Terminal transferase-dependent PCR (TDPCR) for in vivo UV photofootprinting of vertebrate cells.
Terminal transferase-dependent PCR (TDPCR) is a versatile, sensitive method for detecting DNA lesions such as those generated by the footprinting agents commonly used to detect in vivo protein-DNA interactions. Data similar to those obtained by ligation-mediated PCR (LMPCR) are obtained, but one advantage of TDPCR is that no special enzymes are needed other than terminal deoxynucleotide transferase, T4 DNA ligase, and thermostable DNA polymerases. A detailed TDPCR protocol is given for using UV photofootprinting to detect in vivo footprints and chromatin fine structure in vertebrate cells. One version of the protocol makes use of nonradioactive labeling by near-infrared fluorochromes and detection by a LI-COR DNA sequencing instrument. Sensitivity similar to that of (32)P-labeling is obtained, but with superior band resolution and quantitation.
Chen
H H
HH
Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. hchen@coh.org
Kontaraki
J
J
Bonifer
C
C
Riggs
A D
AD
D000818:Animals; D018983:DNA Footprinting / Q000379:methods*; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D006801:Humans; D008860:Microscopy, Ultraviolet / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*
D004253:DNA Nucleotidylexotransferase