COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

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Deteksi Transgen (Glu-1Dx5) pada Populasi Padi (Oryza sativa L.) Putative Transgenik Kultivar Fatmawati

Agrikultura ◽

10.24198/agrikultura.v21i1.985 ◽

2010 ◽

Vol 21 (1) ◽

Author(s):

Nono Carsono ◽

Sri Nurlianti ◽

Inez Nur Indrayani ◽

Ade Ismail ◽

Tri Joko Santoso ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Oryza Sativa ◽

Dna Polymerase ◽

Genomic Dna ◽

Oryza Sativa L ◽

Dna Purification ◽

Coding Region ◽

Chain Reaction ◽

Taq Dna Polymerase ◽

Polymerase Chain

Transformasi gen Glu-1Dx5, pengendali utama karakter elastisitas dan daya mengembang adonan dari gandum, telah berhasil ditransfer ke dalam genom tanaman padi kultivar Fatmawati dengan menggunakan penembakan partikel, dengan tujuan untuk memperbaiki kualitas adonan tepung beras. Galur-galur harapan telah diperoleh, tetapi karena telah mengalami penyerbukan sendiri selama 1-2 generasi yang menyebabkan transgen mengalami segregasi, maka diperlukan upaya pendeteksian transgen pada populasi putative transgenik ini. Upaya ini dapat dilakukan, antara lain dengan menggunakan teknik Polymerase Chain Reaction (PCR) yang memungkinkan perbanyakan fragmen DNA yang spesifik (gen) secara cepat dalam jumlah banyak. Percobaan ini bertujuan untuk mendapatkan tanaman padi transgenik yang memiliki gen Glu-1Dx5 pada dua generasi yang sedang bersegregasi. DNA genom dari 149 tanaman padi (generasi T1 sebanyak 14 tanaman, generasi T2 sebanyak 134 tanaman, dan satu tanaman non-transgenik) telah diekstraksi menggunakan Genomic DNA Purification Kit dari Fermentas. Plasmid pK+Dx5 digunakan sebagai positif kontrol, selain itu digunakan juga enzim Taq DNA polymerase dari Go Green Taq® Master Mix (Promega) dan 2 primer spesifik yang mengamplifikasi coding region dari Glu-1Dx5 (2,5 kb). Hasil percobaan menunjukkan, tanaman padi yang memiliki gen Glu-1Dx5 pada generasi T2-7 sebanyak 26 tanaman, T2-11 : 12 tanaman, T2-12 : 3 tanaman, T2-40 : 3 tanaman dan T2-45 : 5 tanaman. Seluruh tanaman generasi T1 tidak memiliki insert. Hasil ini menunjukkan bahwa gen Glu-1Dx5 sudah terintegrasi ke dalam genom tanaman padi kultivar Fatmawati dan diwariskan dari satu generasi ke generasi berikutnya.

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Development of rapid direct PCR assays to identify downy and powdery mildew and grey mould in Vitis vinifera tissues

OENO One ◽

10.20870/oeno-one.2014.48.4.1697 ◽

2014 ◽

Vol 48 (4) ◽

pp. 261 ◽

Cited By ~ 1

Author(s):

Katia Gindro ◽

Nicole Lecoultre ◽

Luca Molino ◽

Jean-Pierre De Joffrey ◽

Sylvain Schnee ◽

...

Keyword(s):

Vitis Vinifera ◽

Downy Mildew ◽

Fungal Pathogens ◽

Pcr Amplification ◽

Microscopic Analysis ◽

Grey Mould ◽

Dna Purification ◽

Direct Pcr ◽

Fungal Propagules ◽

Pcr Method

Aims: The development of a rapid and reliable direct PCR method to detect fungal propagules in grapevine tissues without prior DNA purification steps, and illustration of its potential use with different examples.Methods and results: Different grapevine samples crushed in the presence of polyvinylpolypyrrolidone (PVPP) were used as templates for direct PCR amplification with primers specifie for Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Vitis vinifera. Sequencing of the PCR products confirmed the specificity of the amplifications. The sensitivity tested using conidia/sporangia dilution series was high, ranging from five sporangia for P. viticola to one conidium for E. necator. The potential of this technique is illustrated through the study of four epidemiological questions. Fungal propagules were observed in dormant buds using microscopy, but the responsible species could not be identified. Direct PCR revealed the presence of E. necator and B. cinerea in 29 % and 65 % of the buds, respectively. Downy mildew could be detected in asymptomatic leaves sampled in fields after potentially infectious events. In bunch, microscopic analysis of rachis sections showed the presence of hyphae growing in the green tissue. Direct PCR identified the presence of P. viticola.Conclusion: A direct PCR method without DNA purification was demonstrated to be a simple and reliable method for the detection and identification of fungal pathogens in grapevine tissues. This method, together with microscopy, is a very interesting tool that can be used to study various epidemiological problems in the grapevine, including important unanswered questions such as the route of infection that leads to brown rot caused by downy mildew.Significance and impact of the study: Direct PCR was shown to be a simple and versatile technique for the study of epidemiological questions in the grapevine. This technique could be extended to other pathosystems with minor adaptations.

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A universal method for direct PCR amplification of plant tissues

Analytical Methods ◽

10.1039/c6ay03156k ◽

2017 ◽

Vol 9 (11) ◽

pp. 1800-1805 ◽

Cited By ~ 4

Author(s):

Yuping Li ◽

Huanhuan Zhao ◽

Xuefen Yan ◽

Meng Li ◽

Peng Chen ◽

...

Keyword(s):

Pcr Amplification ◽

Dna Purification ◽

Plant Tissues ◽

Universal Method ◽

Direct Pcr ◽

Modern Biology

PCR is a vital tool in modern biology; however, it can be costly owing to the price of commercial DNA purification kits.

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Increased complexity of wild-type adeno-associated virus–chromosomal junctions as determined by analysis of unselected cellular genomes

Journal of General Virology ◽

10.1099/vir.0.82880-0 ◽

2007 ◽

Vol 88 (6) ◽

pp. 1722-1732 ◽

Cited By ~ 17

Author(s):

Horace R. Drew ◽

Linda J. Lockett ◽

Gerald W. Both

Keyword(s):

Genomic Dna ◽

Molecular Mechanisms ◽

Pcr Amplification ◽

Pcr Primers ◽

Wild Type ◽

Chromosome 19 ◽

Direct Pcr ◽

Adeno Associated Virus ◽

Junction Formation ◽

New Generation

Adeno-associated virus (AAV) undergoes preferential Rep-mediated integration into the AAVS1 region of human chromosome 19 during latent infection, at least in highly-selected cell cultures. However, integration at the level of the whole eukaryotic genome in unselected cells has not yet been monitored for AAV as it has been for retro- and lentiviruses. Here we have used ligation-mediated PCR (LMPCR) to monitor the formation of AAV–chromosome junctions within unselected genomic DNA after infection. Our analyses show that, in the absence of selection, the complexity of junction formation is much greater than for selected cells. Sequencing of more than 50 authentic LMPCR clones showed that AAV formed junctions with many different chromosomal sites via DNA micro-homologies that frequently involved GGTC motifs located within the AAV p5 element. One site at position 280 was preferred. Even greater complexity was found when unselected junctions identified by LMPCR were analysed by direct PCR amplification and cloning of genomic DNA. No clones containing AAV–AAVS1 chromosome 19 junctions were identified among the LMPCR clones, although they were readily obtained using chromosomal PCR primers, suggesting that junctions with AAVS1 constituted only a small portion of the total. Thus, we have identified an additional means by which AAV sequences may join to human chromosomes, although the detailed molecular mechanisms remain to be elucidated. These data may have implications for the design of new-generation AAV vectors.

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470 PB 339 DNA AMPLIFICATION USING TAQ POLYMERASE AND STOFFEL FRAGMENT IN POLYPLOID SWEETPOTATO

HortScience ◽

10.21273/hortsci.29.5.498e ◽

1994 ◽

Vol 29 (5) ◽

pp. 498e-498

Author(s):

Mario I. Buteler ◽

Don R. LaBonte ◽

James H. Oard

Keyword(s):

Single Dose ◽

Dna Polymerase ◽

Genomic Dna ◽

Dna Amplification ◽

Temperature Treatment ◽

Polymorphic Band ◽

Taq Polymerase ◽

Map Construction ◽

Genome Map

RAPD and the single dose polymorphic band (SDPB) are powerful tools for genome map construction of higher polyploids, such as hexaploid sweetpotato. Duplication in the genome of higher polyploids results in fewer polymorphisms per primer screened than one would expect in diploids. The Stoffel fragment (Sf) is suggested as an alternative to the most commonly used Taq DNA polymerase to maximize the number of polymorphisms. Genomic DNA from two sweetpotato varieties, `Excel' and `Beauregard', and F1 progeny was isolated using a modified CTAB procedure. The DNA was assayed with twelve primers from Operon Technologies groups A and F. Each enzyme was tested with and without a ramp temperature treatment between the annealing and the extension temperatures. Results are based on three separate amplifications and electrophoretic runs. Band reproducibility was better using Sf than Taq; unfortunately, resolution was lower making bands difficult to score. 8.4% more scorable bands and 20.3% more storable polymorphisms were obtained with Taq. The ramp treatment did not alter results using Sf, but did improve the reproducibility of Taq and ease scoring. The number of bands and their location were the same.

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Selection of the most suitable method for the extraction of DNA from foods and feeds for species identification

Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis ◽

10.11118/actaun201058020169 ◽

2010 ◽

Vol 58 (2) ◽

pp. 169-174

Author(s):

Michaela Nesvadbová ◽

Aleš Knoll ◽

Anna Vašátková

Keyword(s):

Species Identification ◽

Genomic Dna ◽

Pcr Amplification ◽

Degraded Dna ◽

Dna Purification ◽

Pcr Analysis ◽

Isolation System ◽

Dna Yield ◽

Pcr Products ◽

Food And Feed

High quality and purity of DNA isolated from food and feed is essential for species identification and has unpredictable influences an effect of analysis. In this study, the efficiency of eight different methods for DNA isolation was investigated. For DNA extraction, the raw chicken meet, ham, sausages, tinned lunch meat, pate, tinned feeds for dogs, complete granulated feeds for dogs and chicken flour were used. Kits of several different producers, i.e.: NucleoSpin Food (Marchery-Nagel), Wizard Genomic DNA Purification Kit (Promega), Invisorb Spin Food Kit I (Invitek), Wizard SV Genomic DNA Purification System (Promega), JetQuick Tissue DNA Spin Kit (Genomed), RNA Blue (Top-Bio), JetQuick Blood & Cell Culture Kit (Genomed), QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit (Qiagen) were employed in the study. Gel agarose electrophoresis for primary verification of DNA quality was performed. The isolates were subsequently assessed for quantity and quality using by spectrophotometer Nanodrop 2000 (Thermo Scientific). To verify of template usability and quality of isolated DNA, the polymerase chain reaction (PCR) was used.Differences between isolated DNA from tinned products and meat, ham, sausage, granulated dog feed and chicken flour were found. In tinned food and feed, the DNA was more degraded, DNA content and DNA purity was lower and also PCR amplification was the most difficult. Overall DNA yield and quality have important influence on PCR products amplification. The best results were obtained with NucleoSpin Food and JetQuick Tissue DNA Spin Kit. DNA extracted by these methods proved highest yields, purity and template quality in all foods and feeds and the results of PCR analysis are excellent reproducible. Analyses showed that results depended on different food or feed using and different isolation system.The results of this work will be utilized to choose the suitable isolating kit for educational course, which is designed for students and also for following research and analyses.

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Tunability of DNA polymerase stability during eukaryotic DNA replication

10.1101/602086 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jacob S. Lewis ◽

Lisanne M. Spenkelink ◽

Grant D. Schauer ◽

Olga Yurieva ◽

Stefan H. Mueller ◽

...

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Genomic Dna ◽

Dna Polymerases ◽

Polymerase Activity ◽

Basic Principles ◽

Large Numbers ◽

Biochemical Studies ◽

Eukaryotic Dna ◽

Lagging Strand

SummaryStructural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α DNA polymerase activity in replication and show that Pol α primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.

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One enzyme reverse transcription qPCR using Taq DNA polymerase

10.1101/2020.05.27.120238 ◽

2020 ◽

Cited By ~ 2

Author(s):

Sanchita Bhadra ◽

Andre C. Maranhao ◽

Andrew D. Ellington

Keyword(s):

Reverse Transcriptase ◽

Dna Polymerase ◽

Leukemia Virus ◽

Dna Polymerases ◽

Reverse Transcriptase Activity ◽

Taq Polymerase ◽

Taq Dna Polymerase ◽

Transcriptase Activity ◽

The Past ◽

Viral Genomic Rna

ABSTRACTTaq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase, and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCR reactions. We demonstrate the utility of Taq-alone RT-qPCR reactions by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/µL of input viral genomic RNA.

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PrimPol (Primase/Polymerase), replicating enzyme – A mini review

Pak-Euro Journal of Medical and Life Sciences ◽

10.31580/pjmls.v2i4.956 ◽

2020 ◽

Vol 2 (4) ◽

pp. 89-92

Author(s):

Muhammad Amir ◽

Sabeera Afzal ◽

Alia Ishaq

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Genetic Information ◽

Nuclear Dna ◽

De Novo ◽

Dna Polymerases ◽

Test Site ◽

Mitochondrial Dna Replication ◽

Dna Template ◽

Preliminary Phase

Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.

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DMSO Improves the Ski-Slope Effect in Direct PCR

Applied Sciences ◽

10.3390/app11041943 ◽

2021 ◽

Vol 11 (4) ◽

pp. 1943

Author(s):

Joo-Young Kim ◽

Ju Yeon Jung ◽

Da-Hye Kim ◽

Seohyun Moon ◽

Won-Hae Lee ◽

...

Keyword(s):

Dna Sequences ◽

Pcr Amplification ◽

Analytical Techniques ◽

Direct Pcr ◽

Dna Profile ◽

Novel Technologies ◽

Specific Amplification ◽

Polymerase Chain ◽

Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

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