Rapid and unambiguous detection of DNase I hypersensitive site in rare population of cells.
DNase I hypersensitive (DHS) sites are important for understanding cis regulation of gene expression. However, existing methods for detecting DHS sites in small numbers of cells can lead to ambiguous results. Here we describe a simple new method, in which DNA fragments with ends generated by DNase I digestion are isolated and used as templates for two PCR reactions. In the first PCR, primers are derived from sequences up- and down-stream of the DHS site. If the DHS site exists in the cells, the first PCR will not produce PCR products due to the cuts of the templates by DNase I between the primer sequences. In the second PCR, one primer is derived from sequence outside the DHS site and the other from the adaptor. This will produce a smear of PCR products of different sizes due to cuts by DNase I at different positions at the DHS site. With this design, we detected a DHS site at the CD4 gene in two CD4 T cell populations using as few as 2×10(4) cells. We further validated this method by detecting a DHS site of the IL-4 gene that is specifically present in type 2 but not type 1 T helper cells. Overall, this method overcomes the interference by genomic DNA not cut by DNase I at the DHS site, thereby offering unambiguous detection of DHS sites in the cells.
Department of Biochemistry and Microbiology, Marshall University, Huntington, West Virginia, United States of America ; Department of Paediatrics, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia, United States of America ; Centre for Cell Development and Differentiation, Department of Biology, College of Science, Marshall University, Huntington, West Virginia, United States of America.
Wei-Ping
WP
Zeng
Department of Biochemistry and Microbiology, Marshall University, Huntington, West Virginia, United States of America.
Margaret M
MM
McFarland
D000818:Animals; D001483:Base Sequence; D015704:CD4 Antigens / Q000235:genetics; D002452:Cell Count; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D016678:Genome; D015847:Interleukin-4 / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013010:Sonication; D018417:Th1 Cells / Q000378:metabolism*; D018418:Th2 Cells / Q000378:metabolism*
D015704:CD4 Antigens; D015847:Interleukin-4; D004247:DNA; D003850:Deoxyribonuclease I