Zeng Wei-Ping, McFarland Margaret M
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.
Department of Biochemistry and Microbiology, Marshall University, Huntington, West Virginia, United States of America.
PLoS One. 2014 Jan 21;9(1):e85740. doi: 10.1371/journal.pone.0085740. eCollection 2014.
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.
脱氧核糖核酸酶I超敏（DHS）位点对于理解基因表达的顺式调控很重要。然而，现有的在少量细胞中检测DHS位点的方法可能会导致结果不明确。在此，我们描述了一种简单的新方法，其中分离出由脱氧核糖核酸酶I消化产生末端的DNA片段，并将其用作两个聚合酶链反应（PCR）的模板。在第一个PCR中，引物来源于DHS位点上下游的序列。如果细胞中存在DHS位点，由于引物序列之间的模板被脱氧核糖核酸酶I切割，第一个PCR将不会产生PCR产物。在第二个PCR中，一个引物来源于DHS位点外的序列，另一个来源于接头。由于脱氧核糖核酸酶I在DHS位点的不同位置切割，这将产生不同大小的PCR产物涂片。通过这种设计，我们使用低至2×10⁴个细胞在两个CD4 T细胞群体中检测到了CD4基因处的一个DHS位点。我们通过检测白细胞介素4（IL-4）基因的一个DHS位点进一步验证了该方法，该位点特异性存在于2型而非1型辅助性T细胞中。总体而言，该方法克服了在DHS位点未被脱氧核糖核酸酶I切割的基因组DNA的干扰，从而能够明确检测细胞中的DHS位点。
