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An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.

Liu H, Naismith JH - BMC Biotechnol. (2008)

Bottom Line: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange.Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability.The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange) but increased the overall success rates.

View Article: PubMed Central - HTML - PubMed

Affiliation: SSPF, Centre for Biomolecular Science, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK. lh9@st-andrews.ac.uk

ABSTRACT

Background: Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable.

Results: We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis.

Conclusion: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange) but increased the overall success rates.

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PCR amplification for single-site mutagenesis. A) Agarose gel electrophoresis of the PCR reactions indicating the amplification efficiency. The names of the mutants are shown on the top of each lane. B) Transformation and mutation efficiency of each reaction for 3026L/M, 3051L/M, 3327L/M, 3356L/M, FabH2111C/A, FabH2111C/S. Labels for the generated mutants are detailed in the text. 3026L/M(13) and 3026L/M(QC) are control PCR reactions using primer pairs designed as [13] and QuickChange™ protocol respectively.
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Figure 3: PCR amplification for single-site mutagenesis. A) Agarose gel electrophoresis of the PCR reactions indicating the amplification efficiency. The names of the mutants are shown on the top of each lane. B) Transformation and mutation efficiency of each reaction for 3026L/M, 3051L/M, 3327L/M, 3356L/M, FabH2111C/A, FabH2111C/S. Labels for the generated mutants are detailed in the text. 3026L/M(13) and 3026L/M(QC) are control PCR reactions using primer pairs designed as [13] and QuickChange™ protocol respectively.

Mentions: Using the primer pair of 3026L/M, 3051L/M, 3327L/M, 3056L/M, FabH2111C/A, FabH2111C/S (Table 1), six single-site mutagenesis reactions were carried out to substitute residues Leu26, Leu51 in CAG38830, Leu27 and Leu56 in CAG38833 by methionines and Cys111 in FabH2 by an alanine and a serine respectively. As controls one primer pair of 3026L/M designed as recommended in the QuikChange™ manual and another designed as described in [13] were tested. Agarose gel electrophoresis showed the synthesis of the full-length plasmid DNA (Figure 3A). DNA quantification showed that amplifications of 3026L/M, 3051L/M and FabH2111C/S produced approximately 1 μg of the synthesized plasmid DNA respectively in each reaction while amplifications of 3327L/M, 3056L/M and FabH2111C/A produced approximately 500 ng respectively. In comparison with the DNA produced using the primer pairs designed by our new scheme the control PCR reactions produced 35 ng for 3026L/M(13) and about 5 ng for 3026L/M(QC). Transformation of E. coli cells with these DNA resulted in a large number of colonies (Figure 3B) indicating a high PCR amplification efficiency. DNA sequencing showed that in each mutagenesis reaction all four transformants contained the desired mutations (Figure 3B). Under these conditions neither control experiment gave colonies which harbored the desired mutants despite five attempts.


An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.

Liu H, Naismith JH - BMC Biotechnol. (2008)

PCR amplification for single-site mutagenesis. A) Agarose gel electrophoresis of the PCR reactions indicating the amplification efficiency. The names of the mutants are shown on the top of each lane. B) Transformation and mutation efficiency of each reaction for 3026L/M, 3051L/M, 3327L/M, 3356L/M, FabH2111C/A, FabH2111C/S. Labels for the generated mutants are detailed in the text. 3026L/M(13) and 3026L/M(QC) are control PCR reactions using primer pairs designed as [13] and QuickChange™ protocol respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629768&req=5

Figure 3: PCR amplification for single-site mutagenesis. A) Agarose gel electrophoresis of the PCR reactions indicating the amplification efficiency. The names of the mutants are shown on the top of each lane. B) Transformation and mutation efficiency of each reaction for 3026L/M, 3051L/M, 3327L/M, 3356L/M, FabH2111C/A, FabH2111C/S. Labels for the generated mutants are detailed in the text. 3026L/M(13) and 3026L/M(QC) are control PCR reactions using primer pairs designed as [13] and QuickChange™ protocol respectively.
Mentions: Using the primer pair of 3026L/M, 3051L/M, 3327L/M, 3056L/M, FabH2111C/A, FabH2111C/S (Table 1), six single-site mutagenesis reactions were carried out to substitute residues Leu26, Leu51 in CAG38830, Leu27 and Leu56 in CAG38833 by methionines and Cys111 in FabH2 by an alanine and a serine respectively. As controls one primer pair of 3026L/M designed as recommended in the QuikChange™ manual and another designed as described in [13] were tested. Agarose gel electrophoresis showed the synthesis of the full-length plasmid DNA (Figure 3A). DNA quantification showed that amplifications of 3026L/M, 3051L/M and FabH2111C/S produced approximately 1 μg of the synthesized plasmid DNA respectively in each reaction while amplifications of 3327L/M, 3056L/M and FabH2111C/A produced approximately 500 ng respectively. In comparison with the DNA produced using the primer pairs designed by our new scheme the control PCR reactions produced 35 ng for 3026L/M(13) and about 5 ng for 3026L/M(QC). Transformation of E. coli cells with these DNA resulted in a large number of colonies (Figure 3B) indicating a high PCR amplification efficiency. DNA sequencing showed that in each mutagenesis reaction all four transformants contained the desired mutations (Figure 3B). Under these conditions neither control experiment gave colonies which harbored the desired mutants despite five attempts.

Bottom Line: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange.Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability.The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange) but increased the overall success rates.

View Article: PubMed Central - HTML - PubMed

Affiliation: SSPF, Centre for Biomolecular Science, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK. lh9@st-andrews.ac.uk

ABSTRACT

Background: Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable.

Results: We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis.

Conclusion: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange) but increased the overall success rates.

Show MeSH
Related in: MedlinePlus