Ligation-mediated PCR for quantitative in vivo footprinting.
Ligation-mediated polymerase chain reaction (LM-PCR) is a genomic analysis technique for determination of (1) primary DNA nucleotide sequences (2) cytosine methylation patterns (3) DNA lesion formation and repair, and (4) in vivo protein-DNA footprints. However, LM-PCR can be limited by the multiple steps required and the relatively short stretch of sequence (usually <200 bp) that can be analyzed per reaction. We report here a simplified, one-day LM-PCR protocol in which all pipetting steps can be performed by a robotic workstation and which, moreover, provides longer reads (>350 bp) and enhanced signal quality by use of nonradioactive detection and a LI-COR DNA sequencing instrument. Sensitivity comparable to radiolabeling is achieved using oligonucleotide primers that are 5'-end labeled with infrared fluorochromes. We showed that the technique could be used for sensitive and reproducible in vivo photofootprinting of the human phosphoglycerate kinase 1 (PGK1) promoter, as well as providing good Maxam-Gilbert sequence information. The methods described here should allow high-throughput, high-resolution analysis of transcription factor binding and chromatin structure, and also may be useful for sequencing gaps that are refractory to cloning.
Dai
S M
SM
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010-3011, USA.
Chen
H H
HH
Chang
C
C
Riggs
A D
AD
Flanagan
S D
SD
D000818:Animals; D001331:Automation; D016466:CHO Cells; D006224:Cricetinae; D004249:DNA Damage / Q000235:genetics; D018983:DNA Footprinting / Q000379:methods*; D011088:DNA Ligases / Q000378:metabolism*; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D005456:Fluorescent Dyes; D016158:Genes, p53 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007259:Infrared Rays; D010735:Phosphoglycerate Kinase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D012371:Robotics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors; D014466:Ultraviolet Rays
D017931:DNA Primers; D005456:Fluorescent Dyes; D010735:Phosphoglycerate Kinase; D011088:DNA Ligases