DNA amplification in mixed aqueous-organic media: chemical analysis of leading polymerase chain reaction compositions

2021 ◽  
Author(s):  
Jalel Neffati ◽  
Ioanna Petrounia ◽  
Rudy D. Moreira ◽  
Raj Chakrabarti
Keyword(s):  
Sample Preparation ◽  
Mass Spectra ◽  
Matrix Effects ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Polymerase Activity ◽  
Ion Source ◽  
Amplification Efficiency ◽  
Internal Standards ◽  
Polymerase Chain

AbstractPCR amplification of GC-rich regions often leads to low yield and specificity. Addition of PCR-enhancing compounds is employed in order to overcome these obstacles. PCR-enhancing additives are low molecular polar organic compounds that are included as undisclosed co-solvents in commercial PCR buffers. In the interest of transparency and to permit further optimization by researchers of PCR compositions for challenging amplification problems, we studied eight PCR buffers by GC/MS to identify and quantify their co-solvents. Buffer specificity, both rich in water and salified substances, required a suitable sample preparation before injection into the GC/MS system. The aqueous phase of each buffer was replaced by an organic solvent to remove, by precipitation and filtration, salified substances which are detrimental to the GC/MS analysis. This approach has demonstrated the advantage of eliminating both water and salified substances without any loss of co-solvents. The sensitivity of the developed method was demonstrated as the main co-solvents were easily detected, identified and quantified. The methodology for identifying the co-solvents is mainly based on comparison of both library matching of acquired MS spectra with NIST library and experimental mass spectra obtained from authentic chemical standards. For the quantification of each co-solvent, deuterated Internal standards of similar structure to the cosolvents were used to correct the variable recovery caused by sample preparation, matrix effects, and ion source variability. The recovery ratio of the developed method was verified and found to be in the range 90-120 %. We then characterized the effects of specific organic co-solvents identified during PCR amplification -- using DNA melting, polymerase thermostability, polymerase activity and real-time PCR methods -- in order to elucidate their mechanism of action and to permit further optimization of their effects on amplification efficiency and specificity.

scholarly journals Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination

2018 ◽  
Vol 13 (3) ◽  
pp. 115
Author(s):  
Dwiyitno Dwiyitno ◽  
Stefan Hoffman ◽  
Koen Parmentier ◽  
Chris Van Keer
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Blue Mussel ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Seafood Products ◽  
Polymerase Chain ◽  
Commercial Kit

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

Optimal conditions and specific characteristics of Vent exo– DNA polymerase in ligation-mediated polymerase chain reaction protocols

2005 ◽  
Vol 83 (2) ◽  
pp. 147-165 ◽  
Author(s):  
François Vigneault ◽  
Régen Drouin
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Pcr Amplification ◽  
Polymerase Activity ◽  
Optimal Dosage ◽  
Chain Reaction ◽  
Thermus Aquaticus ◽  
Thermococcus Litoralis ◽  
Polymerase Chain

An optimized procedure for the ligation-mediated polymerase chain reaction (PCR) technique using Thermococcus litoralis exo– DNA polymerase (Vent exo–) was developed. The optimal dosage of Vent exo– at the primer extension and PCR amplification steps as well as the optimal DNA quantity to use were established. We showed that Vent exo– can efficiently create the blunt-ended termini required for subsequent linker ligation. Vent exo– proves to be more efficient than Pyrococcus furiosus exo– (Pfu exo–) for this task. Vent exo– resolves highly GC-rich sequence substantially better than Thermus aquaticus DNA polymerase (Taq) and with a similar efficiency as Pfu exo–. The DNA/DNA polymerase activity ratio is significantly higher for Vent exo– than for Pfu exo–, which is reflected by the sensibility of Vent exo– in efficiently amplifying genomic DNA. Furthermore, the range of efficiency of Vent exo– demonstrates the importance of conducting evaluative testing to identify the optimal dosage of use of this polymerase to obtain successful PCR amplification. Optimal MgSO4 concentrations to use with Vent exo– were established. Our results show that Vent exo– DNA polymerase produces bands of uniform and strong intensity and can efficiently be used for the analysis of DNA in living cells by ligation-mediated PCR.Key words: Vent exo– DNA polymerase, Pfu exo– DNA polymerase, DNA sequence context, ligation-mediated polymerase chain reaction (PCR), PCR buffer.

Group-specific differentiation of Rhizobium from clover species by PCR amplification of 23S rDNA sequences

1998 ◽  
Vol 44 (11) ◽  
pp. 1102-1105
Author(s):  
Mesfin Tesfaye ◽  
F Brian Holl
Keyword(s):  
Root Nodule ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Specific Detection ◽  
Rdna Sequences ◽  
Rhizobium Spp ◽  
Polymerase Chain ◽  
Inoculation Group ◽  
Identification Tool

Two 20-bp primers that provide group-specific detection of Rhizobium spp. by polymerase chain reaction (PCR) have been used to differentiate strains that belong to different effectiveness groups within the Rhizobium-Trifolium cross-inoculation group. The target for DNA amplification was a 370-bp fragment of the 23S rDNA region. Analysis of additional root-nodule forming, as well as root-associated bacterial species by PCR-primer assay revealed that variability within this 20-bp segment of the 23S rDNA region may be widespread and provide an effective identification tool. Our data suggest that strains of Rhizobium isolated from the perennial clover Trifolium semipilosum may be phylogenetically more closely related to Rhizobium etli.Key words: Rhizobium, Trifolium, detection, PCR, rDNA.

Detection ofTrypanosoma congolenseandTrypanosoma bruceisubspecies by DNA amplification using the polymerase chain reaction

Parasitology ◽  
1989 ◽  
Vol 99 (1) ◽  
pp. 57-66 ◽  
Author(s):  
D. R. Moser ◽  
G. A. Cook ◽  
Diane E. Ochs ◽  
Cheryl P. Bailey ◽  
Melissa R. McKane ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Large Scale ◽  
Nuclear Dna ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Detection Methods ◽  
Chain Reaction ◽  
African Trypanosomes ◽  
Polymerase Chain

SUMMARYThe nuclear DNA ofTrypanosoma congolensecontains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA ofTrypanosoma brucei brucei(Sloofet al.1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite ofT. congolenseorT. bruceispp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor inLeishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected withT. congolenseand/orT. bruceispp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

scholarly journals Molecular Bird Sexing on Fischeri Lovebird (Agapornis fischeri) by Using Polymerase Chain Reaction

2020 ◽  
Vol 20 ◽  
pp. 04003
Author(s):  
Azalea Dyah Argarini ◽  
Herjuno Ari Nugroho ◽  
Medania Purwaningrum ◽  
Aris Haryanto
Keyword(s):  
Sex Chromosome ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Total Sample ◽  
Z Chromosome ◽  
Agarose Gel Electrophoresis ◽  
Chain Reaction ◽  
Male And Female ◽  
Pcr Products ◽  
Polymerase Chain

Fischeri Lovebird (Agapornis fischeri) found originally in Africa which has spread to many countries. In Indonesia, Fischeri Lovebird is popular as a pet animal. This lovebird is a monomorphic bird, so it is difficult to differentiate morphologically between male and female birds. In general, a male lovebird has ZZ homozygotes, whereas females' lovebird has ZW heterozygous of their sex chromosome. These sex chromosomes set used as study targets for molecular bird sexing of many species of birds because this method is effective and simple to perform. This method targeted to amplify the Chromodomain Helicase DNA-binding (CHD) gene, which found into the sex chromosome of male and female birds. The objective of this study was to rapid molecular bird sexing of Fischeri Lovebird by using PCR methods. Research samples were collected from feather calamus of A. fischeri. The total sample was 11 feathers from A. fischeri. which were collected three to six feathers for each lovebird. Then the research was followed by DNA extraction from calamus feathers, DNA amplification by PCR and agarose gel electrophoresis of PCR products and visualization of PCR predicts by UV-Transilluminator in darkroom. It concluded that PCR amplification using NP, MP and P2 primers produced double DNA bands in size of 400 bp on Z chromosome and bp on W chromosome for female Fischeri Lovebird, whereas for male Fischeri Lovebird only produced a single DNA band in size of 400 bp on Z chromosome. From eleven samples of Fischeri Lovebird showed a total of five females and six male Fischeri Lovebirds.

scholarly journals Deteksi Vibrio harveyi dengan Metode Amplifikasi DNA pada Gen toxR

2020 ◽  
Vol 1 (1) ◽  
pp. 29-37
Author(s):  
Calvin Wijaya Johan ◽  
Sulistyo Emantoko Dwi Putra ◽  
Ernest Suryadjaja
Keyword(s):  
False Positive ◽  
Vibrio Harveyi ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain ◽  
Toxr Gene ◽  
Positive Results

Abstrak -Infeksi hewan akuakultur oleh Vibrio harveyi dapat menyebabkan kematian serta kerugian ekonomi. Kemampuan untuk mendeteksi bakteri tersebut secara dini dapat mencegah penyebarannya dalam akuakultur. Penelitian ini bertujuan untuk mengembangkan metode untuk mendeteksi Vibrio harveyi melalui amplifikasi gen toxR. Amplifikasi DNA dilakukan dengan dua metode, yakni amplifikasi isotermal termediasi loop (loop-mediated isothermal amplification, LAMP) dan reaksi berantai polimerase (PCR). Amplifikasi menggunakan metode LAMP menunjukan perlu dilakukan optimasi protokol maupun desain primer untuk mencegah perolehan hasil false positive. Amplifikasi menggunakan metode PCR menghasilkan produk berukuran 229 pasang basa yang spesifik pada Vibrio harveyi dengan batas deteksi hingga 0,526 ng.µL-1 (setara 2,09 × 106 CFU.mL-1). Kata kunci: Akuakultur, LAMP, PCR, toxR, Vibrio harveyi Abstract -Vibrio harveyi infection in aquacultures may cause death and economical loss. Rapid detection of this bacteria may prevent its dispersal in aquacultures. The goal of this research was to develop method in detection of Vibrio harveyi via amplification of toxR gene. DNA amplification was carried out with two methods, loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Amplification with LAMP suggest optimization of either protocol or primer design was needed to prevents false positive results. Amplification with PCR yields 229 bp-length product specific to Vibrio harveyi with detection limit up to 0.526 ng.µL-1 (equals to 2.09 × 106 CFU.mL-1). Keywords: Aquaculture, LAMP, PCR, toxR, Vibrio harveyi

scholarly journals Using Polymerase Chain Reaction-based DNA Amplification to Fingerprint North American Potato Cultivars

HortScience ◽  
1996 ◽  
Vol 31 (1) ◽  
pp. 130-133 ◽  
Author(s):  
B. Sosinski ◽  
D.S. Douches
Keyword(s):  
Polymerase Chain Reaction ◽  
North American ◽  
Genetic Relationships ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Chain Reaction ◽  
Arbitrary Primers ◽  
Polymerase Chain ◽  
Tuber Type

DNA from 46 North American potato (Solanum tuberosum L.) cultivars was examined using the polymerase chain reaction (PCR) with 16 arbitrary primers of 10 nucleotide length (10 mers) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) in delineating cultivars, both sexually derived and clonal variants. The 16 primers yielded 43 useful polymorphisms that were evaluated according to the presence or absence of fragments of equal size. All cultivars were discriminated with as few as 10 primers. The russet sport of Burbank was distinguished from a white-skinned clone by one band. More primers (29) were examined to identify a band polymorphism among six Russet Burbank clonal variants. When the cultivars were grouped by tuber type (excluding the russet clonal variants), three to four primers discriminated these commonly grown cultivars. Determination of cultivar integrity was accomplished with PCR amplification, regardless of tissue source (leaf vs. tuber) for DNA extraction. Cluster analysis based on RAPD markers was performed to examine pedigree relationships of the cultivars. Genetic relationships correlated with some pedigrees; however, many exceptions were noted.

scholarly journals A Disposable and Multi-Chamber Film-Based PCR Chip for Detection of Foodborne Pathogen

Sensors ◽  
2018 ◽  
Vol 18 (9) ◽  
pp. 3158 ◽  
Author(s):  
Nam Bae ◽  
Sun Lim ◽  
Younseong Song ◽  
Soon Jeong ◽  
Seol Shin ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Pathogen Detection ◽  
Detection System ◽  
Foodborne Pathogen ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Adhesive Film ◽  
Thermal Transfer ◽  
Polymerase Chain ◽  
Structurally Stable

Since the increment of the threat to public health caused by foodborne pathogens, researches have been widely studied on developing the miniaturized detection system for the on-site pathogen detection. In the study, we focused on the development of portable, robust, and disposable film-based polymerase chain reaction (PCR) chip containing a multiplex chamber for simultaneous gene amplification. In order to simply fabricate and operate a film-based PCR chip, different kinds of PCR chambers were designed and fabricated using polyethylene terephthalate (PET) and polyvinyl chloride (PVC) adhesive film, in comparison with commercial PCR, which employs a stereotyped system at a bench-top scale. No reagent leakage was confirmed during the PCR thermal cycling using the film PCR chip, which indicates that the film PCR chip is structurally stable for rapid heat cycling for DNA amplification. Owing to use of the thin film to fabricate the PCR chip, we are able to realize fast thermal transfer from the heat block that leads to short PCR amplification time. Moreover, using the film PCR chip, we could even amplify the target pathogen with 10 CFU mL−1. The artificially infected milk with various concentration of Bacillus cereus was successfully amplified on a single film PCR chip. On the basis of the reliable results, the developed film PCR chip could be a useful tool as a POCT device to detect foodborne pathogens via genetic analysis.

scholarly journals Optimisation of microfluidic polymerase chain reaction devices

2021 ◽  
Vol 321 ◽  
pp. 01007
Author(s):  
Foteini Zagklavara ◽  
Peter K. Jimack ◽  
Nikil Kapur ◽  
Osvaldo M. Querin ◽  
Harvey M. Thompson
Keyword(s):  
Polymerase Chain Reaction ◽  
Pressure Drop ◽  
Dna Amplification ◽  
Response Surfaces ◽  
Channel Structure ◽  
Design Parameters ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Polyharmonic Splines

The invention and development of Polymerase Chain Reaction (PCR) technology have revolutionised molecular biology and molecular diagnostics. There is an urgent need to optimise the performance of these devices while reducing the total construction and operation costs. This study proposes a CFD-enabled optimisation methodology for continuous flow (CF) PCR devices with serpentine-channel structure, which enables the optimisation of DNA amplification efficiency and pressure drop to be explored while varying the width (W) and height (H) of the microfluidic (μ) channel. This is achieved by using a surrogate-enabled optimisation approach accounting for the geometrical features of a μCFPCR device by performing a series of simulations using COMSOL Multiphysics 5.4®. The values of the objectives are extracted from the CFD solutions, and the response surfaces are created using polyharmonic splines. Genetic algorithms are then used to locate the optimum design parameters. The results indicate that there is the possibility of improving the DNA concentration and the pressure drop in a PCR cycle by ~2.1 % ([W, H] = [400 μm, 50 μm]) and ~95.2 % ([W, H] = [400 μm, 80 μm]) respectively, by modifying its geometry.

scholarly journals Towards PCB-Based Miniaturized Thermocyclers for DNA Amplification

Micromachines ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 258
Author(s):  
Georgia D. Kaprou ◽  
Vasileios Papadopoulos ◽  
Christos-Moritz Loukas ◽  
George Kokkoris ◽  
Angeliki Tserepi
Keyword(s):  
Power Consumption ◽  
Large Scale ◽  
Printed Circuit Board ◽  
Dna Amplification ◽  
Circuit Board ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Seamless Integration ◽  
Printed Circuit ◽  
Polymerase Chain

In recent years, printed circuit board (PCB)-based microfluidics have been explored as a means to achieve standardization, seamless integration, and large-scale manufacturing of microfluidics, thus paving the way for widespread commercialization of developed prototypes. In this work, static micro polymerase chain reaction (microPCR) devices comprising resistive microheaters integrated on PCBs are introduced as miniaturized thermocyclers for efficient DNA amplification. Their performance is compared to that of conventional thermocyclers, in terms of amplification efficiency, power consumption and duration. Exhibiting similar efficiency to conventional thermocyclers, PCB-based miniaturized thermocycling achieves faster DNA amplification, with significantly smaller power consumption. Simulations guide the design of such devices and propose means for further improvement of their performance.

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