scholarly journals Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Peter McInerney ◽  
Paul Adams ◽  
Masood Z. Hadi
Keyword(s):  
Error Rate ◽  
Direct Sequencing ◽  
Dna Polymerases ◽  
Pcr Amplification ◽  
Error Rates ◽  
High Fidelity ◽  
Pcr Cloning ◽  
Hot Start ◽  
Pcr Products ◽  
Mutation Spectra

As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.

scholarly journals Construction of a highly error-prone DNA polymerase for developing organelle mutation systems

2020 ◽  
Vol 48 (21) ◽  
pp. 11868-11879 ◽  
Author(s):  
Junwei Ji ◽  
Anil Day
Keyword(s):  
Dna Polymerase ◽  
Error Rate ◽  
Dna Polymerases ◽  
Plant Mitochondria ◽  
Error Rates ◽  
Wild Type ◽  
High Error Rate ◽  
Wild Type Enzyme ◽  
Organelle Dna

Abstract A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3′–5′ exonuclease function resulted in a modest 5–8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.

General Taq PCR Master Mix -- CHEM 384/584 v2

2022 ◽  
Author(s):  
Ken Christensen
Keyword(s):  
Pcr Amplification ◽  
Blue Dye ◽  
Extension Rate ◽  
Dna Templates ◽  
Sample Handling ◽  
Pcr Screening ◽  
Colony Pcr ◽  
Hot Start ◽  
Direct Loading ◽  
Pcr Products

SapphireAmp Fast PCR Master Mix contains a hot start PCR enzyme, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent as a 2X premix. SapphireAmp Fast PCR Master Mix is optimized for fast PCR and offers a rapid extension rate (10 sec. per kb). The inclusion of blue dye and a density reagent allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR. SapphireAmp Fast PCR Master Mix is ideal for fast colony PCR screening. Fast colony PCR amplification of a 5 kb insert can be completed in approximately 1 hr 15 min. Furthermore, it is possible to amplify fragments up to 6 kb from genomic DNA templates.

scholarly journals Allelic status of PavCNR12 gene in Ukrainian sweet cherry (Prunus avium L.) cultivars

2017 ◽  
Vol 15 (1) ◽  
pp. 40-46 ◽  
Author(s):  
Ya. I. Ivanovych ◽  
R. A. Volkov
Keyword(s):  
Sweet Cherry ◽  
Prunus Avium ◽  
Direct Sequencing ◽  
Fruit Size ◽  
Pcr Amplification ◽  
Promoter Regions ◽  
Allelic Status ◽  
Pcr Products ◽  
Economically Valuable Traits ◽  
Sweet Cherry Cultivars

Aim. In recent decades, Ukrainian breeders have created a large number of sweet cherry cultivars. Further progress in the breeding of sweet cherry requires a broad involvement of molecular methods. Especially important is the development of methods for the identification of genes / alleles that control economically valuable traits. The goal of the study was to develop a new method for discrimination of alleles of the PavCNR12 gene, which controls the fruit size in sweet cherry, and to reveal the allelic status of PavCNR12 in Ukrainian sweet cherry cultivars. Methods. The SNP-polymorphisms in the promoter regions of the PavCNR12-1, -2 and -3 alleles was detected applying comparison of published sequences. PCR amplification of the region was conducted, the obtained PCR products were cut by TaiI restriction endonuclease and separated by electrophoresis in a polyacrylamide gel. The identity of PCR products was confirmed by direct sequencing. Results. A new convenient method for the identification of allelic variants of the PavCNR12 gene using CAPS-markers is proposed. Using the method the allelic status of PavCNR12 in 56 sweet cherry cultivars of Ukrainian and foreign breeding was elucidated. Conclusions. A significant prevalence of the desirable allele PavCNR12-1 over the alleles PavCNR12-2 and -3 was found among the studied cultivars.Keywords: Ukrainian sweet cherry cultivars, genetic control of fruit size, alleles of PavCNR12 gene, CAPSmarkers, Prunus avium.

Perceptual Characteristics of Consonant Production in Apraxia of Speech and Aphasia

2019 ◽  
Vol 28 (4) ◽  
pp. 1411-1431 ◽  
Author(s):  
Lauren Bislick ◽  
William D. Hula
Keyword(s):  
Error Rate ◽  
Error Rates ◽  
Group Differences ◽  
Diagnostic Process ◽  
Apraxia Of Speech ◽  
Contextual Variables ◽  
Error Type ◽  
Type Distribution ◽  
Phonetic Features ◽  
Syllable Position

Purpose This retrospective analysis examined group differences in error rate across 4 contextual variables (clusters vs. singletons, syllable position, number of syllables, and articulatory phonetic features) in adults with apraxia of speech (AOS) and adults with aphasia only. Group differences in the distribution of error type across contextual variables were also examined. Method Ten individuals with acquired AOS and aphasia and 11 individuals with aphasia participated in this study. In the context of a 2-group experimental design, the influence of 4 contextual variables on error rate and error type distribution was examined via repetition of 29 multisyllabic words. Error rates were analyzed using Bayesian methods, whereas distribution of error type was examined via descriptive statistics. Results There were 4 findings of robust differences between the 2 groups. These differences were found for syllable position, number of syllables, manner of articulation, and voicing. Group differences were less robust for clusters versus singletons and place of articulation. Results of error type distribution show a high proportion of distortion and substitution errors in speakers with AOS and a high proportion of substitution and omission errors in speakers with aphasia. Conclusion Findings add to the continued effort to improve the understanding and assessment of AOS and aphasia. Several contextual variables more consistently influenced breakdown in participants with AOS compared to participants with aphasia and should be considered during the diagnostic process. Supplemental Material https://doi.org/10.23641/asha.9701690

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

Correction: “Influence of Selection Bias on the Test Decision – A Simulation Study”

2014 ◽  
Vol 53 (05) ◽  
pp. 343-343
Keyword(s):  
Selection Bias ◽  
Simulation Study ◽  
Error Rate ◽  
Type I Error ◽  
Block Size ◽  
Error Rates ◽  
Type I ◽  
Type I Error Rate ◽  
Representation Error ◽  
Numeric Representation

We have to report marginal changes in the empirical type I error rates for the cut-offs 2/3 and 4/7 of Table 4, Table 5 and Table 6 of the paper “Influence of Selection Bias on the Test Decision – A Simulation Study” by M. Tamm, E. Cramer, L. N. Kennes, N. Heussen (Methods Inf Med 2012; 51: 138 –143). In a small number of cases the kind of representation of numeric values in SAS has resulted in wrong categorization due to a numeric representation error of differences. We corrected the simulation by using the round function of SAS in the calculation process with the same seeds as before. For Table 4 the value for the cut-off 2/3 changes from 0.180323 to 0.153494. For Table 5 the value for the cut-off 4/7 changes from 0.144729 to 0.139626 and the value for the cut-off 2/3 changes from 0.114885 to 0.101773. For Table 6 the value for the cut-off 4/7 changes from 0.125528 to 0.122144 and the value for the cut-off 2/3 changes from 0.099488 to 0.090828. The sentence on p. 141 “E.g. for block size 4 and q = 2/3 the type I error rate is 18% (Table 4).” has to be replaced by “E.g. for block size 4 and q = 2/3 the type I error rate is 15.3% (Table 4).”. There were only minor changes smaller than 0.03. These changes do not affect the interpretation of the results or our recommendations.

scholarly journals Presence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with reduced susceptibility to fluoroquinolones isolated from human salmonellosis in Gyeonggi-do, South Korea from 2016 to 2019

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sohyun Lee ◽  
Nanjoo Park ◽  
Sujung Yun ◽  
Eunseon Hur ◽  
Jiwon Song ◽  
...  
Keyword(s):  
South Korea ◽  
Pcr Amplification ◽  
Public Health Problem ◽  
Test Method ◽  
Quinolone Resistance ◽  
Human Salmonellosis ◽  
Pcr Products ◽  
Antimicrobial Susceptibility Tests ◽  
Susceptibility Tests ◽  
Sequence Types

AbstractNon-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene (qnrA, qnrB, or qnrS), and three Salmonella strains carried two PMQR genes (qnrS and aac(6′)-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,[5]12:i:- (32.4%, 11/34) and Salmonella Typhimurium (29.4%, 10/34) were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.

Reducing Culture Reporting Errors in the Microbiology Laboratory

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S131-S132
Author(s):  
Kathryn Hogan ◽  
Beena Umar ◽  
Mohamed Alhamar ◽  
Kathleen Callahan ◽  
Linoj Samuel
Keyword(s):  
Real Time ◽  
Error Rate ◽  
Reporting System ◽  
Error Rates ◽  
Tracking Errors ◽  
Corrective Measures ◽  
Safety Awareness ◽  
Before And After ◽  
Set Up ◽  
System Errors

Abstract Objectives There are few papers that characterize types of errors in microbiology laboratories and scant research demonstrating the effects of interventions on microbiology lab errors. This study aims to categorize types of culture reporting errors found in microbiology labs and to document the error rates before and after interventions designed to reduce errors and improve overall laboratory quality. Methods To improve documentation of error incidence, a self-reporting system was changed to an automatic reporting system. Errors were categorized into five types Gram stain (misinterpretations), identification (incorrect analysis), set up labeling (incorrect patient labels), procedures (not followed), and miscellaneous. Error rates were tracked according to technologist, and technologists were given real-time feedback by a manager. Error rates were also monitored in the daily quality meeting and frequently detected errors were discussed at staff meetings. Technologists attended a year-end review with a manager to improve their performance. To maintain these changes, policies were developed to monitor technologist error rate and to define corrective measures. If a certain number of errors per month was reached, technologists were required to undergo retraining by a manager. If a technologist failed to correct any error according to protocol, they were also potentially subject to corrective measures. Results In 2013, we recorded 0.5 errors per 1,000 tests. By 2018, we recorded only 0.1 errors per 1,000 tests, an 80% decrease. The yearly culture volume from 2013 to 2018 increased by 32%, while the yearly error rate went from 0.05% per year to 0.01% per year, a statistically significant decrease (P = .0007). Conclusion This study supports the effectiveness of the changes implemented to decrease errors in culture reporting. By tracking errors in real time and using a standardized process that involved timely follow-up, technologists were educated on error prevention. This practice increased safety awareness in our micro lab.

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