scholarly journals Development of a recombinant Taq DNA polymerase enzyme expressed using a synthetic gene and its comparison with a commercial enzyme

2021 ◽  
pp. 43-50
Author(s):  
Yuliana ◽  
Uus Saepuloh ◽  
Suryani
Keyword(s):  
Dna Polymerase ◽  
Production Efficiency ◽  
Target Genes ◽  
Thermophilic Bacteria ◽  
Expression System ◽  
Dna Amplification ◽  
Thermostable Enzyme ◽  
Synthetic Gene ◽  
Taq Polymerase ◽  
Taq Dna Polymerase

Taq DNA polymerase is a thermostable enzyme widely used for DNA amplification in the PCR technique. It was initially characterized and isolated from thermophilic bacteria, Thermus aquaticus. It was difficult to developed in this enzyme using a native host system. Therefore, the development of the recombinant Taq DNA polymerase expressed using a synthetic gene is important to improve production efficiency. In this study, we developed the in house Taq DNA polymerase recombinant based on a codon-optimized using E. coli expression system. We cloned 2685 bp of the Taq DNA polymerase gene in the pET151/D-TOPO vector. The gene was synthesized and the expression was analyzed with SDS-PAGE technique which indicated with a 100.9 kDa specific target protein. The concentration and activity of this purified enzyme were 5.17 mg/mL and 4.647 U/µL, respectively. The application of this enzyme to the PCR technique showed that this enzyme could amplify the target genes from 200 bp to 3500 bp amplicons with a minimum DNA concentration template 10 ng/µL. This assumes that the in house recombinant Taq DNA polymerase based on synthetic genes is successfully expressed, purified, and was functional and comparable to the commercial Taq polymerase.

scholarly journals Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples

2009 ◽  
Vol 37 (5) ◽  
pp. e40-e40 ◽  
Author(s):  
M. B. Kermekchiev ◽  
L. I. Kirilova ◽  
E. E. Vail ◽  
W. M. Barnes
Keyword(s):  
Dna Polymerase ◽  
Whole Blood ◽  
Dna Amplification ◽  
Soil Samples ◽  
Pcr Inhibitors ◽  
Taq Dna Polymerase

scholarly journals The reproducibility of RAPD profiles: Effects of PCR components on RAPD analysis of four centaurium species

2012 ◽  
Vol 64 (1) ◽  
pp. 191-199 ◽  
Author(s):  
Marijana Skoric ◽  
B. Siler ◽  
Tijana Banjanac ◽  
Jasmina Zivkovic ◽  
Slavica Dmitrovic ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Polymorphism ◽  
Reliable Method ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Taq Polymerase ◽  
Taq Dna Polymerase ◽  
Final Volume ◽  
Template Dna ◽  
Diversity Studies

Random amplified polymorphic DNA (RAPD) analysis is a simple and reliable method used to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of the assay. In this study, we analyzed the effects of different concentrations of primer, magnesium chloride, template DNA and Taq DNA polymerase to develop and standardize a RAPD protocol for Centaurium species. The optimized PCR reaction mixture included: 50 ng of DNA extracted using a CTbased protocol, 2.5 mM MgCl2, 7.5 pmol primer and 2 U of Taq polymerase in a final volume of 25 ?l. Each of the five primers used in experiments (OPB11, OPB15, OPB18, OPF05 and OPH02) generated reproducible and distinguishable fingerprinting patterns of four Centaurium species. The obtained optimized RAPD protocol and the selected primers are useful for our further work in the genetic diversity studies of Centaurium species.

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

HortScience ◽  
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.

scholarly journals Modification Of Taq DNA Polymerase with Improved Elongation Ability

2021 ◽  
Author(s):  
A. Sarsen ◽  
Zh. Akishev ◽  
M. Saginova ◽  
B. Sultankulov ◽  
B. Khassenov
Keyword(s):  
Dna Polymerase ◽  
Fusion Gene ◽  
Thermophilic Bacterium ◽  
Optimal Conditions ◽  
Thermus Aquaticus ◽  
T7 Promoter ◽  
Taq Polymerase ◽  
Taq Dna Polymerase ◽  
Diagnostic Practice ◽  
Polymerase I

Thermostable polymerases play a significant role in molecular biology and diagnostic practice. The most famous and demanded is Polymerase I from the thermophilic bacterium Thermus aquaticus (Taq-pol). This polymerase at one time made a kind of revolution in the polymerase chain reaction. In this work, we attempted to modify this polymerase by attaching an additional Sso7d protein from Sulfolobus solfataricus to Taq-pol, which provides additional binding to the double-stranded DNA of the template. Sso7d-Taq fusion gene was expressed in BL21(DE3) cells. Optimal conditions were selected for maximum production of modified Sso7d-Taq polymerase. The optimal conditions for the intracellular accumulation of Sso7d-Taq polymerase: activation of the T7 promoter when the optical density of the culture reaches OD600 = 0.8-1.0 by adding IPTG at a concentration of 0.2 mM, followed by incubation of the culture at 37°C for 20-24 hours. Recombinant Sso7d-Taq polymerase has been purified and tested by PCR for thermal stability and elongation time. It was found that the Sso7d-Taq enzyme withstands 5 hour incubation at 95°C and 75 minute incubation at 98°C. Comparative analysis with unmodified Taq DNA polymerase showed that the Sso7d-Taq enzyme reduces the elongation rate by several times - up to 15-13 seconds per 1 kbp. The results obtained indicate the prospects of using Sso7d-Taq DNA polymerase in scientific research and diagnostic practice.

scholarly journals One enzyme reverse transcription qPCR using Taq DNA polymerase

2020 ◽  
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.

scholarly journals Human Saliva and Dried Saliva Spots as Source of DNA for PCR based HLA Typing using a Combination of Taq DNA Polymerase and AccuPrimeTaq Polymerase

2019 ◽  
Vol 17 (2) ◽  
pp. 113-127
Author(s):  
Prerana Madhusudhana MURTHY ◽  
Anupama Cheleri NEDUVAT ◽  
Cheemalamarri VEENADHAR ◽  
Sudarson SUNDARRAJAN ◽  
Sriram PADMANABHAN
Keyword(s):  
Dna Polymerase ◽  
Internal Control ◽  
Genomic Dna ◽  
Target Genes ◽  
Coagulation Factor ◽  
Potential Method ◽  
Human Saliva ◽  
Hla Typing ◽  
Taq Dna Polymerase ◽  
Dna Yield

Genomic DNA extracted from human saliva samples showed high inter-subject variations in DNA yield, compelling the need to explore a methodology for the accurate quantitation of the extracted genomic DNA. Quantitative assessment of DNA extracted from saliva was achieved using human coagulation factor XIII as an internal control for subsequent downstream applications of amplification of human leucocyte antigen (HLA) genes by PCR. The PCR signals for the HLA target genes, namely, HLA-A, -B, -C , DPB1, DQB1, and DRB1 of exons 2 and 3, improved greatly with the use of a combination of Taq DNA polymerase and AccuPrimeTaq DNA polymerase.  We also describe a new method of using dried saliva spots (DSS) as an alternate source of genomic DNA for HLA typing. PCR-based typing of DNA from human saliva offers a potential method for HLA typing and amplification, and typing of DNA, thus presented, could be applied in forensic science to saliva samples recovered from crime scenes.

scholarly journals MARCADORES RAPD PARA MAPEAMENTO GENÉTICO E SELEÇÃO DE HÍBRIDOS DE CITROS

2001 ◽  
Vol 23 (3) ◽  
pp. 477-481 ◽  
Author(s):  
ROBERTO PEDROSO DE OLIVEIRA ◽  
MARIÂNGELA CRISTOFANI ◽  
MARCOS ANTÔNIO MACHADO
Keyword(s):  
Dna Polymerase ◽  
Citrus Reticulata ◽  
Taq Dna Polymerase ◽  
Citrus Reticulata Blanco

Os marcadores moleculares apresentam várias aplicações no melhoramento de plantas, permitindo uma série de análises genéticas. Este trabalho foi realizado com o objetivo de estabelecer marcadores RAPD para serem utilizados em estudos de mapeamento genético e na seleção de híbridos entre tangerina-'Cravo' (Citrus reticulata Blanco) e laranja-'Pêra' (C. sinensis (L.) Osbeck). Extraiu-se DNA de folhas dos parentais e de seis híbridos F1. As reações de amplificação foram preparadas em 13 uL de solução, constituída por tampão 1x GIBCO BRL; soluções 1,54 mM de MgCl2 e 0,2 mM de cada dNTP; 15 ng de cada 'primer'; 1,5 unidade de 'Taq DNA Polymerase' e 15 ng de DNA genômico. As reações foram realizadas em termocicladores programados para 36 ciclos de 1 min a 92ºC, 1 min a 36ºC, 2 min a 72ºC e 10 min de extensão a 72ºC. Foram testados 'primers' decâmeros arbitrários dos 'kits' A, AB, AT, AV, B, C, D, E, G, H, M, N, P, Q, R e U da Operon, sendo selecionados 113 por apresentarem polimorfismo, com número de marcadores variando de 1 a 6 por 'primer'. Esses 'primers' amplificaram 201 (23,13%) bandas polimórficas, aplicáveis no mapeamento genético e seleção de híbridos. A freqüência de 'primers' com 1; 2; 3; 4; 5 e 6 bandas polimórficas foi de 49,5%, 33,6%, 9,7%, 4,4%, 1,8% e 1,0%, respectivamente.

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

Agrikultura ◽  
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.

scholarly journals Efforts towards crystallization of Taq DNA polymerase mutants (967.4)

2014 ◽  
Vol 28 (S1) ◽  
Author(s):  
Alfredo Valencia ◽  
Jacqueline Kroll ◽  
Matthew Sazinsky ◽  
Aaron Leconte
Keyword(s):  
Dna Polymerase ◽  
Taq Dna Polymerase ◽  
Polymerase Mutants
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