New PDF release: Analytical Biochemistry

By David Holme, Hazel Peck

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Introduction The vast bulk of the genome of eukaryotic cells exists as a highly regular array of nucleosomes. A single nucleosome comprises 146 bp of DNA wrapped around a histone core particle. Arrays of nucleosomes usually exist as condensed chromatin fibers with a nucleosome repeat length of about 200 bp. The DNA within condensed chromatin is for the most part relatively inaccessible to proteins, such as nucleases. However, about 1% of the genome exists as discrete regions of decondensed chromatin termed DNaseI hypersensitive (DH) sites (1) which provide greatly increased access to factors that interact with DNA.

30 V for 15 h). 3. Select samples for further analysis that exhibit a distribution of DNA fragments between about 5 and 30 kb in length (see Note 21). 38 Cockerill An example of this screening procedure is shown in Fig. 2A, and a subsequent Southern-blot analysis of DH sites within a BamHI fragment of DNA is shown in Fig. 2B. The optimal DNaseI-digested samples are marked with an asterisk. An ideal DNaseI digestion is seen in lane 1, where 4 µg/mL DNaseI leaves most of the DNA greater than 20 kb in length, with a tail of DNA trailing below the main band (see Fig.

Nuclei are digested for just a few minutes at room temperature with a range of 1–10 µg/mL of DNaseI, and then lysed in SDS to stop the digestion. The optimum range of DNaseI has to be determined empirically, and the viscosity of the nuclear lysates provides the best indication of the extent of DNaseI digestion. The DNA is then purified, digested with restriction enzymes, and analyzed by Southern-blot hybridization. The indirect end-labeling detection method (Fig. 1) employs a short hybridization probe that abuts one end of the restriction enzyme fragment under investigation and reveals specific DNaseI cleavage sites that occur at discrete distances from that end.

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Analytical Biochemistry by David Holme, Hazel Peck

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