Fig. 1 Gel electrophoresis of PCR products
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RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany
1.RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany
Published: 2012-04 ,
Received: 06 June 2011 ,
Revised: 01 December 2011 ,
Accepted: 29 September 2011
Cite this article
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 (Biomedicine & Biotechnology) 13(4):244-247(2012)
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 (Biomedicine & Biotechnology) 13(4):244-247(2012) DOI: 10.1631/jzus.B1100180.
RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany
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.
QuickChange mutagenesis is a method for achieving site-directed mutagenesis (SDM) in a plasmid, by substitution, deletion, or insertion of nucleotides in the target plasmid. The method is fast, straightforward, and essential in functional studies, genetic engineering, and biochemistry. QuickChange mutagenesis employs polymerase chain reaction (PCR) using oligonucleotide primer pairs that carry the desired mutation, deletion, or insertion. There are many approaches for mutagenesis using PCR (Kunkel,
The high-fidelity Pfu DNA polymerase commonly used in SDM can amplify a DNA template up to 20 kb (recommended by Fermentas, Germany). Indeed, using Pfu DNA polymerase for SDM of a plasmid less than 10 kb can be very efficient (Laible and Boonrod,
Polymerase | Template plasmid DNA (ng) | Volume (µl) | ||||||
---|---|---|---|---|---|---|---|---|
Forward primer 10 pmol | Reverse primer 10 pmol | Buffer | MgCl2 50 mmol/L | dNTPs 10 mmol/L | DNA polymerase | Final volume by adding H2O | ||
Pfu | 70 | 1 | 1 | 5 | 0 | 1 | 1 | 50 |
KOD Hot Start | 70 | 1 | 1 | 5 | 3 | 1 | 1/0.5 | 50 |
Pfu polymerase (2.5 U/µl) purchased from Fermentas, Germany; KOD Hot Start polymerase (1.0 U/µl) purchased from Novagen, Germany
Polymerase | Denaturated temperature/time | Annealing temperature/time | Extension temperature/time | Holding temperature (°C) | |
---|---|---|---|---|---|
Template | Activated enzyme | ||||
Pfu | 95 °C/1 min | 95 °C/30 s | 55 °C/1 min | 72 °C/12 min | 4 |
KOD Hot Start | 94 °C/2 min | 98 °C/10 s | 57 °C/30 s | 68 °C/6 min | 4 |
The PCR products were electrophoresed in a 1% Tris-acetate-ethylenediaminetetraacetic acid (TAE) gel and visualized under ultraviolet (UV) light. In addition, the PCR products were digested with DpnI restriction enzyme and then were transformed into Escherichia coli competent cells. The results revealed that the reaction using desalted primers in combination with either Pfu or KOD Hot Start polymerase did not yield any PCR products (Fig.
Fig. 1 Gel electrophoresis of PCR products
(a) Comparison of the efficiencies of two DNA polymerases using desalted and HPLC-purified primers. The plasmid pKB-DRD1-GFP-DRM-Red (16 kb) was mutated using 1 µl of either Pfu or KOD Hot Start DNA polymerase in combination with either desalted or HPLC-purified primers. The reactions were performed as described in Tables
To confirm that the plasmids were mutated, plasmids were prepared from 10 different colonies and analyzed by NcoI restriction digestion as described by Zhang et al. (
We conclude that KOD Hot Start DNA polymerase in combination with HPLC-purified primers is the key factor for achieving SDM in a big plasmid. To confirm that this combination can be applied to SDM with more than single base changes, we performed other sets of mutagenesis trials in which 5 or 8 bases were substituted. The results revealed that all mutants were successful and 99% of the tested transformants were correctly mutated (data not shown).
To reduce time and costs of the reaction, we investigated the reaction conditions by reducing the number of cycles and the amount of the polymerase.
The pKB-DRD1-GFP-DRM-Red plasmid was used as a template and the reactions were performed as described in Tables
In conclusion, KOD-polymerase in combination with HPLC-purified primers is efficient for achieving SDM in big plasmids (up to 16 kb). Only 0.5 µl of the enzyme and six cycles of reaction are sufficient for the PCR reaction.
We thank Dr. Guenther BUCHHOLZ (RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Germany) for valuable suggestions and Dr. Michael WASSENEGGER (RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Germany) for providing the plasmid.
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