14 Weirnuclease inhibitors. RNases are dealt with in much more detail else-
where in this volume (Chapter 13).
3.5. "Degradative" Uses of DNase I
Stock solutions ofDNase I should be made up at 2 mg/mL in 10 mM
Tris-HC1, pH 7.5, 50 mM NaC1, 10 mM MgCI2, 1 mM dithiothreitol,
and 50% glycerol, and stored frozen in small aliquots. Removal of
DNA from preparations can be accomplished by incubation with 20-
50 ~tg/mL DNase I in the presence of 50 mM Tris-HC1, pH 7.5, 10 mM
MgC12, at 37°C for 60 min.
A method for preparing random oligonucleotide fragments from calf
thymus DNA for use in the oligolabeling protocol can be found in (9).
3.6. Nick Translation
The alternative method for producing radiolabeled probes other
than oligolabeling is nick translation. In this method DNase I is used
to nick the DNA to be labeled creating free 3'OH groups. Escherichia
coli DNA polymerase I catalyzes the addition of nucleotides to the
3'OH termini and the 5'-3' exonuclease activity of the same enzyme
removes nucleotides from the 5' end thus translating the nick. Linear,
supercoiled, nicked, or gapped circular ds DNA molecules can be labeled
to specific activities of > 108 cpm/~tg with 32p labeled deoxynucleotide
triphosphates. Good protocols for the method are given in refs. 10 and
11 and Chapter 3. Meinkoth and Wahl (10) pointed out that, at saturating
levels of nucleotide triphosphates, the size of the labeled fragments is
determined by the DNase concentration: Fragments of 500-1500 nucle-
otides long produce optimal signal:noise ratios in the subsequent
hybridization. The necessary concentration of DNase required can be
determined by doing a titration using from 0-100 pg/mL DNase in the
reactions and running the labeled DNA on acrylamide gels (9). DNase
concentration, which gives approx 30% incorporation of label, is optimal.
- DNA Footprinting by DNase I Protection
DNase I has been used in the past to dissect the structure of chroma-
tin and to isolate nucleosomes (e.g., see 12,13), but in recent years
molecular biologists have become interested in the proteins that bind
to DNA and possibly regulate the expression of genes. The isolation
and characterization of such transactivating factors can be accom-
plished by using the powerful technique of DNA footprinting. In this
- DNA Footprinting by DNase I Protection