Ampligase® Thermostable DNA Ligase

• 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.

Ampligase® Thermostable DNA Ligase catalyzes 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 1 hour 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.¹⁰ This exceptional thermostability permits extremely high hybridization 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.

Benefits

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

Figure 1 (click to enlarge). 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.

Figure 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 25 µl of 1X 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.

Unit Definition: One unit of Ampligase DNA Ligase catalyzes the ligation of 50% of the cos sites in 1 µg 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.⁴

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

Ampligase 10X Reaction Buffer: 200 mM Tris-HCl (pH 8.3), 250 mM KCl, 100 mM MgCl₂, 5 mM 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 Sma I-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.

References

1. Landegren, U. et al. (1988) Science 242, 229.
2. Wu, D.Y. and Wallace, R.B. (1989) Genomics 4, 560.
3. Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88, 189.
4. Birkenmeyer, L. and Armstrong, A.S. (1992) J. Clin. Micro. 30, 3089.
5. Dille, B.J. et al. (1993) J. Clin. Micro. 31, 729
6. Nakazawa, H. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 360.
7. Sirugo, G. and Kidd, K. (1995) Epicentre Forum 2(3), 1.
8. Moore, D. and Michael, S. (1995) Epicentre Forum 2(4), 4.
9. Rouwendal, G.J. et al. (1993) BioTechniques 15, 68.
10. Schalling, M. et al. (1993) Nature Genetics 4, 135.
11. Hardenbol, P. et al. (2003) Nature Biotechnology 21, 673.