Positional mapping relative to the NM using a higher DNA template concentration. Positional mapping relative to the NM of specific target sequences along the 162 kbp rat albumin gene-family genomic region by PCR using a higher DNA template concentration. Nucleoids from G0 rat hepatocytes were treated with DNase I (0.5 U/ml) for different times (Figure 2A). The residual NM-bound DNA in the partially digested samples was directly used as template for PCR amplification of the chosen target sequences (a - o). For these experiments the nuclear matrix-bound DNA template was increased six-fold (from 10 to 60 ng) and the DNA polymerase concentration was doubled (from 0.7 to 1.25 U) in order to facilitate the amplification of target sequences embedded within the NM. The specific amplicons were resolved in 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). C, 0' digestion-time control. The amplicons were scored either as positive or negative as a function of endonuclease digestion time and for each topological zone relative to the NM, depending on whether or not they were detected by a digital image-analysis system (Kodak 1D Image Analysis Software 3.5) using the default settings. Topological zones relative to the NM: D, distal; P, proximal; VC, very close; E, embedded within the NM. (-) Negative control (no template); (+) positive control (pure genomic DNA as template). For detailed analysis of results see Table 4.
Rivera-Mulia et al. BMC Cell Biology 2011 12:3 doi:10.1186/1471-2121-12-3