SI Nuclease

SI nuclease j Degrades single-stranded DNA. Removal of "hairpin" in synthesis of cDNA RNA mapping studies (both 5 and 3 ends). 

On the basis of Scheme 5, we recently realized label-free sequence-specific DNA detection with SNP selectivity with the aid of SI nuclease [59]. In this assay, IBr and TO are chosen as the energy donor and acceptor, respectively. 

Martin-Bertram 1981,1982 Martin-Bertram et al. 1983 for DNA isolated from y-irradiated yeast cells see Andrews et al. 1984). While such lesions, detected by SI nuclease ( Sl-nuclease-sensitive sites ), are not observed with A phage DNA y-irradiated in aqueous solutions (Martin-Bertram et al. 1984), they become prominent when the solutions is highly scavenged (Junker et al. 1984). Under such conditions, the damage caused by the direct effect plays a role because the indirect effect is largely suppressed (Ward 1981). 

Andrews J, Martin-Bertram El, Elagen U (1984) SI nuclease-sensitive sites in yeast DNA an assay for radiation-induced base damage. Int J Radiat Biol 45 497-504 Antoku S (1983) Radiosensitization and radioprotection of E. coli by thiourea in nitrous oxide saturated suspensions. Int J Radiat Biol 43 451-458... 

Junker B, Martin-Bertram H, Hagen U (1984) Distribution of Si-nuclease sensitive sites and doublestrand breaks iny-irradiated A-DNA. Int J Radiat Biol 46 675-679 Kan Y, Schuster GB (1999a) Radical cation transport and reaction in triplex DNA long-range guanine damage. J Am Chem Soc 121 11608-11614... 

Fig. 10. Incremental truncation libraries (Ostermeier et al., 1999b). Plasmid DNA is digested with two restriction enzymes one that produces a 3 recessed end (A which is susceptible to Exo III digestion) and the other that produces a 5 recessed end (B which is resistant to Exo III digestion). Digestion with Exonuclease III proceeds under conditions in which the digestion rate is slow enough so that the removal of aliquots at frequent intervals results in a library of deletions of all possible lengths from one end of the fragment. The ends of the DNA can be blunted by treatment with SI nuclease and Klenow so that unimolecular ligation results in the desired incremental truncation library.

Unavoidable terminal heterogeneity as a consequence of using DNAase I or SI nuclease... 

An alternative to the hydroxyapatite procedure for discrimination between single-stranded and duplex DNA is the SI nuclease procedure.29 30... 

According to this method, DNA is treated with SI nuclease after hybridization and then precipitated. SI nuclease digests single-stranded DNA but not double-stranded DNA. The rigor of enzyme treatment can have a dramatic effect on the effective criterion, and it is important to establish strict control in SI nuclease assays. 

SI Nuclease Method. Sutton25 and Crosa et al.26 demonstrated that DNA hybridization mixtures could be freed of single-stranded DNA by hydrolysis with SI endonuclease. Double-stranded hybrids are then removed from the reaction mix by precipitation with trichloroacetic acid (TCA) or by collection on Whatman (Clifton, NJ) DE-81 filters.27... 

Following reassociation, place a 100-//1 sample from each hybridization vial into a polypropylene tube containing 1 ml of 50 mM sodium acetate-0.3 M NaCl-0.5 raM ZnCl2 buffer (pH 4.6) and 50 fA of 0.5 mg/ml denatured sheared calf thymus DNA. Mix the contents and add 50/d (1 unit//d) of SI nuclease. Mix and incubate for 1 h at 50°. 

RNase P, which removes the 5 leader from tRNA transcripts, contains an RNA moiety in bacteria, eucarya, and archaea. The enzyme from Haloferax volcanii has been purified and the 345-nucleotide RNA portion used to generate a probe for cloning of the corresponding gene [123]. The sequence can be folded into a structure similar to that of bacterial RNase P RNAs. SI nuclease and primer extension localize the 5 end of the transcript adjacent to four potential archaeal promotor sequences. An in vitro transcript corresponding to the native RNA plus twenty 5 and nine 3 flanking nucleotides did not by itself exhibit RNase P activity (as bacterial RNase P RNAs do), under a variety of conditions. It was, however, able to reconstitute an active enzyme in combination with the protein moiety from Bacillus subtilis. This indicates that in Haloferax volcanii the RNase P RNA is the catalytic part of the enzyme but it may require some structural help [123]. 

Nucleases are enzymes that hydrolyze one or more phos-phodiester bonds in nucleic acid polymers. Nucleases may require a free hydroxyl end (exonucleases), with specificity for the 3 or 5 end, or may act only on internal bonds (endonucleases). For example, some probe techniques are based on 5 -exonuclease activity that cleaves nucleic acids between two fluorescent labels. Nucleases can be DNA- or RNA-specific and may act on only double- or single-stranded polymers. For example, DNAse I digests double-stranded DNA (dsDNA) and SI nuclease acts only on smgle-stranded DNA (ssDNA). DNase I can be used to specifically degrade DNA in nucleic acid mixtures when only RNA is of interest. RNAses are very stable enzymes that are common laboratory contaminants. 

Antisense RNA amphfication (aRNA) Target T4 DNA polymerase Klenow SI nuclease T7 polymerase No... 

SI nuclease. An enzyme which digests only single-stranded nucleic acids. 

Fig. 5 Ordered fragmentation of pDNA induced by the complexation with PEG-PLL (12-17). SI nuclease, known to cleave looped DNA strand, is applied to stoichiometrically prepared PIC micelle, in which DNA is condensed in toroid and rod configurations. The condensed DNA is cleaved into seven fragments composed of 10/12, 9/12, 8/12, 6/12, 4/12, 3/12, 2/12 of the original length...

Logical extensions of this method are the SI nuclease protection assay (Reyes and Wallace, 1987) and the simultaneous quantification of several mRNA species by solution hybridization with oligonucleotides (Section 12.3) (O Donovan et al., 1991). 

Urea, in solution hybridization, has several advantages over formamide (Dutton and Chovnick, 1987) (i) mild temperatures (50-55°C) provide stringent conditions for both RNA DNA and DNA DNA hybridization (ii) the solubility of nucleic acid (particularly at high concentrations) is greater in urea than in formamide (iii) urea is more stable. In their procedure, Dutton and Chovnick (1987) hybridized the target and probe in solution, digested the free JS probe with SI nuclease and collected the hybrids on glass fiber... 

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