不同分子量PVP对PCR扩增反应效率的影响

江汉大学学报（自然科学版） ›› 2012, Vol. 40 ›› Issue (2): 69-73.

不同分子量PVP对PCR扩增反应效率的影响

李佳，林燕，姚晶，蒋琳，李正，梁勇

江汉大学医学院，湖北武汉430056

收稿日期:2011-11-23 出版日期:2012-04-20 发布日期:2013-11-07
通讯作者: 梁勇(1976—)，男，副教授，博士，研究方向:生态毒理学。
作者简介:李佳(1981—)，女，讲师，硕士，研究方向:生态毒理学。
基金资助:
国家自然科学基金项目(20907017,20890112

Study on the Effects of Polyvinylpyrrolidone in Different Molecular-weight on the Efficiency of Polymerase Chain Reaction

LI Jia,LIN Yan,YAO Jing,JIANG Lin,LI Zheng,LIANG Yong

School of Medicine,Jianghan University,Wuhan 430056,Hubei,China

Received:2011-11-23 Online:2012-04-20 Published:2013-11-07

摘要/Abstract

摘要： 以人为设计的110bp单链DNA为模板，研究不同分子量聚乙烯基吡咯烷酮(Polyvinyl-pyrrolidone，PVP)对聚合酶链式反应(polymerasechainreaction，PCR)的影响。实验结果表明0.5％的PVPK－30、PVP8000、PVP58000和PVP630000均可显著提高PCR效率，并且这4种化合物提高PCR效率的能力，依次为PVP630000＞PVP58000＞PVPK－30和PVP8000;然而，在相同浓度时，PVPK－40却显著抑制PCR扩增反应;另外，溶解曲线的实验结果显示，0.5％PVP630000可显著降低PCR产物的Tm值，表明PVP630000可通过与DNA相互作用，降低DNA结构稳定性，使得模板更易在PCR的变性阶段解链，最终提高PCR效率。上述研究结果表明，不同分子量的PVP对PCR扩增效率的影响不同，与其分子量大小密切相关。

关键词: 不同分子量PVP, 荧光定量PCR, DNA构象

Abstract: The effect of different molecular－weight Polyvinylpyrrolidone (PVP) on PCR efficiency has been researched into by undertaking the 110 bp, single－stranded DNA amplification with quantitative real－time polymerase chain reaction (QPCR). The results indicates that 0.5％ PVP K－30, PVP 8000, PVP 58000 and PVP 630000 could effectively enhance the PCR efficiency, and the ability of different molecular－weight PVP to improve PCR efficiency is PVP 630000, PVP 58000, PVP K－30 and PVP 8000, respectively. However, PVP K－40 could significantly inhibit the generation of PCR product. In addition, the results of melting curve showed that 0.5％ PVP 630000 could dramatically reduce the Tm value of PCR product. It suggestes that PVP 630000 could interact with DNA template to reduce the stability of DNA conformation, promote the unwinding of DNA template in the denaturation stage of PCR, then enhance the PCR efficiency. The results reveal that the effects of different PVP on the PCR efficiency is closely related to the molecular－weight.

Key words: different molecular－weight PVP, PCR efficiency, DNA conformation

中图分类号:

李佳，林燕，姚晶，蒋琳，李正，梁勇. 不同分子量PVP对PCR扩增反应效率的影响[J]. 江汉大学学报（自然科学版）, 2012, 40(2): 69-73.

LI Jia,LIN Yan,YAO Jing,JIANG Lin,LI Zheng,LIANG Yong. Study on the Effects of Polyvinylpyrrolidone in Different Molecular-weight on the Efficiency of Polymerase Chain Reaction[J]. Journal of Jianghan University(Natural Science Edition), 2012, 40(2): 69-73.

参考文献

[1]Cawthon R M.Telomere length measurement by a novel monochrome multiplex quantitative PCR method[J].Nucleic Acids Research, 2009, 37(3)e21－e21.
［2]Hellgren O, WaldenstrÖm J, Bensch S.A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood[J].Journal of Parasitology,2004,90(4)797－802.
［3]Whiley D M, Bialasiewicz S, Bletchly C, et al.Detection of novel influenza A (H1N1) virus by real－time RT－PCR[J].Journal of Clinical Virology, 2009, 45(3)203－204.
［4]Roux K H.Optimization and troubleshooting in PCR[J].Cold Spring Harbor Protocols, 2009, 4(5) 185－194.
［5]Li L Y, Li Q, Yu Y H, et al.A primer design strategy for PCR amplification of GC－rich DNA sequences[J].Clinical Biochemistry, 2011, 44 692－698.
［6]Chakrabarti R, Schutt C E.The enhancement of PCR amplification by low molecular－weight sulfones[J].Gene, 2001, 274(1)293－298.
［7]Kang J, Soog Lee M, Gorenstein D G.The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine Application to in vitro combinatorial selection of aptamers[J].Journal of Biochemical and Biophysical Methods,2005,64(2)147－151.
［8]Musso M, Bocciardi R, Parodi S, et al.Betaine, dimethyl sulfoxide, and 7－deaza－dGTP, a powerful mixture for amplification of GC－rich DNA sequences[J].The Journal of Molecular Diagnostics,2006,8(5)544－550.
［9]Chin W W L, Heng P W S, Thong P S P, et al.Improved formulation of photosensitizer chlorin e6 polyvinylpyrrolidone for fluorescence diagnostic imaging and photodynamic therapy of human cancer[J].European Journal of Pharmaceutics and Biopharmaceutics, 2008, 69(3)1083－1093.
［10]Haaf F, Sanner A, Straub F.Polymers of N－Vinylpyrrolidone synthesis, characterization and uses[J].Polymer Journal, 1985, 17143－152.
［11]Yu D G, Shen X X, Branford－White C, et al.Oral fast－dissolving drug delivery membranes prepared from electrospun polyvinylpyrrolidone ultrafine fibers[J].Nanotechnology, 2009, 20(5)055104.
［12]Koonjul P K, Brandt W F, Lindsey G G, et al.Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA[J].Nucleic Acids Research, 1999, 27(3) 915－916.
［13]Fuhrman J A, Liang X, Noble R T.Rapid detection of enteroviruses in small volumes of natural waters by real－time quantitative reverse transcriptase PCR[J].Applied and Environmental Microbiology, 2005, 71(8) 4523－4530.
［14]Monpoeho S, Dehee A, Mignotte B, et al.Quantification of enterovirus RNA in sludge samples using single tube real－time RT－PCR[J].Biotechniques, 2000, 29 88－93.
［15]Lee C H, Mizusawa H, Kakefuda T.Unwinding of double－stranded DNA helix by dehydration[J].Proceedings of the National Academy of Sciences, 1981, 78(5) 2838－2842.
［16]Cheng S, Fockler C, Barnes W M, et al.Effective amplification of long targets from cloned inserts and human genomic DNA[J].Proceedings of the National Academy of Sciences, 1994, 91(12) 5695－5699.
［17]Varadaraj K, Skinner D M.Denaturants or cosolvents improve the specificity of PCR amplification of a G＋C－rich DNA using genetically engineered DNA polymerases[J].Gene, 1994, 140(1)1－5.