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Ligation-mediated PCR for quantitative in vivo footprinting

Nature Biotechnology volume 18, pages 1108–1111 (2000)Cite this article

Abstract

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 footprints1,2,3,4. 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.

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Figure 1: Flowchart of automated LM-PCR.
Figure 2: Quantitative analysis of UV photofootprinting.

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Acknowledgements

We thank Dr. G. Holmquist for encouragement and support, and J. Flanagan for help with editing the manuscript. Jin Zhou supplied Maxam–Gilbert HeLa DNA for these studies. This work was supported in part by grants GM50575 (to A.D.R.), AG15695 (to S.D.F.), and CA69449 (G. Holmquist) from the National Institutes of Health.

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Authors and Affiliations

  1. Division of Biology, Beckman Research Institute of the City of Hope, Duarte, 91010-3011, CA

    Shu-Mei Dai, Hsiu-Hua Chen, Cheng Chang & Arthur D. Riggs

  2. Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, 91010-3011, CA

    Steven D. Flanagan

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  1. Shu-Mei Dai
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Correspondence to Steven D. Flanagan.

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Dai, SM., Chen, HH., Chang, C. et al. Ligation-mediated PCR for quantitative in vivo footprinting. Nat Biotechnol 18, 1108–1111 (2000). https://doi.org/10.1038/80323

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