A Strong Strand Displacement Activity of Thermostable DNA Polymerase Markedly Improves the Results of DNA Amplification

Ignatov, Konstantin B.; Barsova, Ekaterina V.; Fradkov, Arkady F.; Blagodatskikh, Konstantin A.; Kramarova, Tatiana V.; Kramarov, V. M.

doi:10.2144/000114198

Cited by 57 publications

(43 citation statements)

References 15 publications

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“… * Encyclo (Evrogen JSC), Kapa HiFi PCR Kit (Kapa Biosystems), Phusion High-Fidelity DNA Polymerase (NEB), SD-HS 29 , SNP-detect, HS Taq, Tersus in buffer 1 (Mg 2+ 3.5 mM, Ph 8.5), Tersus in buffer 2 (Mg 2+ 2.5 mM, Ph 8.0) (Evrogen JSC), TruSeq Custom Enrichment Kit (Illumina), KTN (KlenTaq N polymerase) 30 .…”

Section: Resultsmentioning

confidence: 99%

A high-throughput assay for quantitative measurement of PCR errors

Shagin

Shagina

Zaretsky

et al. 2017

Sci Rep

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The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.

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Section: Resultsmentioning

confidence: 99%

A high-throughput assay for quantitative measurement of PCR errors

Shagin

Shagina

Zaretsky

et al. 2017

Sci Rep

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“…Sequence gaps between the respective contigs were determined by PCR and sequencing using primers located near the ends of the contigs. The approach to extend the sequences at the 5′ and 3′ ends is described in the supplementary methods using a mutant Taq polymerase (SD polymerase) with high strand-displacement activity [16]. From the analysis of the 5′ end of the genome, it can be inferred, based on primer binding and extension, that positive sense viral genomes are packaged into virus particles.…”

Section: Resultsmentioning

confidence: 99%

Identification of a novel bovine copiparvovirus in pooled fetal bovine serum

et al. 2020

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A novel parvovirus was identified as a cell culture contaminant by metagenomic analysis. Droplet digital PCR (ddPCR) was used to determine viral loads in the cell culture supernatant and further analysis, by ddPCR and DNA sequencing, demonstrated that fetal bovine serum (FBS) used during cell culture was the source of the parvovirus contamination. The FBS contained ~ 50,000 copies of the novel parvovirus DNA per ml of serum. The viral DNA was resistant to DNAse digestion. Near-full length sequence of the novel parvovirus was determined. Phylogenetic analysis demonstrated that virus belongs to the Copiparvovirus genus, being most closely related to bovine parvovirus 2 (BPV2) with 41% identity with the nonstructural protein NS1 and 47% identity with the virus capsid protein of BPV2. A screen of individual and pooled bovine sera identified a closely related variant of the novel virus in a second serum pool. For classification purposes, the novel virus has been designated bovine copiparvovirus species 3 isolate JB9 (bocopivirus 3-JB9).

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“…For example, an alternative method that includes denaturation of the template DNA at 95°C for 5 min prior to the reaction has been reported to increase sensitivity by as much as 200-fold, although others report no change (32,33). A thermostable stranddisplacing DNA polymerase that would enable denaturation after enzyme addition for ease of reaction assembly has recently been developed and may increase the efficiency and fidelity of LAMP assays in general (34).…”

Section: Discussionmentioning

confidence: 99%

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens

et al. 2015

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Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia (CAP) across patient populations of all ages. We have developed a loop-mediated isothermal amplification (LAMP) assay that enables rapid, low-cost detection of M. pneumoniae from nucleic acid extracts and directly from various respiratory specimen types. The assay implements calcein to facilitate simple visual readout of positive results in approximately 1 h, making it ideal for use in primary care facilities and resourcepoor settings. The analytical sensitivity of the assay was determined to be 100 fg by testing serial dilutions of target DNA ranging from 1 ng to 1 fg per reaction, and no cross-reactivity was observed against 17 other Mycoplasma species, 27 common respiratory agents, or human DNA. We demonstrated the utility of this assay by testing nucleic acid extracts (n ‫؍‬ 252) and unextracted respiratory specimens (n ‫؍‬ 72) collected during M. pneumoniae outbreaks and sporadic cases occurring in the United States from February 2010 to January 2014. The sensitivity of the LAMP assay was 88.5% tested on extracted nucleic acid and 82.1% evaluated on unextracted clinical specimens compared to a validated real-time PCR test. Further optimization and improvements to this method may lead to the availability of a rapid, cost-efficient laboratory test for M. pneumoniae detection that is more widely available to primary care facilities, ultimately facilitating prompt detection and appropriate responses to potential M. pneumoniae outbreaks and clusters within the community. Mycoplasma pneumoniae is a human pathogen that accounts for an estimated 15% to 20% of cases of community-acquired pneumonia (CAP) (1, 2). An atypical bacterial pneumonia, M. pneumoniae infection can result in both pulmonary and extrapulmonary manifestations, including pharyngitis, encephalitis, Stevens-Johnson syndrome, septic arthritis, and pericarditis, among others (3). Ongoing endemic M. pneumoniae infection is overlaid with cyclical epidemics of disease, typically occurring every 3 to 5 years (1). Although symptoms of M. pneumoniae infection are generally mild or subclinical, the organism accounts for up to 33% of hospitalizations among children and adults with bacterial pneumonia (4). In the absence of a diagnostic result, empirical therapy can be misdirected, resulting in inappropriate use of antibiotics and continued spread of M. pneumoniae in the community. This may promote the evolution of antibiotic resistance and leads to negative economic impact due to health care costs and missed school and work days.Traditional diagnostics for M. pneumoniae infections exist but are underused due to their cost, time-intensive nature, or requirement for specialized expertise and equipment. M. pneumoniae is fastidious and slow-growing, with isolation from liquid culture requiring up to 8 weeks and often resulting in low yield (5, 6). Appropriate interpretation of serological data requires paired acute-phase and convalescent-phase serum samples, constraining their use to retrosp...

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A Strong Strand Displacement Activity of Thermostable DNA Polymerase Markedly Improves the Results of DNA Amplification

Cited by 57 publications

References 15 publications

A high-throughput assay for quantitative measurement of PCR errors

A high-throughput assay for quantitative measurement of PCR errors

Identification of a novel bovine copiparvovirus in pooled fetal bovine serum

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens

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