Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

Drum, Matthias; Kranaster, Ramon; Ewald, Christina; Blasczyk, Rainer; Marx, Andreas

doi:10.1371/journal.pone.0096640

Cited by 26 publications

(26 citation statements)

References 81 publications

(97 reference statements)

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“…As shown in Supplemental Figure S7B, the SNGD method successfully edited both target loci. We further confirmed gene editing by PCR using high singlenucleotide discrimination (HiDi) DNA polymerase that efficiently discriminates primers with a mismatch at the 3 ′ end (Drum et al 2014). The edited alleles, but not non-edited alleles, can be amplified by HiDi PCR using the edited allelespecific PCR primer.…”

Section: Sngd Can Edit Endogenous Locimentioning

confidence: 63%

Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair

et al. 2017

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CRISPR/Cas9, which generates DNA double-strand breaks (DSBs) at target loci, is a powerful tool for editing genomes when codelivered with a donor DNA template. However, DSBs, which are the most deleterious type of DNA damage, often result in unintended nucleotide insertions/deletions (indels) via mutagenic nonhomologous end joining. We developed a strategy for precise gene editing that does not generate DSBs. We show that a combination of single nicks in the target gene and donor plasmid (SNGD) using Cas9D10A nickase promotes efficient nucleotide substitution by gene editing. Nicking the target gene alone did not facilitate efficient gene editing. However, an additional nick in the donor plasmid backbone markedly improved the gene-editing efficiency. SNGD-mediated gene editing led to a markedly lower indel frequency than that by the DSB-mediated approach. We also show that SNGD promotes gene editing at endogenous loci in human cells. Mechanistically, SNGD-mediated gene editing requires long-sequence homology between the target gene and repair template, but does not require CtIP, RAD51, or RAD52. Thus, it is considered that noncanonical homology-directed repair regulates the SNGD-mediated gene editing. In summary, SNGD promotes precise and efficient gene editing and may be a promising strategy for the development of a novel gene therapy approach.

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Section: Sngd Can Edit Endogenous Locimentioning

confidence: 63%

Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair

et al. 2017

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“…Residues for mutation were chosen by their proximity with either primer or template DNA during the catalyzed reaction, as seen in Fig 1 . Similar studies on sequence family A Thermus aquaticus DNA polymerase focused solely on interactions along the primer backbone [ 37 ]. However, our study also includes amino acids which contact the template strand.…”

Section: Resultsmentioning

confidence: 99%

“…The sites at which amino acid exchanges led to increased KOD pol selectivity contact the primer (R606) as well as the template (R501) (see Fig 1 ). Our previous studies based on rational design, identified solely residues that interact with the primer 3’ end as crucial for tuning selectivity [ 37 ]. Our approach in the presented study also included residues which contact both primer and template at the active site as well as distal from the active site.…”

Section: Discussionmentioning

confidence: 99%

“…Several studies have identified DNA polymerases with decreased selectivity [ 28 – 32 ]. DNA polymerases with increased fidelity were also generated [ 33 – 37 ], as well as others with altered substrate tolerances [ 38 – 41 ].…”

Section: Introductionmentioning

confidence: 99%

“…The DNA polymerase from Thermococcus kodakaraensis is a prominent member of sequence family B and our recent studies have shown the potential of engineering this DNA polymerase to be able to recognize mismatched base pairs [ 49 ]. Some of our previous works have shown that the combination of site specific mutagenesis and screening techniques in order to generate tailor-made enzymes to explore the scope of altered DNA polymerase selectivity for both biotechnological as well as diagnostic applications is a promising approach [ 37 , 49 – 51 ].…”

Section: Introductionmentioning

confidence: 99%

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Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity

Huber

Marx

2017

PLoS ONE

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Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3'-5'-exonuclease deficient variant of Thermococcus kodakaraensis DNA polymerase (KOD pol) and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges-R501C, R606Q, and R606W-which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.

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The Effect of γ Phosphate Modified Deoxynucleotide Substrates on PCR Activity and Fidelity

et al. 2023

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Controlling PCR fidelity is an important issue for molecular biology and high-fidelity PCR is essential for gene cloning. In general, fidelity control is achieved by protein engineering of polymerases. In contrast, only a few studies have reported controlling fidelity using chemically modified nucleotide substrates. In this report, we synthesized nucleotide substrates possessing a modification on P γ and evaluated the effect of this modification on PCR fidelity. One of the substrates, nucleotide tetraphosphate, caused a modest decrease in Taq DNA polymerase activity and the effect on PCR fidelity was dependent on the type of mutation. The use of deoxyadenosine tetraphosphate enhanced the A : T!G : C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G : C!A : T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.

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Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

Cited by 26 publications

References 81 publications

Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair

Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair

Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity

The Effect of γ Phosphate Modified Deoxynucleotide Substrates on PCR Activity and Fidelity

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