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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 deletions. 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 for VraRNHISD, VraRC5D and VraRN3D, cloned vraR genes with its N-terminal His tag removal, five residues from the C-terminus and three residues from the N-terminus deleted respectively.
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Figure 4: PCR amplification for single-site deletions. 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 for VraRNHISD, VraRC5D and VraRN3D, cloned vraR genes with its N-terminal His tag removal, five residues from the C-terminus and three residues from the N-terminus deleted respectively.

Mentions: Using the primer pairs of VRARN3, VRARC5 and VRARDHIS (Table 1), we successfully generated a vraR clone with three residues deleted from its N-terminus, a clone with five residues deleted from its C-terminus and a clone with its His tag removed from the N-terminus. PCR amplifications of these mutants again showed high amplification efficiency (Figure 4A). Transformation of E. coli cells with these PCR products produced more than 100 colonies (Figure 4B). Sequencing the plasmid DNA showed that in each reaction all four isolated recombinants contained the desired deletions.


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 deletions. 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 for VraRNHISD, VraRC5D and VraRN3D, cloned vraR genes with its N-terminal His tag removal, five residues from the C-terminus and three residues from the N-terminus deleted respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: PCR amplification for single-site deletions. 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 for VraRNHISD, VraRC5D and VraRN3D, cloned vraR genes with its N-terminal His tag removal, five residues from the C-terminus and three residues from the N-terminus deleted respectively.
Mentions: Using the primer pairs of VRARN3, VRARC5 and VRARDHIS (Table 1), we successfully generated a vraR clone with three residues deleted from its N-terminus, a clone with five residues deleted from its C-terminus and a clone with its His tag removed from the N-terminus. PCR amplifications of these mutants again showed high amplification efficiency (Figure 4A). Transformation of E. coli cells with these PCR products produced more than 100 colonies (Figure 4B). Sequencing the plasmid DNA showed that in each reaction all four isolated recombinants contained the desired deletions.

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