Open AccessTerminal transferase-dependent PCR: A versatile and sensitive method for in vivo footprinting and detection of DNA adducts
Author(s) -
J. Komura,
Arthur D. Riggs
Publication year - 1998
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/26.7.1807
Subject(s) - biology , primer extension , terminal deoxynucleotidyl transferase , primer (cosmetics) , microbiology and biotechnology , dna footprinting , dna , footprinting , oligonucleotide , transferase , genomic dna , ribonucleotide , sequencing by ligation , dna nanoball sequencing , primer dimer , biochemistry , genetics , genomic library , gene , nucleotide , polymerase chain reaction , multiplex polymerase chain reaction , enzyme , gene expression , promoter , chemistry , apoptosis , organic chemistry , tunel assay , base sequence
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.
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