DNase digestion

Select slides with optimized proteinase K digestion and follow the steps below for DNase digestion. Prepare in an Eppendorf tube 1.0 pL RNasin (RNase inhibitor), 11.5 pL UV-irradiated dH20, 37.5 U DNase (RQl RNase free DNase, Promega Inc., Madison, Wl)/(1 U/pL) Total (per slide) 50.0 pL. 

During the middle phase the composition of the 3 -P terminal and of the 5 -OH terminal and penultimate nucleotides of oligonucleotides obtained from calf thymus DNA by acid DNase digestion, in the incubation conditions of Fig. 1, is practically constant. Some results obtained in this phase are given in Table III. Purine nucleotides form about 75% of the 3 -terminals and of the 5 -OH penultimates with a predominance of G in... 

Glucosylated oligonucleotides obtained from T4 phage DNA by acid DNase digestion are resistant to spleen exonuclease (28). It has been reported that acetylation of the 2 -OH groups of tRNA completely inhibits the action of the enzyme, whereas venom exonuclease is not affected (29). The naturally occurring methylation of sugars and bases in tRNA does not seem to hinder the action of spleen exonuclease. 

Using RNA core as the substrate, EDTA and sulfhydryl reagents are activators Mg2+, Mn2+, and, more effectively, Cu2+, Hg2+, and Zn2+ are inhibitors arsenite and fluoride are weak inhibitors (14) Deoxy-ribonucleoside-3 -phosphates are competitive inhibitors of the activity on acid DNase digests. 

Region protected against DNase digestion by bound RNA polymerase ... 

A simple approach would be to take small aliquots of the sample and treat them with the enzymes ribo-nuclease (RNase) or deoxyribonuclease (DNase). Digestion of the sample by one of these enzymes would indicate whether the sample is RNA or DNA. Another method is to treat a small sample with alkali, which degrades RNA to mononucleotides, but only denatures DNA to the single-stranded form. 

It is extremely important to control the quality of the two DNase digestions to ensure that the RNA template is free of residual EMCV plasmid DNA. Presence of EMCV DNA would result in false-positive results or background Amp-RT signal. To check for absence of EMCV DNA, the RNA template is subjected to PCR amplification and probing by Southern blot hybridization to 32P-labeled EmcPl probe. 

These results Indicate that the chromatin structure of nontranscribed DNA Is more condensed, and therefore more protected from DNase digestion, than that of transcribed DNA. In condensed chromatin, the DNA Is largely Inaccessible to DNase 1 because of Its close association with histones and other less abundant chromatin proteins. In contrast, actively transcribed DNA Is much more accessible to DNase 1 digestion because It Is present In the extended, beads-on-a-string form of chromatin. 

DNA contamination is diCBcult to remove from S. aureus RNA preparations. The protocol below uses a single on column DNase digestion followed by two consecutive RNA clean-up assays. The RNeasy silica-membrane removes DNA therefore, additional clean-up steps are used to aid in removal of contaminating S. aureus DNA. 

DNase digestion of nucleoli Extrachromosomal nucleoli Extrachromosomal nucleoli... 

Locusta migratoria DNase digestion of nucleoli Kunz (1967a)... 

Optional. For DNase digestion after antigen selection (an alternative to Protocol 2, step 3) add 2-5 U of DNase I in 50 p,l of 1 X DNase I digestion buffer to the antigen-selected, washed beads. Incubate at 37 C for 10 min. Remove DNase by washing the beads three times with 50 xl PTM and once with 50 irl sterilized distilled H2O. Resuspend beads in 10 p.1 sterilized distilled H2O ready for RT-PCR (Protocol 5). The beads can be stored either at 4 C or -20 °C. 

Pancreatic DNase. Digestion of DNA by pancreatic DNase shows a considerable lag time. During this time, no apparent decrease of molecular weight of the DNA is observed. The enzyme, an endonuclease (Fig. 3.8) first splits only one chain (haplotomic break) randomly, and only when the opposite chain is broken at the same (or a nearby) position (again randomly) do smaller fragments appear. Eventually, complete digestion will occur, yielding 5 -phosphate termini. 

Nucleosome Smallest repeat unit of chromatin nucleo-protein, containing 145 bp of DNA wrapped around a histone octamer core (2 subunits each of histone H2A, H2B, H3, and H4) along with linker DNA of variable length. Mild treatment of chromatin with DNase digests the linker and generates nucleosome fragments of different repeat lengths ( ladder ). 

Protocol 23.1 describes a standard fly miniprep that requires very few flies and produces high-quality DNA. This protocol can also be used to isolate RNA when RNase-ffee conditions are utilized an extra step must be taken to rid the sample of genomic DNA (e.g., RNase-ffee DNase digestion). Genomic DNA prepared as in Protocol 23.1 is then digested with restriction enzymes (Protocol 23.2) and ligated (Protocol 23.3), and used for either plasmid rescue or inverse PCR as described below. 

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