Duplex-Specific NucleaseBioCat is pleased to offer Evrogen´s novel Duplex-Specific Nuclease (DSN) enzyme purified from Kamchatka crab hepatopancreas. DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for non-perfectly matched duplexes of the same length.
DSN acquires its enzymatic activity in the presence of Mg2+ ions (at least 5 mM is required for most applications) and is inhibited by EDTA. The pH and temperature optima for activity are 7-8 and 55-65°C, respectively. The nuclease is stable at a wide range of pH (from 4 to 12) and temperatures below 60°C. Moreover, 60% of DSN activity remains after incubation at 70°C for 30 min, and 40 % - after incubation at 80°C. In addition, DSN is tolerant to proteinase K treatment (at 37°C).
DSN exhibited strong cleavage preference for ds DNA substrates and little activity against ss DNA (Figure 1). No significant cleavage activity on RNA substrates is observed. However, the nuclease effectively cleaves DNA molecules in DNA-RNA hybrid duplexes. Analysis of DSN action on synthetic oligonucleotide substrates revealed that the enzyme discriminates between perfectly matched short DNA-DNA duplexes (8-12 bp) and duplexes of the same length with at least one mismatch (Figure 2).
Figure 1. Determination of crab nuclease preference for specific structural features of DNA substrates.
(A) Action of crab nuclease on ss phage M13 DNA and ds λ DNA. Lanes 1, 2 – negative controls, incubation without nuclease. 1 – phage M13 DNA alone, 2 - mixture containing phage M13 DNA and λ DNA. Lanes 3; 4 – digestion of phage M13 and λ DNA mixture by crab nuclease at 70°C for 1.5 min (line 3) and 5 min (line 4). The reaction was performed in a total volume of 10 μl comprising 1 x DSN buffer, 0.06 Kunitz units DSN, 150 ng λ DNA and 50 ng M13 DNA. To prevent ds structure formation in phage M13 DNA, the reaction mixture was incubated at 70°C for 1, 5 or 5 min. The digestion products were visualized on a 0,9% agarose gel, following ethidium bromide staining.
(B) Action of crab nuclease on synthetic ss and ds 20-mer DNA substrates. Red line - ds-DNA; blue line - ss-DNA. Oligonucleotide (5´-tgggcagtgctcgcttagtg-3´) labeled with a fluorescent donor at the 5´ end and a fluorescent quencher at the 3´ end were used as ss DNA. To generate ds substrate, this labeled oligonucleotide was mixed with equimolar amount of complementary non-labeled oligonucleotide. The cleavage reaction was performed in a total volume of 20 μl comprising 1 x DSN buffer, 0.6 Kunitz units DSN, and 0.3 μM oligonucleotide substrate. DNase activity was evaluated by estimating the change in fluorescence intensity of the reaction mixture during incubation with DSN. Fluorescence intensity was measured on a spectrofluorimeter Cary Eclypse (Varian). Fluorescence intensity was measured at 570 nm (with excitation at 550 nm). The relative fluorescence increase in the oligonucleotide substrate, RFI, was defined as RFI= (Fi-Fo/Fmax-Fo) x 100%, where Fi is the fluorescence intensity of a substrate after incubation with nuclease, Fo is the substrate fluorescence in the absence of enzyme, and Fmax represents the fluorescence of 100% cleaved substrate. For kinetic graph construction, three identical experiments were performed and the average values and standard deviations were plotted.
Figure 2. Results of the DSN action on one mismatch-containing (A, B) and perfectly matched (C) DNA duplexes. Duplexes was formed by 5-carboxyfluorescein (Fl)-5´-gccctatagt-3´-TAMRA signal probe and complementary targets: A - 5´-actcactataCggcgaat-3´; B - 5´-actcactataggTcgaat-3´; C - 5´-actcactatagggcgaat-3´. The duplexes were incubated with DSN at 35°C for 15 min. Emission spectra were obtained on the spectrofluorimeter, with excitation at 480 nm. Blue line - substrate fluorescence in the absence of enzyme; green line - substrate fluorescence after incubation with DSN.
1. Shagin D.A., Rebrikov D.V., Kozhemyako V.B., Altshuler I.M., Shcheglov A.S., Zhulidov P.A., Bogdanova E.A., Staroverov D.B., Rasskazov V.A., Lukyanov S. (2002) A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. Genome Res. 12, 1935-1942.
2. Zhulidov P.A., Bogdanova E.A., Shcheglov A.S., Vagner L.L., Khaspekov G.L., Kozhemyako V.B., Matz M.V., Meleshkevitch E., Moroz L.L., Lukyanov S.A., Shagin D.A. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acid Res., 2004, 32: e37.
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