Duplex-specific nucleases


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Background

The rTDSN and rZDSN degrade double-stranded DNA and DNA in DNA-RNA hybrid duplex with high selectivity, while display little cleaving activities to single-stranded DNA. The rTDSN and rZDSN are the thermostable enzymes with the optimum temperature around 65°C (maximum activity at 67°C) and 60°C, respectively. The rTDSN and rZDSN are tolerant to proteinase K digestion.

Application

The duplex-specific nuclease activity is applicable to various purposes, for example, methods for SNP analysis [1], cDNA normalization [2], subtraction [3], and quantitative telomeric overhang determination [4].

rTDSN (recombinant Taraba crab duplex-specific nuclease)

Optimum Temperature: 67°C

CSR_BTN102_1
Figure 1. The rTDSN exhibit a strong preference for double double-stranded DNA substrate
In the case of the rTDSN-added reaction mixture (+) the double-stranded DNA substrate λ DNA (void arrow) alone was degraded, while the single-stranded DNA substrate M13 mp18 single strand DNA (black arrow) was not degraded. On the other hand, when the dilution buffer was added instead of the enzyme (-) both of the substrate were not degraded. The rTDSN hardly acts on the single-stranded DNA, it specifically degrades the double-stranded DNA alone.

CSR_BTN102_2 CSR_BTN102_3
Figure 2. Temperature dependence of rTDSN activity.
DNase activities toward dsDNA were measured by modified Kunitz assay at various temperatures. Measured activity (U/ml) and relative activity (%) at each temperature were shown. The rTDSN showed high activity within the range of from about 20 Ž to 70 Ž, its optimum activity temperature was about 67 Ž, and it showed an activity of 70% or more of the maximum activity at from about 60 Ž to 70 Ž.
Figure 3. Kinetics of thermal inactivation of rTDSN.
After heat treatment of rTDSN at indicated temperatures, the relative activities of the rTDSN were determined. The DNase activities toward dsDNA were measured at 25Ž. The rTDSN retained more than 90% activity after incubation at 50 Ž or 60 Ž for 30 minutes. The activity of the enzyme treated at 70 Ž decreased to be about 60% after a lapse of time of 30 minutes. The activity of the enzyme treated at 80 Ž disappeared after 5 minutes.

rZDSN (recombinant Zuwai crab duplex-specific nuclease)

Optimum Temperature: 60°C

CSR_BZN102_1
Figure 1
(a) The rZDSN exhibit a strong preference for double double-stranded DNA substrate
In the case of the rZDSN-added reaction mixture (+) the double-stranded DNA substrate λ DNA (void arrow) alone was degraded, while the single-stranded DNA substrate M13 mp18 single strand DNA (black arrow) was not degraded. On the other hand, when the dilution buffer was added instead of the enzyme (-) both of the substrate were not degraded. The rZDSN hardly acts on the single-stranded DNA, it specifically degrades the double-stranded DNA alone.
(b) Time course of digestion of single-stranded DNA and double-stranded DNA
The reaction mixture was incubated at 60 Ž for 0, 3, 10 or 30 minutes. The double-stranded DNA substrate λ DNA (void arrow) alone was degraded with incubation time, while the single-stranded DNA substrate M13 mp18 single strand DNA (black arrow) was not degraded even after 30 minutes.

CSR_BZN102_2 CSR_BZN102_3
Figure 2. Temperature dependence of rZDSN activity.
DNase activities toward dsDNA were measured by modified Kunitz assay at various temperatures. Measured activity (U/ml) and relative activity (%) at each temperature were shown. The rZDSN showed high activity within the range of from about 20 Ž to 63 Ž, its optimum activity temperature was about 60 Ž, and it showed an activity of 70% or more of the maximum activity at from about 55 Ž to 63 Ž.
Figure 3. Kinetics of thermal inactivation of rZDSN.
After heat treatment of rZDSN at indicated temperatures, the relative activities of the rZDSN were determined. The DNase activities toward dsDNA were measured at 25Ž. The rZDSN retained more than 80% activity after incubation at 50 Ž or 60 Ž for 30 minutes. The rZDSN retained about 60% activity after incubation at 63 Ž for 30 min. The activity of the enzyme treated at 70 Ž decreased to be about 40% of before the heat treatment after the lapse of time of 15 minutes and decreased to be about 10% after the lapse of time of 30 minutes. The activity of the enzyme treated at 80 Ž disappeared after 5 minutes.
References

1) Shagin DA, et al., Genome Res, 12(12), 1935-1942 (2002).

2) Zhulidov PA, et al., Nucleic Acids Res, 32(3), e37 (2004).

3) Peng RH, et al., Anal Biochem, 372(2), 148-155 (2008).

4) Zhao Y, et al., Nucleic Acids Res, 36(3), e14 (2008).

5) Hirakawa Y, Ohiso I, Expression of duplex-specific nuclease derived from
Paralithodes camtschaticus by insect cells-baculovirus system. The 11th
Annual Meeting of Japanese Society for Marine Biotechnology (2008).

6) Hirakawa Y, Ohiso I, Establishment of production strategy of recombinant
red king crab duplex-specific nuclease using baculovirus expression system.
Joint Annual Meeting of the Molecular Biology Society of Japan and the
Japanese Biochemical Society, 2008 (BMB2008).

7) Hirakawa Y, Ohiso I, Cloning, expression, and characterization of
thermostable duplex-specific nuclease from snow crab (Chionoecetes opilio).
The 32nd Annual Meeting of the Molecular Biology of Japan (MBSJ2009).

Product List

Note : Product "CSR-BTN102" "CSR-BTN103" cannot be sold to USA because of the patent issue.


Product Name Cat# Quantity Price

rTDSN (Taraba crab duplex-specific nuclease) DataSheet ,

CSR-BTN102

50 UNIT N/A

rTDSN (Taraba crab duplex-specific nuclease) DataSheet ,

CSR-BTN103

100 UNIT N/A

rZDSN (Zuwai crab duplex-specific nuclease) DataSheet ,

CSR-BZN102

50 UNIT N/A

rZDSN (Zuwai crab duplex-specific nuclease) DataSheet ,

CSR-BZN103

100 UNIT N/A

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.



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