Primase-based whole genome amplification

Li, Ying; Kim, Hyun‐Jin; Zheng, Chunhong; Chow, Wing Huen A.; Lim, Jeonghwa; Keenan, Brendan T.; Pan, Xiaojing; Lemieux, Bertrand; Kong, Huimin

doi:10.1093/nar/gkn377

Cited by 40 publications

(45 citation statements)

References 43 publications

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“…Whole genome amplification by isothermal methods was reported lately by Li et al [166]. The so called primase-based whole genome [157]…”

Section: Various Other Methodsmentioning

confidence: 99%

“…No need of added primers, no thermocycling nor prior heat-denaturation make that principle very promising for the transfer into microfluidic devices. The reaction has initially shown an over thousand-fold amplification after 1 h at 37°C [166]. By using circular DNA as template, a 10 8 fold amplification of as low as 100 initial target strands has been achieved.…”

Section: Various Other Methodsmentioning

confidence: 99%

“…By using circular DNA as template, a 10 8 fold amplification of as low as 100 initial target strands has been achieved. In addition to amplifying total genomic DNA, pWGA can also be used for detection and quantification of contaminant DNA in a sample when combined with a fluorescent reporter dye [166]. The commercialization is covered by BioHelix (Beverly, MA, USA).…”

Section: Various Other Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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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“…Whole genome amplification by isothermal methods was reported lately by Li et al [166]. The so called primase-based whole genome [157]…”

Section: Various Other Methodsmentioning

confidence: 99%

Section: Various Other Methodsmentioning

confidence: 99%

Section: Various Other Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

View full text Add to dashboard Cite

show abstract

“…Recently, another type of DNA amplification using T7 gp4 primase was developed to remove the requirement of adding primers [105]. T7 gp4 contains the primase activity, Fig.…”

Section: Helicase-dependent Dna Amplificationmentioning

confidence: 99%

“…The T7 DNA polymerase can synthesize DNA by extending from the short RNA primers. In this method, referred to as primase-based whole genome amplification (pWGA) [105], which utilizes the dual activities of T7 gp4 as helicase and primase, dsDNA templates are denatured and primers are generated without addition of DNA primers and thermocycling. After T7 gp4 denatures the dsDNA template and synthesizes short RNA primers, the T7 DNA polymerase subsequently extends the strand with the aid of E .coli thioredoxin and T7 gp2.5.…”

Section: Helicase-dependent Dna Amplificationmentioning

confidence: 99%

Isothermal DNA amplification in vitro: the helicase-dependent amplification system

Jeong

Park

Kim

2009

Cell. Mol. Life Sci.

121

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Since the development of polymerase chain reaction, amplification of nucleic acids has emerged as an elemental tool for molecular biology, genomics, and biotechnology. Amplification methods often use temperature cycling to exponentially amplify nucleic acids; however, isothermal amplification methods have also been developed, which do not require heating the double-stranded nucleic acid to dissociate the synthesized products from templates. Among the several methods used for isothermal DNA amplification, the helicase-dependent amplification (HDA) is discussed in this review with an emphasis on the reconstituted DNA replication system. Since DNA helicase can unwind the double-stranded DNA without the need for heating, the HDA system provides a very useful tool to amplify DNA in vitro under isothermal conditions with a simplified reaction scheme. This review describes components and detailed aspects of current HDA systems using Escherichia coli UvrD helicase and T7 bacteriophage gp4 helicase with consideration of the processivity and efficiency of DNA amplification.

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Programmable High‐Speed and Hyper‐Efficiency DNA Signal Magnifier

Zhang

Yin

et al. 2021

Advanced Science

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Herein, a programmable dual-catalyst hairpin assembly (DCHA) for realizing the synchronous recycle of two catalysts is developed, displaying high reaction rate and outstanding conversion efficiency beyond traditional nucleic acid signal amplifications (NASA). Once catalyst I interacts with the catalyst II, the DCHA can be triggered to realize the simultaneous recycle of catalysts I and II to keep the highly concentrated intermediate product duplex I-II instead of the steadily decreased one in typical NASA, which can accomplish in about only 16 min and achieves the outstanding conversion efficiency up to 4.54 × 10 8 , easily conquering the main predicaments of NASA: time-consuming and low-efficiency. As a proof of the concept, the proposed DCHA as a high-speed and hyper-efficiency DNA signal magnifier is successfully applied in the rapid and ultrasensitive detection of miRNA-21 in cancer cell lysates, which exploits the new generation of universal strategy for the applications in biosensing assay, clinic diagnose, and DNA nanobiotechnology.

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Primase-based whole genome amplification

Cited by 40 publications

References 43 publications

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

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

Isothermal DNA amplification in vitro: the helicase-dependent amplification system

Programmable High‐Speed and Hyper‐Efficiency DNA Signal Magnifier

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