CLC number: Q319
On-line Access: 2024-08-27
Received: 2023-10-17
Revision Accepted: 2024-05-08
Crosschecked: 2011-12-01
Cited: 5
Clicked: 6556
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.
@article{title="Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids",
author="Bogdan Munteanu, Mario Braun, Kajohn Boonrod",
journal="Journal of Zhejiang University Science B",
volume="13",
number="4",
pages="244-247",
year="2012",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1100180"
}
%0 Journal Article
%T Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids
%A Bogdan Munteanu
%A Mario Braun
%A Kajohn Boonrod
%J Journal of Zhejiang University SCIENCE B
%V 13
%N 4
%P 244-247
%@ 1673-1581
%D 2012
%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
VL - 13
IS - 4
SP - 244
EP - 247
%@ 1673-1581
Y1 - 2012
PB - Zhejiang University Press & Springer
ER -
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.
[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.
Open peer comments: Debate/Discuss/Question/Opinion
<1>