A Rapid and Accurate Method for Tth DNA Polymerase Activity Assay

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

Abstract

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

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.

Figures/Tables 8

Fig. 1 Hairpin probe

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

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

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

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

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

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

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

References 20

[1]	Carballeira N, Nazabal M, Brito J, et al. Purification of a thermostable DNA polymerase from Thermus thermophilus HB8, useful in the polymerase chain reaction[J]. Biotechniques, 1990,9(3):276-281. pmid: 2223065
[2]	Myers TW, Gelfand DH. Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase[J]. Biochemistry, 1991,30(31):7661-7666. doi: 10.1021/bi00245a001 URL
[3]	Perez-Ruiz M, Torres C, et al. Determination of HCV RNA concentration by direct quantitation of the products from a single RT-PCR[J]. J Virol Methods, 1997,69(1-2):113-124. doi: 10.1016/S0166-0934(97)00155-9 URL
[4]	Poddar SK, Sawyer MH, Connor JD. Optimized PCR amplification of influenza A virus RNA using Tth DNA polymerase, incorporating uracil N glycosylase(UNG)in a single tube reaction[J]. J Clin Lab Anal, 1997,11(6):323-327. pmid: 9406050
[5]	Laskus T, Radkowski M, Wang LF, et al. Detection of hepatitis G virus replication sites by using highly strand-specific Tth-based reverse transcriptase PCR[J]. J Virol, 1998,72(4):3072-3075. pmid: 9525631
[6]	Katcher HL, Schwartz I. A distinctive property of Tth DNA polymerase:enzymatic amplification in the presence of phenol[J]. Biotechniques, 1994,16(1):84-92. pmid: 8136148
[7]	Abu Al-Soud W, Radstrom P. Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples[J]. Appl Environ Microbiol, 1998,64(10):3748-3753. doi: 10.1128/AEM.64.10.3748-3753.1998 URL
[8]	Poddar SK, Sawyer MH, Connor JD. Effect of inhibitors in clinical specimens on Taq and Tth DNA polymerase-based PCR amplification of influenza A virus[J]. J Med Microbiol, 1998,47(12):1131-1135. pmid: 9856650
[9]	Moreno R, Haro A, Castellanos A, et al. High-level overproduction of His-tagged Tth DNA polymerase in Thermus thermophilus[J]. Appl Environ Microbiol, 2005,71(1):591-593. doi: 10.1128/AEM.71.1.591-593.2005 URL
[10]	李华, 杨玲玲, 杜红旗. 重组Tth DNA聚合酶基因的克隆表达及活性鉴定[J]. 河南大学学报:自然科学版, 2019,49(3):311-317.
	Li H, Yang LL, Du HQ. Cloning expression and activity assaying of recombinant Tth DNA polymerase gene[J]. Journal of Henan University:Natural Science, 2019,49(3):311-317.
[11]	Terpe K. Overview of thermostable DNA polymerases for classical PCR applications:from molecular and biochemical fundamentals to commercial systems[J]. Appl Microbiol Biotechnol, 2013,97(24):10243-10254. doi: 10.1007/s00253-013-5290-2 URL
[12]	El-Deiry WS, Downey KM, So AG. Molecular mechanisms of manganese mutagenesis[J]. Proc Natl Acad Sci U S A, 1984,81(23):7378-7382. pmid: 6095289
[13]	Cai D, Behrmann O, Hufert F, et al. Direct DNA and RNA detection from large volumes of whole human blood[J]. Sci Rep, 2018,8(1):3410. doi: 10.1038/s41598-018-21224-0 URL
[14]	Tang F, Liu S, Li QY, et al. Location analysis of 8-oxo-7, 8-dihydroguanine in DNA by polymerase-mediated differential coding[J]. Chem Sci, 2019,10(15):4272-4281. doi: 10.1039/C8SC04946G URL
[15]	Aye SL, Fujiwara K, et al. Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication[J]. Biochem Biophys Res Commun, 2018,499(2):170-176. doi: 10.1016/j.bbrc.2018.03.098 URL
[16]	全国工具酶标准化工作组. GB/T 35542-2017, Taq DNA聚合酶[S]. 北京: 中国标准出版社, 2017.
	National Technical Committee of Standardization for Research Use Enzyme. GB/T 35542-2017, Taq DNA Polymerase[S]. Beijing: Standards Press of China, 2017.
[17]	Yu L, Hu G, Howells L. Fluorescence-based, high-throughput DNA polymerase assay[J]. Biotechniques, 2002,33(4):938-941. doi: 10.2144/02334pf02 URL
[18]	Ma C, Tang Z, Wang K, et al. Real-time monitoring of DNA polymerase activity using molecular beacon[J]. Anal Biochem, 2006,353(1):141-143. doi: 10.1016/j.ab.2006.02.006 URL
[19]	Dorjsuren D, Wilson DM 3rd, et al. A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases[J]. Nucleic Acids Res, 2009,37(19):e128. doi: 10.1093/nar/gkp641 URL
[20]	Seville M, West AB, Cull MG, et al. Fluorometric assay for DNA polymerases and reverse transcriptase[J]. Biotechniques, 1996,21(4):664-672. doi: 10.2144/96214st04 URL