scholarly journals Simera: Modelling the PCR Process to Simulate Realistic Chimera Formation

2016 ◽  
Author(s):  
Ben Nichols ◽  
Christopher Quince
Keyword(s):  
Experimental Data ◽  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Chain Reaction ◽  
Future Studies ◽  
Target Dna ◽  
Detection Software ◽  
Polymerase Chain ◽  
Diversity Studies ◽  
Principal Method

AbstractPolymerase Chain Reaction (PCR) is the principal method of amplifying target DNA regions and, as such, is of great importance when performing microbial diversity studies. An unfortunate side effect of PCR is the formation of unwanted byproducts such as chimeras. The main goal of the work covered in this article is the development of an algorithm that simulates realistic chimeras for use in the evaluation of chimera detection software and for investigations into the accuracy of community structure analyses. Experimental data has helped to identify factors which may cause the formation of chimeras and has provided evidence of how influential these factors can be. This article makes use of some of this evidence in order to build a model with which to simulate the PCR process. This model helps to better explain the formation of chimeras and is therefore able to provide aid to future studies that intend to use PCR.

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

2006 ◽  
Vol 52 (5) ◽  
pp. 451-461 ◽  
Author(s):  
S S Hynes ◽  
O Chaudhry ◽  
M A Providenti ◽  
M L Smith
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Environmental Persistence ◽  
Fungal Strains ◽  
Target Dna ◽  
Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

scholarly journals Evaluation of Resistance to Rhabdocline Needlecast in Douglas Fir Variety Shuswap, with Quantitative Polymerase Chain Reaction

2010 ◽  
Vol 100 (4) ◽  
pp. 337-344 ◽  
Author(s):  
M. Catal ◽  
G. C. Adams ◽  
D. W. Fulbright
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Douglas Fir ◽  
Chain Reaction ◽  
Disease Symptoms ◽  
Seed Sources ◽  
Release Times ◽  
Spore Release ◽  
Target Dna ◽  
Polymerase Chain

A quantitative polymerase chain reaction assay was developed that could detect DNA of Rhabdocline pseudotsugae and R. oblonga among DNA of Douglas fir needles to a limit as low as three copies of target DNA. Differential infection rates of two varieties (seed sources) of Douglas fir interplanted in a field were studied in relation to staggered bud breaks. Infection of Douglas fir var. San Isabel corresponded to ascospore release times for Rhabdocline spp., whereas infection of var. Shuswap Lake did not occur throughout the spore release period during 2 years of study, despite abundant inoculum and adequate moisture during bud break. Rhabdocline spp. DNA was never detected in Shuswap Lake and disease symptoms were not observed in any year. We provide evidence that Shuswap Lake is resistant and probably immune to Rhabdocline spp. infection and Rhabdocline needlecast under Michigan conditions.

scholarly journals Performance of a Commercial Polymerase Chain Reaction Test for EndocervicalChlamydia trachomatisInfection in a University Hospital Population

1998 ◽  
Vol 6 (5) ◽  
pp. 224-229
Author(s):  
C. H. Livengood III ◽  
K. A. Boggess ◽  
J. W. Wrenn ◽  
A. P. Murtha
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Test ◽  
Laboratory Evaluation ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Predictive Values ◽  
Target Dna ◽  
Wide Range ◽  
Polymerase Chain ◽  
Pcr Test

Objectives:To examine the accuracy of a commercial polymerase chain reaction (PCR) test (Amplicor CTR, Roche Diagnostic Systems, Branchburg NJ) for identification of endocervical chlamydial infections through both laboratory evaluation and among a diverse teaching hospital patient population.Methods:Testing of reliable threshold inocula and reproducibility were carried out using laboratory stock organisms. Paired endocervical samples from patients with a wide range of indications were tested by PCR and an established culture procedure, and discrepant pairs were further analyzed to determine true results.Results:Laboratory evaluation suggested that one copy of target DNA from a viable organism consistently yielded a positive result, and test reproducibility was very good, with an overall coefficient of variation of 15%. Compared to true results in 1,588 paired clinical samples from 1,489 women with a 10% prevalence of infection, the PCR test and culture yielded respective sensitivities of 87.4% and 78.0%, and negative predictive values of 98.6% and 97.6%. Specificity and positive predictive value for both tests were 100%. Cost per specimen was nearly identical at $18.84 and $18.88 respectively. Polymerase inhibitors and organisms lacking target DNA were not found in false-negative PCR samples.Conclusion:This commercial PCR test is accurate, cost-competitive, and much faster than culture for diagnosis of endocervical chlamydia infections in our population of intermediate prevalence of chlamydial infection.

scholarly journals HIV-1 Preintegration Complexes Preferentially Integrate into Longer Target DNA Molecules in Solution as Detected by a Sensitive, Polymerase Chain Reaction-based Integration Assay

2001 ◽  
Vol 276 (50) ◽  
pp. 46946-46952 ◽  
Author(s):  
Alexei Brooun ◽  
Douglas D. Richman ◽  
Richard S. Kornbluth
Keyword(s):  
Polymerase Chain Reaction ◽  
Host Cell ◽  
Polymerase Chain Reaction Amplification ◽  
Chain Reaction ◽  
Preintegration Complex ◽  
Target Dna ◽  
Host Cell Factors ◽  
A Cell ◽  
Polymerase Chain ◽  
Exonuclease Digestion

After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using λ exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs.

scholarly journals Deconstructing the Polymerase Chain Reaction II: an improved workflow and effects on artifact formation and primer degeneracy

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7121
Author(s):  
Ankur Naqib ◽  
Silvana Poggi ◽  
Stefan J. Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Microbial Community ◽  
Annealing Temperature ◽  
Degenerate Primer ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Polymerase Chain ◽  
Dna Template ◽  
Microbial Dna

Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias. In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

Polymerase chain reaction (PCR) v1

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Plasmid Construction ◽  
Target Dna ◽  
Dna Fragment ◽  
Polymerase Chain

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

Polymerase chain reaction (PCR) v2

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Plasmid Construction ◽  
Target Dna ◽  
Dna Fragment ◽  
Polymerase Chain

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

Polymerase Chain Reaction Detection of the Modified cry1Ac Gene in Transgenic Bt (Bollgard®) Cotton

2005 ◽  
Vol 40 (2) ◽  
pp. 206-210
Author(s):  
Yu Cheng Zhu ◽  
John J. Adamczyk
Keyword(s):  
Polymerase Chain Reaction ◽  
Transgenic Cotton ◽  
Polymerase Chain Reaction Detection ◽  
Chain Reaction ◽  
Cry1ac Gene ◽  
Future Studies ◽  
Cotton Plants ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Bacterium Bacillus

Transgenic cotton (Gossypium hirsutum L.) containing a modified cry1Ac gene from the soil bacterium Bacillus thuringiensis Berliner has been widely adopted for suppression of lepidopterous pests. As of 2004, over 90% of the cotton acreage in the mid-southern United States contained this modified cry1Ac gene. We developed a technique using the polymerase chain reaction (PCR) for routine detection of the cry1Ac gene in transgenic cotton plants. A total of eight cry1Ac genes were aligned for the PCR primer design. A DNA fragment was amplified from transgenic cotton, sequenced, and confirmed to be a portion of the cry1Ac gene. A total of 150 cotton plants representing four cultivars were examined for the presence of the cry1Ac gene. Results demonstrated that all of these cotton plants harbored the cry1Ac gene (i.e., 100% purity). This PCR technique can be used for future studies involving the expression of cry1Ac gene as well as corresponding protein expression.

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