Comparison of real-time PCR DHS with Southern blotting
As 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.
Figure 3. The upper panel shows a Southern blot DHS map of the human STIL promoter in K562 cells using a 3′ probe (grey box labelled ‘P’) to analyse the BglII-restricted fragment (cut −4 kb and +4.2 kb relative to the STIL transcription start site). This revealed a core DHS ∼500 bp wide. The lower panel represents the mean ± SD real-time PCR quantification of enrichments from three biological replicates of K562 material relative to known genomic standards, using 10 separate primer sets that together amplify ∼1200 bp centred around the STIL transcription start site. The black bar represents the approximate location of the DHS, as identified by the Southern blot experiment in the upper panel. The real-time PCR amplification profile closely represents the Southern blot profile, although a central dip in real-time PCR signal is observed over the site of transcription factor binding.