一种快速精确测定Tth DNA聚合酶活性的方法

doi:10.13560/j.cnki.biotech.bull.1985.2020-0953

摘要/Abstract

摘要：

Tth DNA聚合酶是一种同时具备DNA聚合酶和逆转录酶活性的耐热型工具酶,但在生产和改造过程中,缺乏准确、快速、简便测定酶活的方法。利用高灵敏度双链特异性核酸染料PicoGreen和特殊设计的发夹型探针,建立了一种测定Tth DNA聚合酶活性的新方法。该方法的标准曲线线性关系良好,决定系数R²达到0.99以上,灵敏度和准确度高,操作简单,测定结果与商业化产品标定的活力相符,经荧光定量PCR验证能够反映真实使用环境下的酶活。该方法还可推广到测定其他DNA聚合酶活性,将为DNA聚合酶的生产和改造提供有效的检测手段。

关键词: Tth DNA聚合酶, 酶活性测定, 荧光染料, 发夹型探针

Abstract:

Tth DNA polymerase is a thermostable enzyme with both DNA polymerase and reverse transcriptase activities. Traditional methods for activity assay are discontinuous,time consuming,and laborious. We developed a new method for activity assay of Tth DNA polymerase using a highly sensitive double-stranded nucleic acid specific dye PicoGreen,and a specially designed hairpin probe. A good linearity of the standard curve can be established by this method,with the coefficient of determination R² > 0.99. This method was of high sensitivity and accuracy,and the results by it were consistent with the activity of commercial products. The verification from fluorescence quantitative PCR confirmed that this method may reveal the enzyme activity in real environment. The method can also be extended to other DNA polymerase activity assay,which will provide an effective tool for the production and transformation of DNA polymerase.

Key words: Tth DNA polymerase, enzyme activity assay, fluorescence dye, hairpin probe

陈晓雨, 张建, 张新亚, 唐雨婷, 邵钰晨, 罗志丹, 卢辰. 一种快速精确测定Tth DNA聚合酶活性的方法[J]. 生物技术通报, 2021, 37(5): 281-286.

CHEN Xiao-yu, ZHANG Jian, ZHANG Xin-ya, TANG Yu-ting, SHAO Yu-chen, LUO Zhi-dan, LU Chen. A Rapid and Accurate Method for Tth DNA Polymerase Activity Assay[J]. Biotechnology Bulletin, 2021, 37(5): 281-286.

图/表 8

图1 发夹型探针示意图

Fig. 1 Hairpin probe

表1 标准曲线探针工作溶液配方

Table 1 Probe working solution formulations for standard curve

序号 No.	H1/μL	H2/μL	PicoGreen/μL	TE缓冲液 TE Buffer/μL
1	10	0	0.5	89.5
2	8	2	0.5	89.5
3	6	4	0.5	89.5
4	4	6	0.5	89.5
5	2	8	0.5	89.5
6	0	10	0.5	89.5

表2 Tth DNA聚合酶活性检测反应体系

Table 2 Reaction system for Tth DNA polymerase activity assay

组分Group	体积Volume/μL
Tth酶稀释液Tth dilution	1
10× PCR buffer for Tth	2
H1底物溶液 H1 substrate solution	10
dNTP（每种2.5 mmol/L） dNTP（25 mmol/L each）	1
ddH₂O	加至20

图2 荧光增加值与H2含量的标准曲线

Fig. 2 Standard curve of fluorescence intensity added and H2 content

图3 不同稀释倍数的rTth聚合酶在不同反应时间下的荧光增加值

Fig. 3 Fluorescence intensity added of rTth polymerase with different dilution ratios at different reaction times

表3 商业化Tth DNA聚合酶活性测试结果

Table 3 Results of commercial Tth DNA polymerase activity assay

稀释倍数 Dilution	荧光增加值 Added fluorescence intensity	H2生成量 H2 generated/pmol	稀释液酶活 Diluted enzyme activity/（U·μL^-1）	原液酶活 Original enzyme activity/（U·μL^-1）
2000	11898	1.10	2.74 × 10^-3	5.48
4000	5905	0.53	1.33 × 10^-3	5.31

图4 重组表达Tth野生型与突变体蛋白的酶活测定与荧光定量PCR验证 A：不同稀释倍数的野生型Tth蛋白在不同反应时间下的荧光增加值;B：不同稀释倍数的I640F突变体蛋白在不同反应时间下的荧光增加值;C：使用商业化rTth、野生型Tth和I640F突变体（均为原液）对相同模板进行荧光定量PCR的扩增曲线

Fig. 4 Activity assay and fluorescence quantitative PCR validation of recombinant WT Tth and its mutant polymerase A: Fluorescence intensity added of wild-type Tth protein at different dilutions for different reaction times. B: Fluorescence intensity added of I640F mutant at different dilutions for different reaction times. C: Amplification curves of fluorescent quantitative PCR using original solutions of commercial rTth, wild-type Tth and I640F mutant against the same template

表4 测得酶活与荧光定量PCR Ct值对比

Table 4 Comparison of measured enzyme activity and fluorescence quantitative PCR Ct values

	测得酶活 Measured enzyme activity/（U·μL^-1）	Ct值 Ct value
rTth	5.60	13.40 ± 0.15
野生型WT	0.27	17.68 ± 0.16
I640F	0.53	16.69 ± 0.13

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