Enzymes for Molecular Biology: Enzymes for Molecular Biology

Ampligase® Thermostable DNA Ligase catalyses NAD-dependent ligation of adjacent 3"-hydroxylated and 5"-phosphorylated termini in duplex DNA structures that are stable at high temperatures. Derived from a thermophilic bacterium, the enzyme is stable and active at much higher temperatures than conventional DNA ligases. Its half-life is 48 hours at 65°C and greater than 1hour at 95°C.

Ampligase® DNA Ligase has been shown to be active for at least 500 thermal cycles (94°C/80°C) or 16 hours of cycling. This exceptional thermostability permits extremely high hybridisation stringency and ligation specificity. Ampligase® DNA Ligase has no detectable activity in ligating blunt-ended DNA and has no activity on RNA or RNA:DNA hybrids.

Unit Definition:

One unit of Ampligase® DNA Ligase catalyses the ligation of 50% of the cos sites in 1µg of lambda DNA in 1 minute at 45°C in 1X Ampligase® Reaction Buffer.

Note:

One unit of Ampligase® DNA Ligase is equivalent to at least 15 of the "cohesive end units" or "nick ligation units" defined elsewhere.

Storage Buffer:

50% glycerol containing 50mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1mM EDTA, 1mM DTT, and 0.1% Triton® X-100.

Ampligase® 10X Reaction Buffer:

200 mM Tris-HCl (pH 8.3), 250mM KCl, 100mM MgCl2, 5mM NAD, and 0.1% Triton® X-100.

Quality Control:

Ampligase® Thermostable DNA Ligase is assayed for the absence of blunt-end ligation activity by ligating SmalI-digested bacteriophage lambda DNA at 45°C; ligation occurs only at cos sites as determined by gel electrophoresis. Ampligase® DNA Ligase is free of detectable exo- and endonuclease and RNase activities.

Figure 1. Schematic of mutation discovery and screening using ligation amplification. The existence of a point mutation at the site of ligation interferes with oligonucleotide ligation, resulting in no ligation product. The lack of an amplification product indicates the presence of a point mutation at the ligation site. Oligos can also be designed so ligation occurs in the presence of the mutant template

2. Ligation Amplification using Ampligase® DNA Ligase. Oligonucleotides A, B, C, and D from Figure 1 (25 pmoles each) were incubated as in Figure 1 with 5 units of Ampligase DNA Ligase in 25µl of 1 X Ampligase Reaction Buffer, with ( ) or without (-) 0.25 pmole DNA template. The reactions were cycled 40 times for 1 minute at 94°C and 2 minutes at 65°C, then separated by electrophoresis and visualized with ethidium bromide. In the presence of template DNA, the target sequence is amplified by formation of the ligation products A B and C D.

Note: Some applications in which this product may be used may be covered by patents or patent applications applicable in certain countries. Because purchase of this product does not include a license to perform any patented application, users of this product may be required to obtain a license depending upon the particular application and country in which the product is used.

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200

Protocols for: Ampligase® Range

Due to the constant updating of the protocols by the manufacturer we have provided a direct link to Lucigen’s product page, where the latest protocol is available.

Please note this will open a new page or window on your computer.

Ampligase® Thermostable DNA Ligase Protocol

(catalogue number A8101 / A30201 / A0110K / A0125K / A3210K / A3225K / A0102K / A32250 / A32750 / A3202K / A1905B / A3201S)

Please note: all protocols off site are the responsibility of the products supplier

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200

References

1. Schalling, M. et al. (1993) Nature Genetics 4, 135.

1. Landegren, U. et al. (1988) Science 242, 229.

1. Wu, D.Y. and Wallace, R.B. (1989) Genomics 4, 560.

1. Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88, 189.

1. Birkenmeyer, L. and Armstrong, A.S. (1992) J. Clin. Micro. 30, 3089.

1. Dille, B.J. et al. (1993) J. Clin. Micro. 31, 729.

1. Nakazawa, H. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 360.

1. Sirugo, G. and Kidd, K.K. (1995) EPICENTRE Forum 2 (3), 1.

1. Moore, D.S. and Michael, S.F. (1995) EPICENTRE Forum 2 (4), 4.

1. Rouwendal, G.J. et al. (1993) BioTechniques 15, 68.

1. Hardenbol, P. et al. (2003) Nature Biotechnology 21, 673.

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200

Note: Some applications in which this product may be used may be covered by patents or patent applications applicable in certain countries. Because purchase of this product does not include a license to perform any patented application, users of this product may be required to obtain a license depending upon the particular application and country in which the product is used.

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200

Applications

• Ligation amplification (Ligase Chain Reaction, LCR)^1-7
• Repeat Expansion Detection (RED).⁷
• Simultaneous mutagenesis of multiple sites.^8,9
• Other ligation-based detection methods.

Benefits

• High thermostability allows ligation using high-stringency hybridization conditions.
• High specificity and stringency permits sensitive detection of SNPs.¹¹

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200