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Ampligase® Thermostable DNA Ligase催化NAD-依赖的双链DNA的3'-羟基化和5'-磷酸化末端的连接反应，对热稳定。该酶来源于嗜热细菌，与传统DNA连接酶相比，该酶具有更高的活性和稳定性。65°C时该酶半衰期为48小时，95°C时在1小时以上。
Ampligase DNA Ligase在500个以上热循环后（16个小时循环）.10 这种高稳定性保证了极高的杂交严谨性和连接特异性。Ampligase DNA Ligase无平端DNA连接活性，无RNA或RNA:DNA杂交体连接活性。
优点
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高热稳定性，保证可以使用更严谨的杂交温度。
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高特异性和严谨性能够敏感检测SNPs.11
活力单位定义: 1个活力单位的Ampligase DNA Ligase指在1X Ampligase Reaction缓冲液中，45°C条件下1分钟能够催化1 µg的lambda DNA中的50% cos位点发生连接。
Note:一个单位的Ampligase DNA Ligase等价于至少15个别处定义的 "cohesive end units"或 "nick ligation units"。4
储存缓冲液: 50%甘油含有50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT和0.1% Triton® X-100。
Ampligase 10X反应缓冲液: 200 mM Tris-HCl (pH 8.3), 250 mM KCl, 100 mM MgCl2, 5 mM NAD和0.1% Triton® X-100。
质量控制: Ampligase Thermostable DNA Ligase的活性通过在45°C条件下连接Sma I消化后产生的平端噬菌体DNA进行检测；电泳检测连接只发生在cos位点。Ampligase DNA Ligase不含DNA外切、内切核酸酶和RNA酶活性。
References
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Landegren, U. et al. (1988) Science242, 229.
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Wu, D.Y. and Wallace, R.B. (1989) Genomics 4, 560.
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Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88, 189.
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Birkenmeyer, L. and Armstrong, A.S. (1992) J. Clin. Micro. 30, 3089.
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Dille, B.J. et al. (1993) J. Clin. Micro.31, 729
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Nakazawa, H. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 360.
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Sirugo, G. and Kidd, K. (1995) Epicentre Forum2(3), 1.
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Moore, D. and Michael, S. (1995) Epicentre Forum2(4), 4.
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Rouwendal, G.J. et al. (1993) BioTechniques15, 68.
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Schalling, M. et al. (1993) Nature Genetics4, 135.
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Hardenbol, P. et al. (2003) Nature Biotechnology21, 673.
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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 ?l 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.
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