Quantification of DNaseI-sensitivity by real-time PCR: quantitative analysis of DNaseI-hypersensitivity of the mouse β-globin LCR

摘要: We employ real-time PCR to allow us quantify the sensitivity of chromatin digestion by DNaseI. This approach has three clear advantages over more conventional use Southern hybridization assay: accuracy quantification is improved; resolution assay enhanced, designing primers amplify small amplicons it possible analyze sequences both co-incident and proximal sites DNaseI-hypersensitivity; less material needed, as little 5 ng treated genomic DNA. applied this method in an analysis structure previously described mouse β-globin locus control region (LCR) using fetal liver cells. The four hypersensitive canonical LCR, HS1 HS4, are shown have kinetics consistent with these being nucleosome-free vivo. A different pattern was seen for HS6, a recently “weak” site. site also rapidly lost but proved resistant, we interpreted show that only forming portion erythroid finding implies vivo LCR structurally heterogeneous. Sequences third intermediate sensitivity, unfolded still bound continual nucleosomal array. Our results demonstrate potential achieve accurate detailed mapping from amounts tissue samples.