247:DNA; D004253:DNA Nucleotidylexotransferase