Polymerase chain reaction (PCR) techniques for site-directed mutagenesis

doi:10.1016/0734-9750(92)91451-J

Biotechnology Advances

Volume 10, Issue 4, 1992, Pages 535-547

https://doi.org/10.1016/0734-9750(92)91451-J Get rights and content

Abstract

Polymerase chain reaction (PCR) technology has revolutionized the process of isolating and amplifying segments of DNA. One powerful application of PCR is its use in precise site-directed mutagenesis (SDM). SDM provides an elegant tool for scientists and engineers to explore biocatalytic mechanisms and processes to understand the structural-functional relationships of enzymes and other proteins. This article reviews techniques and methodology used in site-directed mutagenesis of genes by PCR.

References (29)

S.N. Ho et al.
Site-directed mutagenesis by overlap extension using the polymerase chain reaction
Gene
(1989)
D.H. Jones et al.
Site-specific mutagenesis and DNA recombination by using PCR to generate recombinant circles in vitro or by recombination of linear PCR products in vivo
Methods: A Companion to Methods in Enzymology
(1991)
H. Kadowaki et al.
Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis
Gene
(1989)
O. Landt et al.
A general method for rapid site-directed mutagenesis using the polymerase chain reaction
Gene
(1990)
R.M. Nelson et al.
A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction
Anal. Biochem.
(1989)
I.P. Street et al.
Isopentenyl diphosphate isomerase
Site-directed mutagenesis of cys139 using “counter” PCR amplification of an expression plasmid
Tetrahedron
(1991)
P. Caffrey et al.
An acyl carrier protein thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea
Eur. J. Biochem.
(1991)
J.M. Clark
Nucl. Acids Res.
(1988)
R.T. D'Aquila et al.
Nucl. Acids Res.
(1991)
K.J. Friedman et al.
Detecting multiple Cystic Fibrosis mutations by polymerase chain reaction-mediated site-directed mutagenesis
Clin. Chem.
(1991)

L. Hall et al.

A rapid and efficient method for site-directed mutagenesis by PCR, using biotinylated universal primers and streptavidin-coated magnetic beads

Prot. Eng.

(1991)

A. Hemsley et al.

A simple method for site-directed mutagenesis using the polymerase chain reaction

Nucl. Acids Res.

(1989)

W. Ito et al.

A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction

Gene

(1991)

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The structure of the bacterial iron-catecholate transporter Fiu suggests that it imports substrates via a two-step mechanism
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The ferric iron uptake (Fiu) transporter from Escherichia coli functions in the transport of iron–catecholate complexes across the bacterial outer membrane, providing the bacterium with iron, which is essential for growth. Recently it has become clear that Fiu also represents a liability for E. coli because its activity allows import of antimicrobial compounds that mimic catecholate. This inadvertent import suggests the potential utility of antimicrobial catechol siderophore mimetics in managing bacterial infections. However, to fully exploit these compounds, a detailed understanding of the mechanism of transport through Fiu and related transporters is required. To address this question, we determined the crystal structure of Fiu at 2.1–2.9 Å and analyzed its function in E. coli. Through analysis of the Fiuo crystal structure, in combination with in silico docking and mutagenesis, we provide insight into how Fiu and related transporters bind catecholate in a surface-exposed cavity. Moreover, through determination of the structure of Fiu in multiple crystal states, we revealed the presence of a large, selectively gated cavity in the interior of this transporter. This chamber is large enough to accommodate the Fiu substrate and may allow import of substrates via a two-step mechanism. This would avoid channel formation through the transporter and inadvertent import of toxic molecules. As Fiu and its homologs are the targets of substrate-mimicking antibiotics, these results may assist in the development of these compounds.
Site-directed insertion: Language equations and decision problems
2019, Theoretical Computer Science
Citation Excerpt :
Site-directed mutagenesis is one of the most important techniques for generating mutations on specific sites of DNA using polymerase chain reaction (PCR) based methods [25].
Site-directed insertion is an overlapping insertion operation that can be viewed as analogous to the overlap assembly or chop operations that concatenate strings by overlapping a suffix and a prefix of the argument strings. We consider decision problems and language equations involving site-directed insertion. By relying on the tools provided by semantic shuffle on trajectories (M. Domaratzki, Developments in Language Theory 2004) we show that one variable equations involving site-directed insertion and regular constants can be solved algorithmically. We consider also maximal and minimal variants of the site-directed insertion operation and the nondeterministic state complexity of site-directed insertion.
RecQL4 tethering on the pre-replicative complex induces unscheduled origin activation and replication stress in human cells
2019, Journal of Biological Chemistry
Citation Excerpt :
For expression of WT or various mutant RecQL4 proteins fused to Orc4 (R-O4), cDNAs encoding WT RecQL4 (amino acid residues 1–1209), C-terminal truncated proteins (1–241 for CD1 and 1–427 for CD2), and N-terminal truncated protein (ND, 248–1209) were amplified by PCR and inserted into the N terminus of the ORC4 gene, which was cloned into the pCDNA3.1(−) plasmid. For alanine or glutamic acid substitution of the CDK phosphorylation sites of RecQL4 (S89, T93 and T139), site-directed mutagenesis was conducted using PCR (56, 57). Mcm10 fused to the N terminus of Orc4 and Orc3 fused to the N terminus of And-1 were also generated by PCR and subcloned into the pCDNA3.1(−) plasmid.
Sequential activation of DNA replication origins is precisely programmed and critical to maintaining genome stability. RecQL4, a member of the conserved RecQ family of helicases, plays an essential role in the initiation of DNA replication in mammalian cells. Here, we showed that RecQL4 protein tethered on the pre-replicative complex (pre-RC) induces early activation of late replicating origins during S phase. Tethering of RecQL4 or its N terminus on pre-RCs via fusion with Orc4 protein resulted in the recruitment of essential initiation factors, such as Mcm10, And-1, Cdc45, and GINS, increasing nascent DNA synthesis in late replicating origins during early S phase. In this origin activation process, tethered RecQL4 was able to recruit Cdc45 even in the absence of cyclin-dependent kinase (CDK) activity, whereas CDK phosphorylation of RecQL4 N terminus was required for interaction with and origin recruitment of And-1 and GINS. In addition, forced activation of replication origins by RecQL4 tethering resulted in increased replication stress and the accumulation of ssDNAs, which can be recovered by transcription inhibition. Collectively, these results suggest that recruitment of RecQL4 to replication origins is an important step for temporal activation of replication origins during S phase. Further, perturbation of replication timing control by unscheduled origin activation significantly induces replication stress, which is mostly caused by transcription-replication conflicts.
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Biotechnology Advances

Abstract

References (29)

Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Gene

Site-specific mutagenesis and DNA recombination by using PCR to generate recombinant circles in vitro or by recombination of linear PCR products in vivo

Methods: A Companion to Methods in Enzymology

Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis

Gene

A general method for rapid site-directed mutagenesis using the polymerase chain reaction

Gene

A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction

Anal. Biochem.

Isopentenyl diphosphate isomerase

Site-directed mutagenesis of cys139 using “counter” PCR amplification of an expression plasmid

Tetrahedron

An acyl carrier protein thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea

Eur. J. Biochem.

Nucl. Acids Res.

Nucl. Acids Res.

Detecting multiple Cystic Fibrosis mutations by polymerase chain reaction-mediated site-directed mutagenesis

Clin. Chem.

A rapid and efficient method for site-directed mutagenesis by PCR, using biotinylated universal primers and streptavidin-coated magnetic beads

Prot. Eng.

A simple method for site-directed mutagenesis using the polymerase chain reaction

Nucl. Acids Res.

A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction

Gene

Cited by (88)

The structure of the bacterial iron-catecholate transporter Fiu suggests that it imports substrates via a two-step mechanism

Site-directed insertion: Language equations and decision problems

RecQL4 tethering on the pre-replicative complex induces unscheduled origin activation and replication stress in human cells

A newly identified ferritin L-subunit variant results in increased proteasomal subunit degradation, impaired complex assembly, and severe hypoferritinemia

A dual gene-specific mutator system installs all transition mutations at similar frequencies in vivo

The anion channel SLAH3 interacts with potassium channels to regulate nitrogen-potassium homeostasis and the membrane potential in Arabidopsis

Research review paperPolymerase chain reaction (PCR) techniques for site-directed mutagenesis

Abstract

Gene

Methods: A Companion to Methods in Enzymology

Gene

Gene

Anal. Biochem.

Tetrahedron

An acyl carrier protein thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea

Eur. J. Biochem.

Nucl. Acids Res.

Nucl. Acids Res.

Detecting multiple Cystic Fibrosis mutations by polymerase chain reaction-mediated site-directed mutagenesis

Clin. Chem.

A rapid and efficient method for site-directed mutagenesis by PCR, using biotinylated universal primers and streptavidin-coated magnetic beads

Prot. Eng.

A simple method for site-directed mutagenesis using the polymerase chain reaction

Nucl. Acids Res.

A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction

Gene

Research review paper
Polymerase chain reaction (PCR) techniques for site-directed mutagenesis