隐–荧光DNA扩增
Amplification of Hidden Fluorescent DNA
DOI: 10.12677/AMB.2023.121006, PDF,    国家自然科学基金支持
作者: 刘自厚：河南农业大学国际教育学院，河南 郑州；刘亮伟：河南农业大学生命科学学院，河南 郑州；农业部农业酶工程重点实验室，河南 郑州
关键词: 隐–荧光DNA；扩增；荧光检测；Hidden Fluorescent DNA； Amplification； Fluorescence Assay
摘要: 目的：隐–荧光(Hf: hidden fluorescent) DNA通过带荧光素及10 nt之内淬灭基团的Hf引物扩增。方法：本研究设计Hf_Pf引物带有dT12_Fam荧光素-dT16_BHQ1淬灭基团，扩增5851 bp HfDNA pET28a-xylanase，探讨Hf_Pf引物Tm (退火温度)适合计算器、HfDNA扩增条件、T5 DNA酶(T5exo)切割产物检测、及T5exo酶切动力学。结果：Hf_Pf Tm计算器为Oligo、IDT，经非等量引物PCR优化扩增HfDNA，激光共聚焦定性检测HfDNA酶切产物Fam荧光、酶标仪定量检测荧光值19,683 a.u。根据HfDNA浓度–荧光值方程，T5exo酶促动力学参数Km 0.1 nM，弥补解离常数KD不足。结论：本研究扩增隐–荧光HfDNA，提供了DNA酶学研究新材料。
Abstract: Objective: Hidden fluorescent (Hf) DNA substrates can be amplified by Hf primer that has a fluorescein and a quencher within 10 nt. Method: The study designed a 20 nt Hf_Pf having the dT12_Fam and the dT16_BHQ1, amplified a 5851 bp HfDNA pET28a-xylanase, determined suitable analyzers for calculating primer Hf_Pf Tm (melting temperature), optimized PCR to amplify the HfDNA substrates, assayed HfDNA-T5exo digestion product fluorescence. Result: Suitable analyzers were Oligo and IDT, and un-equal PCR was optimized to amplify the HfDNA substrates. Instead of the HfDNA substrates, the HfDNA-T5exo digestion products exhibited dT12_Fam fluorescence in quality under a laser scanning confocal microcopy and a 19,683 a.u fluorescence intensity in quantity under a microreader. According to HfDNA concentration-fluorescence intensity function, T5exo kinetics was determined to have a 0.1 nM Km, fulfilling in-adequate of dissociation constant KD. Conclusion: The study amplified HfDNA substrates, provided a new material for assaying DNase enzyme properties.
文章引用：刘自厚, 刘亮伟. 隐–荧光DNA扩增[J]. 微生物前沿, 2023, 12(1): 45-54. https://doi.org/10.12677/AMB.2023.121006

参考文献

[1] Sayers, J.R. and Eckstein, F. (1990) Properties of Overexpressed Phage T5 D15 Exonuclease. Similarities with Escherichia coli DNA polymerase I 5’-3’ Exonuclease. The Journal of Biological Chemistry, 265, 18311-18317. [Google Scholar] [CrossRef] 
[2] Ma, X., Hong, Y., Han, W., et al. (2011) Single-Stranded DNA Binding Activity of XPBI, but Not XPBII, from Sulfolobus tokodaii Causes Double-Stranded DNA Melting. Extremophiles, 15, 67-76. [Google Scholar] [CrossRef] [PubMed]
[3] Joseph, J.W. and Kolodner, R. (1983) Exonuclease VIII of Escherichia coli. II. Mechanism of Action. The Journal of Biological Chemistry, 258, 10418-10424. [Google Scholar] [CrossRef] 
[4] Zhang, J., Xing, X., Herr, A.B. and Bell, C.E. (2009) Crystal Structure of E. coli RecE Protein Reveals a Toroidal Tetramer for Processing Double-Stranded DNA Breaks. Structure, 17, 690-702. [Google Scholar] [CrossRef] [PubMed]
[5] Richardson, C.C. (1966) The 5’-Terminal Nucleotides of T7 Bacteriophage Deoxyribonucleic Acid. Journal of Molecular Biology, 15, 49-61. [Google Scholar] [CrossRef] 
[6] Richardson, C.C., Inman, R.B. and Kornberg, A. (1964) Enzymatic Synthesis of Deoxyribonucleic Acid: XVIII. The Repair of Partially Single-Stranded DNA Templates by DNA Polymerase. Journal of Molecular Biology, 9, 46-69. [Google Scholar] [CrossRef] 
[7] Tolun, G. and Myers, R.S. (2003) A Real-Time DNase Assay (ReDA) Based on PicoGreen Fluorescence. Nucleic Acids Research, 31, e111. [Google Scholar] [CrossRef] [PubMed]
[8] Druml, B., Kaltenbrunner, M., Hochegger, R. and Cichna-Markl, M. (2016) A Novel Reference Real-Time PCR Assay for the Relative Quantification of (Game) Meat Species in Raw and Heat-Processed Food. Food Control, 70, 392-400. [Google Scholar] [CrossRef] 
[9] Carr, A.C. and Moore, S.D. (2012) Robust Quantification of Polymerase Chain Reactions Using Global Fitting. PLOS ONE, 7, e37640. [Google Scholar] [CrossRef] [PubMed]
[10] Kubista, M., Andrade, J.M., Bengtsson, M., et al. (2006) The Real-Time Polymerase Chain Reaction. Molecular Aspects of Medicine, 27, 95-125. [Google Scholar] [CrossRef] [PubMed]
[11] 马纪, 黄国霞, 姚运乾, 等. 光谱法结合凝胶电泳和荧光显微镜研究萘与DNA的相互作用及其应用[J]. 分析科学学报, 2022, 38(5): 582-590.
[12] Wang, Z., Ouyang, H., Tesauro, C., et al. (2018) Real-Time Analysis of Cleavage and Religation Activity of Human Topoisomerase 1 Based on Ternary Fluorescence Resonance Energy Transfer DNA Substrate. Archives of Biochemistry and Biophysics, 643, 1-6. [Google Scholar] [CrossRef] [PubMed]
[13] Kristoffersen, E.L., Jorgensen, L.A., Franch, O., et al. (2015) Real-Time Investigation of Human Topoisomerase I Reaction Kinetics Using an Optical Sensor: A Fast Method for Drug Screening and Determination of Active Enzyme Concentrations. Nanoscale, 7, 9825-9834. [Google Scholar] [CrossRef] 
[14] 柳苏月, 田晶晶, 朱龙佼, 等. 核酸外切酶Ⅲ辅助的DNA铽离子配合物时间分辨荧光检测黄曲霉毒素B1 [J]. 分析化学, 2021, 49(8): 1327-1334.
[15] Marcussen, L.B., Jepsen, M.L., Kristoffersen, E.L., et al. (2013) DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I. Sensors, 13, 4017-4028. [Google Scholar] [CrossRef] [PubMed]
[16] Zhao, B., Tong, Z.X., Zhao, G.J., et al. (2014) Effects of 2’-O-Methyl Nucleotide on Ligation Capability of T4 DNA Ligase. Acta Biochimica et Biophysica Sinica, 46, 727-737. [Google Scholar] [CrossRef] [PubMed]
[17] Li, X., Jin, J., Wang, M., et al. (2022) Abortive Ligation Intermediate Blocks Seamless Repair of Double-Stranded Breaks. International Journal of Biological Macromolecules, 209, 1498-1503. [Google Scholar] [CrossRef] [PubMed]
[18] Saiki, R., Gelfand, D., Stoffel, S., et al. (1988) Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase. Science, 239, 487-491. [Google Scholar] [CrossRef] [PubMed]
[19] Yang, A., Cheng, J., Liu, M., Shangguan, Y. and Liu, L. (2018) Sandwich Fusion of CBM9_2 to Enhance Xylanase Thermostability and Activity. International Journal of Biological Macromolecules, 117, 586-591. [Google Scholar] [CrossRef] [PubMed]
[20] 黄亚威, 杨昂, 上官云杰, 等. 双退火温度PCR扩增DNA [J]. 微生物学报, 2017, 57(8): 1262-1269.
[21] 邵玉强, 金家铖, 李雪刚, 刘亮伟. 大量-高纯-高浓度DNA的制备[J]. 微生物前沿, 2022, 11(2): 67-74.
[22] 上官云杰, 梁亚萍, 杨昂, 等. 同源引物的非等量PCR [J]. 河南科学, 2018, 36(3): 326-333.
[23] Jameson, E.E., Roof, R.A., Whorton, M.R., et al. (2005) Real-Time Detection of Basal and Stimulated G Protein GTPase Activity Using Fluorescent GTP Analogues. Journal of Biological Chemistry, 280, 7712-7719. [Google Scholar] [CrossRef] 
[24] van Oijen, A.M., Glainey, P.C., Crampton, D.J., et al. (2003) Single-Molecule Kinetics of λ Exonuclease Reveal Base Dependence and Dynamic Disorder. Science, 301, 1235-1238. [Google Scholar] [CrossRef] [PubMed]
[25] Bajar, B.T., Wang, E.S., Zhang, S., Lin, M.Z. and Chu, J. (2016) A Guide to Fluorescent Protein FRET Pairs. Sensors, 16, Article No. 1488. [Google Scholar] [CrossRef] [PubMed]
[26] Zhang, R., Kwok, R.T.K., Tang, B. and Liu, B. (2015) Hybridization Induced Fluorescence Turn-on of of AIEgen-Oli- gonucleotide Conjugates for Specific DNA Detection. RSC Advances, 5, 28332-28337. [Google Scholar] [CrossRef] 
[27] Cheng, Y., Stakenborg, T., Dorpe, P.V., et al. (2011) Fluorescence near Gold Nanoparticles for DNA Sensing. Analytical Chemistry, 83, 1307-1314 [Google Scholar] [CrossRef] [PubMed]
[28] Dubertret, B., Calame, M. and Libchaber, A.J. (2001) Single-Mismatch Detection Using Gold-Quenched Fluorescent Oligonucleotides. Nature Biotechnology, 19, 365-370. [Google Scholar] [CrossRef] [PubMed]
[29] Liu, F., Yang, Y., Wan, X., et al. (2022) Space-Confinment-Enhanced Fluorescence Detection of DNA on Hydrogel Particles Array. ACS Nano, 16, 6266-6273. [Google Scholar] [CrossRef] [PubMed]
[30] 郑园霞, 张毅, 李雪刚, 刘亮伟. 荧光Cy5DNA扩增及酶切产物分离-检测[J]. 微生物前沿, 2022, 11(4): 241-250.
[31] Spiliotis, M. (2012) Inverse Fusion PCR Cloning. PLOS ONE, 7, e35407. [Google Scholar] [CrossRef] [PubMed]
[32] 韩来闯, 马闪闪, 刘亚娟, 等. 构建重组质粒的二步PCR方法[J]. 河南科学, 2015, 33(8): 1321-1325.
[33] 刘猛, 刘亚娟, 徐文选, 等. 长片断引物反向 PCR 方法构建重复序列的重组质粒[J]. 河南科学, 2016, 34(4): 501- 505.
[34] Breslauer, K.J., Frank, R., Blöcker, H. and Marky, L.A. (1986) Predicting DNA Duplex Stability from the Base Sequence. Proceedings of the National Academy of Sciences of the United States of America, 83, 3746-3750. [Google Scholar] [CrossRef] [PubMed]
[35] Allawi, H.T. and Santa, L.J. (1997) Thermodynamics and NMR of Internal G∙T Mismatches in DNA. Biochemistry, 36, 10581-10594. [Google Scholar] [CrossRef] [PubMed]
[36] Takagi, M., Nishioka, M., Kakihara, H., et al. (1997) Characterization of DNA Polymerase from Pyrococcus sp. Strain KOD1 and Its Application to PCR. Applied and Environmental Microbiology, 63, 4504-4510. [Google Scholar] [CrossRef] [PubMed]

为你推荐