Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase

Biochemistry ◽  
1991 ◽  
Vol 30 (31) ◽  
pp. 7661-7666 ◽  
Author(s):  
Thomas W. Myers ◽  
David H. Gelfand
Keyword(s):  
Dna Polymerase ◽  
Reverse Transcription ◽  
Thermus Thermophilus ◽  
Dna Amplification

Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium-Activated Thermostable DNA Polymerase

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot4115
Author(s):  
Edward S. Smith ◽  
Alvin K. Li ◽  
Alice M. Wang ◽  
David H. Gelfand ◽  
Thomas W. Myers
Keyword(s):  
High Temperature ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Dna Amplification ◽  
Thermostable Dna Polymerase

scholarly journals Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Wendy Ribble ◽  
Shawn D. Kane ◽  
James M. Bullard
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Thermus Thermophilus ◽  
Buffer System ◽  
Dna Polymerase Iii ◽  
Clamp Loader ◽  
Native Form ◽  
Polymerase Iii ◽  
Essential Components ◽  
Pol Iii

DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX (τ and γ), δ and δ′ components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions.

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 Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR

2016 ◽  
Vol 82 (10) ◽  
pp. 3022-3031 ◽  
Author(s):  
Ayako Fujiwara ◽  
Katsuhiro Kawato ◽  
Saori Kato ◽  
Kiyoshi Yasukawa ◽  
Ryota Hidese ◽  
...  
Keyword(s):  
Noise Reduction ◽  
Dna Polymerase ◽  
Biological Activities ◽  
Genetic Diagnosis ◽  
Gc Content ◽  
Dna Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Thermococcus Kodakarensis

ABSTRACTDNA/RNA helicases, which are enzymes for eliminating hydrogen bonds between bases of DNA/DNA, DNA/RNA, and RNA/RNA using the energy of ATP hydrolysis, contribute to various biological activities. In the present study, theEuryarchaeota-specific helicase EshA (TK0566) from the hyperthermophilic archaeonThermococcus kodakarensis(Tk-EshA) was obtained as a recombinant form, and its enzymatic properties were examined.Tk-EshA exhibited maximal ATPase activity in the presence of RNA at 80°C. Unwinding activity was evaluated with various double-stranded DNAs (forked, 5′ overhung, 3′ overhung, and blunt end) at 50°C.Tk-EshA unwound forked and 3′ overhung DNAs. These activities were expected to unwind the structured template and to peel off misannealed primers whenTk-EshA was added to a PCR mixture. To examine the effect ofTk-EshA on PCR, various target DNAs were selected, and DNA synthesis was investigated. When 16S rRNA genes were used as a template, several misamplified products (noise DNAs) were detected in the absence ofTk-EshA. In contrast, noise DNAs were eliminated in the presence ofTk-EshA. Noise reduction byTk-EshA was also confirmed whenTaqDNA polymerase (a family A DNA polymerase, PolI type) and KOD DNA polymerase (a family B DNA polymerase, α type) were used for PCR. Misamplified bands were also eliminated duringtoxAgene amplification fromPseudomonas aeruginosaDNA, which possesses a high GC content (69%).Tk-EshA addition was more effective than increasing the annealing temperature to reduce misamplified DNAs duringtoxAamplification.Tk-EshA is a useful tool to reduce noise DNAs for accurate PCR.IMPORTANCEPCR is a technique that is useful for genetic diagnosis, genetic engineering, and detection of pathogenic microorganisms. However, troubles with nonspecific DNA amplification often occur from primer misannealing. In order to achieve a specific DNA amplification by eliminating noise DNAs derived from primer misannealing, a thermostableEuryarchaeota-specific helicase (Tk-EshA) was included in the PCR mixture. The addition ofTk-EshA has reduced noise DNAs in PCR.

scholarly journals Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples

1998 ◽  
Vol 64 (10) ◽  
pp. 3748-3753 ◽  
Author(s):  
Waleed Abu Al-Soud ◽  
Peter Rådström
Keyword(s):  
Dna Polymerase ◽  
Biological Samples ◽  
Detection Method ◽  
Dna Polymerases ◽  
Dna Amplification ◽  
Nucleic Acid Amplification ◽  
Full Potential ◽  
Thermus Aquaticus ◽  
Pcr Inhibitors ◽  
Thermostable Dna Polymerase

ABSTRACT The PCR is an extremely powerful method for detecting microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase fromThermus aquaticus by many components found in complex biological samples. In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases. Samples of blood, cheese, feces, and meat, as well as various ions, were added to PCR mixtures containing various thermostable DNA polymerases. The nucleic acid amplification capacity of the nine polymerases, under buffer conditions recommended by the manufacturers, was evaluated by using a PCR-based detection method for Listeria monocytogenes in the presence of purified template DNA and different concentrations of PCR inhibitors. The AmpliTaqGold and the Taq DNA polymerases from Thermus aquaticus were totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture, while the HotTub, Pwo, rTth, andTfl DNA polymerases were able to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity. The DNA polymerase from Thermotoga maritima(Ultma) was found to be the most susceptible to PCR inhibitors present in cheese, feces, and meat samples. When the inhibitory effect of K and Na ions was tested on the nine polymerases, HotTub from Thermus flavus and rTthfrom Thermus thermophilus were the most resistant. Thus, the PCR-inhibiting effect of various components in biological samples can, to some extent, be eliminated by the use of the appropriate thermostable DNA polymerase.

scholarly journals Schnellnachweis von SARS-CoV-2 mit recombinase polymerase amplification

BIOspektrum ◽  
2020 ◽  
Vol 26 (6) ◽  
pp. 624-627
Author(s):  
Ole Behrmann ◽  
Iris Bachmann ◽  
Frank Hufert ◽  
Gregory Dame
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Dna Polymerase ◽  
Fluorescent Probes ◽  
Reverse Transcription ◽  
Nucleic Acid Detection ◽  
Detection Scheme ◽  
Recombination Proteins ◽  
Target Dna ◽  
Rapid Amplification

Abstract The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39–42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

2009 ◽  
Vol 21 (3) ◽  
pp. 350-354 ◽  
Author(s):  
Albert Rovira ◽  
Juan Abrahante ◽  
Michael Murtaugh ◽  
Muñoz-Zanzi Claudia
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Loop Mediated Isothermal Amplification

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

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