this approach has been reported as showing good sensitivity, but a limitation is the large number of pcr reactions required to quantify the calculated loss of signal with significant certainty. in this article, we present an alternative method to identify dhs using real - time pcr by adapting the protocol we have used to map dhs using genomic tiling path arrays ( 11 ). we have previously demonstrated the high sensitivity and specificity of the basic protocol with regard to identifying dhs across large genomic regions ( 11 ). here, we detail the laboratory protocol that permits the rapid comparative mapping of known and candidate dhs between different cell types using real - time pcr. as examples, we include comparative dhs mapping of regulatory elements located across the extended tal1 ( t - cell acute lymphocytic leukaemia - 1, also known as scl ( stem cell leukaemia ) ) locus in human embryonic stem ( hes ) cells and the leukaemia cell line k562. generating a library of dhssthe basic protocol is outlined in figure 1. nuclei are extracted from living cells, then digested on ice for 1 h with a range of dnasei concentrations, as detailed in the ‘ materials and methods ’ sections. following rnasea and proteinasek treatment, the dna is extracted and run on a 1 % agarose gel to check for the size of digested dna. the gel in figure 1a shows the dna from mouse thymocyte nuclei digested with 0, 40 and 120 units of dnasei. maximal enrichment at dhs is usually observed with samples that are not over - digested. in the experiment shown, maximal enrichment at a known control dhs was seen with 40 units dnasei ( real - time pcr quantification shown in figure 2b ). the dna is then blunt - ended using t4 polymerase ( figure 1b ) and ligated with an asymmetric double - stranded linker. after extraction, dna is amplified using a biotinylated linker - specific primer and 35 thermal polymerase cycles, as detailed in ‘ materials and methods ’ section. as the linker will ligate to both ends of digested dna, the amplification will represent a mix of primer extension and pcr, depending on the length of dna amplified. as has been previously reasoned ( 13 ), one double strand of dna in a region of dnasei hypersensitivity is more likely to be digested twice within a short distance than non - hypersensitive dna. this will lead to the preferential amplification of dna from regions of dhs. the amplified dna is then extracted using para - magnetic streptavidin beads, which provides a dna library representative of whole - genome dhs. agarose gel electrophoresis of the dna recovered from the beads confirms that the vast majority of these products are between 300 and 500 base pairs in length ( figure 1b ). real - time pcr quantification of dna within the dhs libraryfigure 2 documents the quantification of specific dna sites from different mouse - cell - derived dna libraries. figure 2a shows the quantification of material from two primer sets, which are within ( primer pair a ) and 3 ′ ( primer pair b ) to the mouse stil promoter. the left - hand panels of figure 2a show the sybr - green real - time fluorescence profiles using serial 5 - fold dilutions of quantified mouse genomic dna standards with primers a and b. a calculated standard curve then permits the quantification of dna from this sequence in library samples. the right - hand panels of figure 2a show the quantification of samples from 0 and 40 units dnasei - treated material using primers a and b. while there is no difference in amplification between the samples using primer pair b, with primer pair a, the 40 units dnasei sample amplifies ∼5. 5 pcr cycles before the 0 units sample. this equates to greater than 40 - fold enrichment at the stil promoter compared with no observed enrichment downstream of the promoter. figure 2b shows the quantifiable differences in enrichment at the porphobilinogen deaminase ( hmbs ) promoter between primary mouse thymocytes and the mouse t - cell line bw5147. this representative primary mouse cell experiment was performed with the dna shown in figure 1a, which confirms that with these cells, maximal enrichment is observed from dna that is not over - digested. the comparison of the thymocytes with the bw5147 cells illustrates another common finding that, in our experience of primary cell experiments, maximal enrichment is often obtained using lower amounts of dnasei compared with cell lines. comparison of real - time pcr dhs with southern blottingas the protocol generates template by primer extending away from a dnasei - digestion site, as well as pcr amplification between dnasei - digestion sites, there is the potential for the genomic size of the real - time dhs to be larger than sites revealed by southern blotting. there is also potential for signal to be lost at the most ‘ open ’ stretch of dna, due to over - digestion with dnasei. these issues are addressed in figure 3. the upper panel shows a southern blot of the human stil ( scl / tal1 interrupting locus ) promoter in k562 cells. the bglii restricted fragment is probed from the 3 ′ end, as shown in the upper panel. this reveals a central dhs ∼500 bp wide, with a suggestion of weaker hypersensitivity for a few hundred base pairs 5 ′ and 3 ′ to the central region. the lower panel shows real - time pcr data from k562 material using 10 primer sets, each generating an amplicon ∼120 bp long. this permits the quantification of enrichments over 1200 bp, centred around the stil transcription start site. the lower panel represents the mean ± sd enrichment from three independent biological replicates from k562 cells. the black bar denotes the location of the ‘ core ’ hypersensitive site as defined by southern blotting. there is good correlation between the location of the dhs between the two techniques. the 5 ′ extension of enrichment seen in the lower panel appears to reflect the weaker 5 ′ extension seen in the southern blot in the upper panel. a dip in enrichment is observed in the lower panel over the previously mapped transcription - factor - binding sites ( 14 ), which may represent over - digestion of the most accessible core region of the dhs. quantification of dhs across the tal1 locus in hes and k562 cellsas examples of relative enrichments at different regulatory elements, we present a comparison of enrichments across the extended tal1 locus from human embryonic stem ( hes ) cells and k562 cells in figure 4b. relative quantification of mrna expression using real - time pcr shows that both of these cell types express stil, but only k562 cells express tal1 ( figure 4a ). tal1 expression is critical to the establishment of haematopoiesis in a developing embryo ( 15 ). post embryogenesis, tal1 expression is maintained in the non - lymphoid haematopoietic system, although in addition, expression is observed in a range of non - haematopoietic tissues, including endothelium and brain ( 16 – 18 ). over the past 10 years, we and others have dissected the regulatory elements that direct the expression of tal1 to different tissues ( 11, 19 – 31 ). these include the 3 ′ haematopoietic stem cell enhancer ( + 19 ) ( 24, 30 ), the 5 ′ endothelial - haematopoietic enhancer ( −4 ) ( 31 ), the 3 ′ erythroid enhancer ( + 40 in mouse, + 50 in human ) ( 28 ), and a number of neuronal elements ( 25, 26 ). the location of the stil promoter has been previously mapped ( 14 ), although no other stil regulatory elements have yet been identified. figure 4b shows the mean ± sd ratio ( dnasei - treated enrichment / dnasei - untreated enrichment ) of quantitative enrichments from two independent hes cell biological replicates and two independent k562 biological replicates, using primer sets from different locations as indicated in the figure. primers indicated in red correspond to known regulatory elements. the hes cell cultures were maintained as detailed in methods. both cell types show significant enrichment at the stil promoter.