scholarly journals One-Enzyme Reverse Transcription qPCR Using Taq DNA Polymerase

Biochemistry ◽  
2020 ◽  
Vol 59 (49) ◽  
pp. 4638-4645
Author(s):  
Sanchita Bhadra ◽  
Andre C. Maranhao ◽  
Inyup Paik ◽  
Andrew D. Ellington
Keyword(s):  
Dna Polymerase ◽  
Reverse Transcription ◽  
Taq Dna Polymerase

Reverse transcription, amplification and sequencing of poliovirus RNA by Taq DNA polymerase

FEBS Letters ◽  
1996 ◽  
Vol 387 (2-3) ◽  
pp. 189-192 ◽  
Author(s):  
V.I. Grabko ◽  
L.G. Chistyakova ◽  
V.N. Lyapustin ◽  
V.G. Korobko ◽  
A.I. Miroshnikov
Keyword(s):  
Dna Polymerase ◽  
Reverse Transcription ◽  
Taq Dna Polymerase

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

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

Gold nanoparticles alter Taq DNA polymerase activity during polymerase chain reaction

RSC Advances ◽  
2013 ◽  
Vol 3 (43) ◽  
pp. 20793 ◽  
Author(s):  
Samir Mandal ◽  
Maidul Hossain ◽  
T. Muruganandan ◽  
Gopinatha Suresh Kumar ◽  
Keya Chaudhuri
Keyword(s):  
Polymerase Chain Reaction ◽  
Gold Nanoparticles ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Chain Reaction ◽  
Taq Dna Polymerase ◽  
Polymerase Chain

Taq DNA Polymerase

1989 ◽  
pp. 17-22 ◽  
Author(s):  
David H. Gelfand
Keyword(s):  
Dna Polymerase ◽  
Taq Dna Polymerase
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