Duplex-specific nuclease

Innovative Biotechnology Company www.evrogen.com Duplex-specific nuclease Product Cat.# Size Duplex-specific nuclease, lyophilized EA1 5 units* Duplex-specific nuclease, lyophilized EA2 units* Duplex-specific nuclease, lyophilized EA3 1 units* *DNAase activity was measured using modified Kunitz assay where unit definition was defined as: the amount of DSN added to 5 µg/ml calf thymus DNA that causes an increase of.1 absorbance units per minute. Activity assay was performed at 25 O C, in 5 mm Tris-HCl buffer, ph 7.15, containing 5 mm MgCl 2. Reagents and storage conditions Reagents included: DSN storage buffer ( µl) 1X DSN master buffer ( µl) 2X DSN stop solution (5 µl) Control template (ng/µl; 2 µl) Reagents required but not included: Glycerol, % Sterile water Agarose gel electrophoresis reagents Storage: Lyophilized duplex-specific nuclease (DSN) must be stored at +4 O C. After dilution in the DSN storage buffer as described in the section DSN dilution, DSN solution must be stored at -2 O C. Other components must be stored at -2 O C. 1

Buffer compositions: DSN storage buffer: 5 mm Tris-HCl, ph 8. 1X DSN master buffer: 5 mm Tris-HCl, ph 8.; 5 mm MgCl 2 ; 1 mm DTT 2X DSN stop solution: 1 mm EDTA DSN description DSN acquires its enzymatic activity in the presence of Mg 2+ 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 O C, respectively. The nuclease is stable at a wide range of ph and temperatures below 6 O C. Moreover, 6% of DSN remains active after incubation at 7 O C for 3 min, and 4% - after incubation at 8 O C (Figure 1). A Digestion efficiency, % 8 6 4 2 1 2 3 4 5 6 7 8 Temperature, O C Figure 1. Dependence of the DSN activity and stability from temperature. (A) Dependence of the DSN activity from temperature. Activity of DNAse on ds DNA substrate was measured using Kunitz assay at different temperature. (B) Kinetics of the thermal denaturation of DSN. DSN was incubated at 5 (1), 6 (2), 7 (3), 8 (4) and 9 (5) O C for 3 min. Activity of DNAse on ds DNA substrate was measured using Kunitz assay at 65 O C. 2 B Activity, % 8 6 4 2 1,2 1 2 3 Time, min 3 4 5

In addition, DSN is tolerant to proteinase K treatment (at 37 O C). DSN exhibited strong cleavage preference for double stranded (ds) DNA substrates and only slight activity against single-stranded (ss) DNA (Figure 2). Moreover, DSN effectively cleaves DNA molecules in DNA-RNA hybrid duplexes. No significant cleavage activity on RNA substrates is observed. 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 3). Purity: DSN was purified from Kamchatka crab hepatopancreas using acetone precipitation and subsequent column chromatography on DEAE-MacroPrep, Phenyl-Agarose, Hydroxyapatite, Heparin- Sepharose, and Sephadex G-75 columns (Ref. 1). Use: Degradation of ds DNA in complex nucleic acids (Ref. 1-3); Discrimination between perfectly matched short DNA-DNA duplexes (8-12 bp) and duplexes of the same length with at least one mismatch (see Ref. 1 for details). DSN dilution Dilute the lyophilized DSN enzyme as follows: 1. Add DSN storage buffer to the lyophilized DSN enzyme on the basis of 5 µl of the buffer for each 1 DSN units. 2. Mix contents by gently flicking the tube. Spin tube briefly in a microcentrifuge. Avoid foaming of the mixture. 3. Incubate the tube at room temperature for 5 min. 4. Add equal amount of % glycerol (to 5% final glycerol concentration) to the tube. 5. Mix contents by gently flicking the tube. Spin the tube briefly in a microcentrifuge. Avoid foaming of the mixture. 6. Store the DSN solution at -2 O C. 3

A λ DNA M13 DNA 1 2 3 4 Figure 2. Determination of DSN preference for specific structural features of DNA substrates. (A) Action of DSN on ss phage M13 DNA and ds phage 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 DSN at 7 O C for 1.5 min (lane 3) and 5 min (lane 4). B 8 6 RFI 4 2 1 2 3 4 5 8 Time, min (B) Action of DSN on synthetic ss (firm line) and ds (dotted line) 2-mer DNA substrates, labeled by fluorescent donor (TAMRA) and quencher (DABCYL) pair. The cleavage reaction was performed at 35 O C for different periods. Fluorescence intensity was measured at 57 nm (with excitation at 55 nm). The relative fluorescence increase in the oligonucleotide substrate, RFI, was defined as RFI= (Fi-Fo/Fmax-Fo) x %, 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 % cleaved substrate. 4

A B C 55 5 45 4 35 3 25 2 15 5 5 55 Wavelength (nm) 55 5 45 4 35 3 25 2 15 5 5 55 Wavelength (nm) 55 5 45 4 35 3 25 2 15 5 5 55 Wavelength (nm) Figure 3. DSN action on one mismatch-containing (A, B) and perfectly matched (C) DNA duplexes. Duplexes 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 ) were incubated with DSN at 35 O C for 15 min. Emission spectra were obtained on the spectrofluorimeter, with excitation at 48 nm. Dotted line - substrate fluorescence in the absence of enzyme; firm line - substrate fluorescence after incubation with DSN. DSN activity testing Important note: We strongly recommend to perform this procedure before enzyme use 1. Combine the following reagents in the order shown: 12 µl Sterile water (not included) 4 µl DSN control template 2 µl 1XDSN master buffer 18 µl Total Volume 2. Mix contents and spin the tube briefly in a microcentrifuge. 3. Aliquot 9 µl of the reaction mixture into each of the two sterile PCR tubes labeled C (control) and E (experimental). 4. Add 1 µl of DSN storage buffer into C-tube. Mix contents and spin the tube briefly in a microcentrifuge. 5

6 5. Add 1 µl of DSN solution into the E-tube. Mix contents by gently flicking the tube. Spin the tube briefly in a microcentrifuge. 6. Overlay the reaction mixture in each tube with drop of mineral oil and spin the tubes briefly in a microcentrifuge. 7. Incubate the tubes in a thermal cycler at 65 O C for 1 min. 8. To inactivate DSN enzyme, add 5 µl of 2X DSN stop solution, mix contents and spin the tube briefly in a microcentrifuge. Place the tubes at room temperature. 9. Electrophorese 5 µl of each reaction mixture alongside.1 µg of 1-kb DNA size markers on a 1.5% agarose/etbr gel in 1X TAE buffer. Using electrophoresis data, estimate condition of your DSN enzyme. For comparison, Figure 4 shows the typical gel profile of "DSN control template" digested by DSN with successful activity and by partially inactive DSN. Reaction conditions 1. Combine the following reagents in the order shown: Y µl 5-5 ng DNA (in sterile water) X µl Sterile water 1 µl 1X DSN master buffer 1 µl DSN solution 1 µl Total Volume 2. Mix contents gently and spin the tube briefly in a microcentrifuge. 3. If necessary, overlay the reaction mixture with a drop of mineral oil and spin the tube briefly in a microcentrifuge. 4. Incubate the tube in a thermal cycler at 65 O C for 7-2 min. Note: Incubation time depends on the complexity of the sample and particular tasks. Depending on your particular needs, incubation temperature can be changed from 35 to 7 O C (see Figure 1); however, incubation time/dsn concentration in the reaction mixture should be optimized additionally. 5. To inactivate DSN enzyme, add 5 µl of 2X DSN stop solution, mix contents and spin the tube briefly in a microcentrifuge. 6. Incubate the tube in a thermal cycler at reaction temperature for 5 min.

M 1 2 3 control unsuccessful well Figure 4. DSN activity testing. Samples containing 2 ng of plasmid DNA were incubated with or without DSN in 1x DSN master buffer for 7 min at 65 O C. Reactions were stopped by EDTA and digestion products were electrophoresed on a 1.5% agarose/etbr gel in 1X TAE buffer. Lane 1 - control DNA (incubation without DSN). Lane 2 - DNA incubated with ill- conditioned DSN enzyme. Lane 3 - successful digestion of DNA by DSN. Lane M - 1 kb DNA size marker. Typical result, indicative of successful DSN activity, should have the following characteristics: 1. Two strong DNA bands should be present in the pattern of the cdna from the C- tube (Fig. 4, lane 1). If a strong difference between the pattern of DNA obtained from the C-tube and the pattern of control cdna showed at Figure 4 (lane 1) occurs, this may indicate that some reagents used are contaminated with nuclease. 2. A low molecular weight DNA should be detected in the E - tube (as in Fig. 4, lane 3). If the pattern of the digested DNA from the E- tube looks like smears of various intensities with or without clear bands (see for example Fig. 4, lane 2), your DSN enzyme is fully or partially inactive. 7

References 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. (22) 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. (24) Simple cdna normalization using kamchatka crab duplex-specific nuclease. Nucleic Acid Res., 32: e37. 3. Zhulidov P.A., Bogdanova E.A., Shcheglov A.S., Shagina I.A., Wagner L.L., Khaspekov G.L., Kozhemyako V.B., Lukyanov S.A., Shagin D.A. (25) A method for the preparation of normalized cdna libraries enriched with full-length sequences. Russian Journal of Bioorganic Chemistry, 31: 17-177. 8 Notice to Purchaser Evrogen DSN Products (the Products) are available to Purchasers for noncommercial non-for-profit research use. For commercial use of the Products please contact Evrogen at license@evrogen.com for license information.