来自栖热菌的Tsc DNA连接酶 Tsc DNA ligase from Thermus scotoductus

 

Thermostable Tsc DNA ligase and Rma DNA ligase (Dlig120) have shown to have favorable properties in comparison to other DNA ligases. For reference please consider the following papers:

 

Housby & Southern 2002: Thermus scotoductus and Rhodothermus marinus DNA ligases have higher ligation efficiencies than Thermus thermophilus DNA ligase. Anal. Biochem. 302:88-94 – link to article

 

Housby JN et al. 2000. Optimised ligation of oligonucleotides by thermal ligases: comparison of Thermus scotoductus andRhodothermus marinus DNA ligases to other thermophilic ligases. Nucl. Acid Res. 28:e10 – link to article

 

Thorbjarnardottir SH et  et al. 1995: Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases. Gene 161:1-6 –  link to article

 

Jonsson Zo et al . 1994: Sequence of the DNA ligase-encoding gene from Thermus scotoductus and conserved motifs in DNA ligases. Gene 151: 177-180 – link to article

 

 

 

Product Dlig119 Tsc DNA ligase is available as 500 units and 2000 units together with 10x reaction buffers.

 

– Shipping within a week, shipping and handling charges additional to price listed below.

 

 

For further information read below or download the product sheet

 

 

 

 

 

Product description

 

Introduction:

 

Tsc DNA ligase catalyzes the NAD-dependent ligation of adjacent 3´-hydroxyl and 5´-phosphate termini in duplex DNA structures. In contrast to T4 DNA ligase, Tsc DNA ligase has no detectable activity on blunt end DNA fragments. Unlike T4 DNA ligase, Tsc DNA ligase shows only minimal ligation activity under optimal temperature conditions for 4 bp as well as 2 bp of cohesive ends. Tsc DNA ligase has no activity on RNA targets. Tsc DNA ligase is isolated and purified from an E.coli strain carrying a plasmid with the cloned DNA ligase gene from the thermophilic bacteria Thermus scotoduc-tus isolated in Iceland (1, 2). The half-life of Tsc ligase is 26 min at 91°C (3). The enzyme has a broad range of reaction temperatures with the lower limit around 15°C and the upper limit determined by the melting temperature (Tm) of the DNA substrate. The enzyme is also active in various DNA polymerase buffers within the pH range of 7-9. Under optimal conditions the rate and extent of oligonucleotide ligation is much higher for Tsc DNA ligase compared to other commonly available thermostable ligases (4,5).

 

Applications:

 

Tsc DNA ligase is an ideal enzyme for applications re-quiring high temperature, high-stringency ligations of double-stranded DNA.Tsc DNA ligase may be applied to:

 

 

 

 

•Ligase Chain Reaction (LCR) (6-8) for amplification of DNA targets.

•Oligonucleotide Ligation Assay (OLA) (9-10) for mutational analysis.

•Repeat Expansion Detection (11) for determining genetic anomalies, such as trinucleotide repeats, research only, not for diagnostic purposes (for e.g. Fragile X, Huntington disease).

•Gene Synthesis (12) from overlapping oligonucleo-tides.

 

Storage:

 Storage and dilution buffer: 20 mM Tris-HCl, 50 mM KCl, 0,1 mM EDTA, 0,1% Triton X-100 (v/v), 1 mM dithiothreitol (DTT), 50% glycerol (v/v), pH 7,6 (25°C). Tsc Ligase is stable when stored at –15°C to -25°C.

 

Reaction conditions:

 1 x reaction buffer (10 x supplied) 20 mM Tris-HCl, 20 mM KCl, 10 mM MgCl2, 0,1% Nonidet P40 (v/v), 0,5 mM NAD, 1 mM DTT, pH 7,5 (25°C).

 

Concentration and unit definition

 Concentration 10 U/μl. One unit of Tsc DNA Ligase catalyzes the ligation of 50% of the cos sites of 1μg BstEII digested λDNA in 1 min at 45°C.

 

 

 Application Protocol:

 

Reaction protocol for oligonucleotide ligation:

 Thaw the components listed below and place them on ice. Vortex briefly and centrifuge all reagents before setting up the reactions. Set up the reaction components in a microfuge tube placed on ice:

 

Component Volume Final conc. 

Reaction buffer (10x) 2,0 μl 1 x 

Oligo 1 X μl 1-30nM 

Oligo 2 X μl 1-30nM 

Template DNA X μl 0,1 ng 

Tsc DNA Ligase 0,5 μl 5 U 

Add sterile H2O Up to 20,0 μl  

TOTAL 20,0 μl  

 

A typical temperature profile is: 94°C 2 min, 94°C 30 sec, 45-65°C 3 min and repeat last two temperatures for 30 cycles. 99°C for 10 min.

 

Activity assay:

 The enzyme assay for unit definition was ligation of cos sites of λDNA digested with BstII.

 

Component Volume Final conc. 

Reaction buffer (10x) 2,0 μl 1 x 

λDNA (BstEII digested) X μl 1 μg 

Tsc DNA Ligase Dilution serie  

Add sterile H2O Up to 20,0 μl  

TOTAL 20,0 μl  

 

Incubate at 45°C for 1-15 min. Stop reaction in dry ice/ethanol bath. Incubate for 10 min at 65°C before analysis on agarose gel (melting of not ligated cos sites)

 

Results are assayed by agarose gel electrophoresis and ethidium bromide staining.

 

Quality Control:

 

 Quality Control:

 Each lot of ThermoPhageTM ssDNA ligase is assayed for activity and for contaminating activities as stated be-low.

 

Absence of DNA endonuclease:

•0,25 μg supercoiled pBR322 DNA is incubated with increasing amounts of Tsc DNA ligase in 25 μl reactions at 37°C for 16 h. >100 U of Tsc DNA ligase show no relaxation of the supercoiled structure of pBR322 DNA.

•0,25 μg of λ-DNA Eco RI/HindIII fragments is incu-bated with Tsc DNA ligase in 25 μl reactions at 37°C and 64°C for 16 h. 100 U of Tsc DNA ligase show no alteration of the banding pattern.

 

Absence of exonuclease:

 Increasing amounts of Tsc DNA ligase are incubated in 50 μl test buffer containing [3H]-labelled DNA at 37°C and 64°C for 4 h. The amount of enzyme, which shows no exonuclease activity is >100 U.

 

Absence of Rnases:

 RNaseAlertTM Lab Test Kit (cat no. 1964) from Ambion was used to detect RNase activity according to the manufacturer protocol. No RNase activity was detected after incubating X U of Tsc DNA ligase at 37°C after 1 hour.

 

 

References:

•Jonsson ZO, et al. 1994. Gene 151: 177-80

•Kristjansson JK et al. 1995. System. Appl. Microbiol. 17: 44-50

•Thorbjarnardottir SH et al. 1995. Gene 161: 1-6

•Housby JN, et al. 2000. Nucleic Acids Research. 28: e10.

•Housby JN, et al. 2001. Anal. Biochem. 302: 88-94

•Landegren U, et al 1988. Science 241: 1077-1080

•Landegren U, et al 1988. Science 242: 229-237

•Barany F, 1991. PCR Meth. and Appl.. 1: 5-16

•Nickerson DA, et al. 1990. Proc. Natl. Acad. Sci. USA 87: 8923-8927

•Baron H, et al. 1996. Nature Biotech 14: 1279-1282

•Schalling M, et al. 1993. Nature Genetics 4: 135

•Sutton JR, et al. 1992. Transgenic Research 1: 228