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Journal of Zhejiang University SCIENCE B 2012 Vol.13 No.4 P.244-247

http://doi.org/10.1631/jzus.B1100180


Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids


Author(s):  Bogdan Munteanu, Mario Braun, Kajohn Boonrod

Affiliation(s):  RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany

Corresponding email(s):   kajohn.boonrod@agroscience.rlp.de

Key Words:  Site-directed mutagenesis (SDM), Mutant, Plasmid


Bogdan Munteanu, Mario Braun, Kajohn Boonrod. Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids[J]. Journal of Zhejiang University Science B, 2012, 13(4): 244-247.

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author="Bogdan Munteanu, Mario Braun, Kajohn Boonrod",
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pages="244-247",
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%T Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids
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%A Mario Braun
%A Kajohn Boonrod
%J Journal of Zhejiang University SCIENCE B
%V 13
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%P 244-247
%@ 1673-1581
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%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1100180

TY - JOUR
T1 - Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids
A1 - Bogdan Munteanu
A1 - Mario Braun
A1 - Kajohn Boonrod
J0 - Journal of Zhejiang University Science B
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SP - 244
EP - 247
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PB - Zhejiang University Press & Springer
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DOI - 10.1631/jzus.B1100180


Abstract: 
QuickChange mutagenesis is the method of choice for site-directed mutagenesis (SDM) of target sequences in a plasmid. It can be applied successfully to small plasmids (up to 10 kb). However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography (HPLC) purified primers, we were able to achieve SDM in big plasmids (up to 16 kb) involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction (PCR) cycles and 0.5 µl of polymerase (instead of 18 PCR cycles and 1.0 µl of enzyme in the standard protocol) were sufficient for the reaction.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[1]Chapnik, N., Sherman, H., Froy, O., 2008. A one-tube site-directed mutagenesis method using PCR and primer extension. Anal. Biochem., 372(2):255-257.

[2]Cormack, B., 1994. Introduction of a point mutation by sequential PCR steps. Curr. Protoc. Mol. Biol., 2:8.5.7-8.5.9.

[3]Edelheit, O., Hanukoglu, A., Hanukoglu, I., 2009. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnol., 9(1):61.

[4]Fujii, S., Akiyama, M., Aoki, K., Sugaya, Y., Higuchi, K., Hiraoka, M., Miki, Y., Saitoh, N., Yoshiyama, K., Ihara, K., et al., 1999. DNA replication errors produced by the replicative apparatus of Escherichia coli. J. Mol. Biol., 289(4):835-850.

[5]Fushan, A.A., Drayna, D.T., 2009. MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR. BMC Biotechnol., 9(1):83.

[6]Ishii, T.M., Zerr, P., Xia, X.M., Bond, C.T., Maylie, J., Adelman, J.P., 1998. Site-directed mutagenesis. Methods Enzymol., 293:53-71.

[7]Kitabayashi, M., Nishiya, Y., Esaka, M., Itakura, M., Imanaka, T., 2002. Gene cloning and polymerase chain reaction with proliferating cell nuclear antigen from Thermococcus kodakaraensis KOD1. Biosci. Biotechnol. Biochem., 66(10):2194-2200.

[8]Kumar, R., Rajagopal, K., 2008. Single-step overlap-primer-walk polymerase chain reaction for multiple mutagenesis without overlap extension. Anal. Biochem., 377(1):105-107.

[9]Kunkel, T.A., 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. PNAS, 82(2):488-492.

[10]Laible, M., Boonrod, K., 2009. Homemade site directed mutagenesis of whole plasmids. J. Vis. Exp., (27):pii:1135.

[11]Li, J., Li, C., Xiao, W., Yuan, D., Wan, G., Ma, L., 2008. Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli. Anal. Biochem., 373(2):389-391.

[12]Liu, H., Naismith, J.H., 2008. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol., 8(1):91.

[13]Mizuguchi, H., Nakatsuji, M., Fujiwara, S., Takagi, M., Imanaka, T., 1999. Characterization and application to Hot Start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase. J. Biochem., 126(4):762-768.

[14]Tseng, W.C., Lin, J.W., Wei, T.Y., Fang, T.Y., 2008. A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal. Biochem., 375(2):376-378.

[15]Zhang, B.Z., Zhang, X., An, X.P., Ran, D.L., Zhou, Y.S., Lu, J., Tong, Y.G., 2009. An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screeing. J. Zhejiang Univ.-Sci. B, 10(6):479-482.

[16]Zheng, L., Baumann, U., Reymond, J.L., 2004. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res., 32(14):e115.

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