Characterization of a Thermostable UvrD Helicase and Its Participation in Helicase-dependent Amplification

Luo, An; Tang, Wen; Ranalli, Tamara A.; Kim, Hyun‐Jin; Wytiaz, Jamie; Kong, Huimin

doi:10.1074/jbc.m503096200

Cited by 148 publications

(126 citation statements)

References 27 publications

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“…Although many proof-of-principal results in this study were obtained using "classical" TaqMan probes (Figures 2, 5, and 6), duplex-stabilizing dNTPs were also found to be effective with Scorpion primers (60) (see Figure 7). Use of base-modified dNTPs is not limited to PCR and could be advantageously used in other amplification schemes performed at elevated temperatures such as nucleic acid sequence-based amplification (NASBA) (61,62) and helicase-dependent amplification (HDA) (63,64).…”

Section: Discussionmentioning

confidence: 99%

Use of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction

Kutyavin¹

2008

Biochemistry

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Several base-modified duplex-stabilizing deoxyribonucleoside 5′-triphosphates (dNTPs) have been evaluated as agents for enhancing the hybridization properties of primers and probes in real-time polymerase chain reaction (PCR). It was shown that pyrimidines substituted at the 5-position with bromine or iodine atoms and methyl or propynyl groups are incorporated into PCR amplicons by Taq DNA polymerase as efficiently as natural dNTPs. The dNTP of 2-aminoadenosine was incorporated somewhat less efficiently than dATP but still supported PCR. Incorporation of these modified nucleotides into the amplified DNA represents a simple and inexpensive way to stabilize duplexes of primers and probes and is particularly effective in improving the amplification and detection of A/T-rich sequences. This technology permits the use of higher PCR annealing temperatures or alternatively a reduction in the length of the oligonucleotide components. Examples of successful application in TaqMan and Scorpion real-time detection assays are provided. Limits of the approach are identified and discussed. For example, application of the 5-bromo and 5-iodo derivatives may be limited to relatively G/C-rich DNA targets and, in particular, to those lacking long runs of adenylate and/or thymidylate. Simultaneous use of base-modified analogues of dATP and dTTP should be avoided in PCR due to "overstabilization" of the amplicon.Structurally modified DNA and RNA have applications in bioengineering, nanotechnology, molecular biology, and medicine. Oftentimes, however, chemical synthesis of modified polynucleotides is difficult and inefficient. Moreover, certain modifications cannot be chemically introduced due to instability. Enzymatic synthesis is an alternative way to prepare DNA or RNA polymers. Relatively minor modifications at the R-phosphate moiety of deoxyribonucleoside 5′-triphosphates (dNTPs) are tolerated well by DNA polymerases (1). Examples include thio (2) and borano phosphate (3, 4) derivatives. DNA polymerases also tolerate certain base modifications in dNTPs as long as normal Watson-Crick base pairing is preserved.

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Section: Discussionmentioning

confidence: 99%

Use of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction

Kutyavin¹

2008

Biochemistry

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“…Furthermore the performance of the HDA was improved [104], leading to longer amplification fragments from initially up to 400 bp to >2000 bp [104]. A sensitivity of as few as 10 copies of bacterial genomic DNA has been presented [103]. This thermophilic HDA system was further developed for diagnostic applications by Goldmeyer et al [106,107].…”

Section: Hda: Helicase-dependent Amplificationmentioning

confidence: 99%

“…This exponential reaction can produce million-fold copies of target DNA in 60 to 120 min [104]. The development of a heat-stable helicase from Thermoanaerobacter tengcongensis allowed driving the reaction at a temperature of 45°C to 65°C [103,105] without the addition of MutL and SSB. Furthermore the performance of the HDA was improved [104], leading to longer amplification fragments from initially up to 400 bp to >2000 bp [104].…”

Section: Hda: Helicase-dependent Amplificationmentioning

confidence: 99%

“…A helicase unwinds the target DNA strand at a temperature of 37 °C to circumvent the heat-induced denaturation step of PCR. The MutL protein stimulates the helicase unwinding, whilst the single-stranded binding (SSB) protein prevents re-hybridization of the separated ssDNA targets [103] (Figure 5). The primers can hybridize to the free ssDNA and a DNA polymerase subsequently extends them.…”

Section: Hda: Helicase-dependent Amplificationmentioning

confidence: 99%

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Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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“…Although HDA is difficult to optimize, it is a highly sensitive and rapid technique. 13 For efficient amplification, we used a concentration of primers that was higher than that of the probes. The reactions were conducted at 65 C in the presence of both a thermostable helicase and a polymerase.…”

Section: Ligation-independent Probe Amplification Is Functionalmentioning

confidence: 99%

Ligation-Independent Mechanism of Multiplex Ligation-Dependent Probe Amplification

Uno

Yanagihara

2014

ANAL. SCI.

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Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for detecting genomic structural variants. The technique is based on hybridization and ligation, followed by amplification of the ligation products. Therefore, ligation is considered a fundamental process that determines the feasibility and fidelity of MLPA. However, despite the widespread use of this technique, its reaction mechanism has not been fully analyzed. Herein, we describe a ligation-independent pathway for MLPA and introduce a ligation-independent probe amplification system that can be used to obtain amplified products without the hybridization and ligation processes. Fragment analysis revealed that the ligation-independent pathway is functional and that the capacity to discriminate single nucleotides with MLPA does not depend on ligation. These findings indicate that the feasibility and fidelity of MLPA do not rely on ligation.

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Characterization of a Thermostable UvrD Helicase and Its Participation in Helicase-dependent Amplification

Cited by 148 publications

References 27 publications

Use of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction

Use of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Ligation-Independent Mechanism of Multiplex Ligation-Dependent Probe Amplification

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