Highly Efficient DNA Synthesis by the Phage ϕ 29 DNA Polymerase

Blanco, Luis; Bernad, António; Lázaro, José Portolés; Martín, G; Garmendia, Cristina; Salas, Margarita

doi:10.1016/s0021-9258(18)81883-x

Cited by 615 publications

(161 citation statements)

References 37 publications

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“…To overcome these bottlenecks, unbiased amplification (not based on PCR) has been commonly used for viral enrichment, as it amplifies all DNA material present in the sample. Multiple displacement amplification (MDA) is the gold standard method for non-PCR-based amplification techniques, where the reaction is based on annealing random hexamer primers to the DNA template [ 54 ]. MDA provides an effective way of amplifying minimal quantities of DNA, but there exist biases associated with this technology.…”

Section: Methodsmentioning

confidence: 99%

Human Papillomavirus Detection by Whole-Genome Next-Generation Sequencing: Importance of Validation and Quality Assurance Procedures

Mühr

Guerendiain

Cuschieri

et al. 2021

Viruses

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Next-generation sequencing (NGS) yields powerful opportunities for studying human papillomavirus (HPV) genomics for applications in epidemiology, public health, and clinical diagnostics. HPV genotypes, variants, and point mutations can be investigated in clinical materials and described in previously unprecedented detail. However, both the NGS laboratory analysis and bioinformatical approach require numerous steps and checks to ensure robust interpretation of results. Here, we provide a step-by-step review of recommendations for validation and quality assurance procedures of each step in the typical NGS workflow, with a focus on whole-genome sequencing approaches. The use of directed pilots and protocols to ensure optimization of sequencing data yield, followed by curated bioinformatical procedures, is particularly emphasized. Finally, the storage and sharing of data sets are discussed. The development of international standards for quality assurance should be a goal for the HPV NGS community, similar to what has been developed for other areas of sequencing efforts including microbiology and molecular pathology. We thus propose that it is time for NGS to be included in the global efforts on quality assurance and improvement of HPV-based testing and diagnostics.

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Section: Methodsmentioning

confidence: 99%

Human Papillomavirus Detection by Whole-Genome Next-Generation Sequencing: Importance of Validation and Quality Assurance Procedures

Mühr

Guerendiain

Cuschieri

et al. 2021

Viruses

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“…Initial indications of spontaneous resolution of these multimers into circular chromosomes under IVTT reaction conditions 17 should otherwise be investigated in more detail. Another bottleneck of the ϕ29 system may be that the processivity of its DNAP (responsible for replicating its 20 kb genome) is not sufficient to enable replication of a single chromosome of the size needed for a synthetic cell 13,14 . As splitting the genome into multiple smaller chromosomes is not preferred (see below; DNA segregation), a potential solution could be to improve the processivity of ϕ29 DNAP by laboratory evolution 5 .…”

Section: Genomementioning

confidence: 99%

Towards a synthetic cell cycle

et al. 2021

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Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle.

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“…Unfortunately, these polymerases remain poorly characterized when compared with commercially available polymerases. The use of highly processive polymerases including phi29 DNA polymerase as a parental polymerase or fusing accessory proteins that tether reported mutant polymerases to templates may improve polymerase processivity; [55,56] however, these polymerase mutants have not been reported. Nonetheless, extensive research is now being undertaken toward enzymatic synthesis of 2' modified oligonucleotides.…”

Section: Resultsmentioning

confidence: 99%

Variety of Nucleotide Polymerase Mutants Aiming to Synthesize Modified RNA

2021

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Significant efforts have been made to develop therapeutic RNA aptamers that exploit synthetic RNA to capture target molecules. However, ensuring RNA aptamers are resistant against intrinsic nucleases remains an issue and restricts their use as therapeutics. Introduction of chemical modifications to the 2' sugar moiety of RNA improves their stability effectively and can be achieved by chemical synthesis using modified phosphoramidites; however, this approach is not suitable for preparing long RNA molecules. Although recombinant nucleotide polymerases can transcribe RNA, these polymerases cannot synthesize modified RNA because they do not recognize 2' modified nucleoside triphosphates. In this review, we focus on several polymerase mutants that tolerate substrates containing modifications of the 2' sugar moiety to synthesize RNA, and the problems that must be overcome to prepare chemically modified RNA with high efficacy by in vitro transcription.

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Highly Efficient DNA Synthesis by the Phage ϕ 29 DNA Polymerase

Cited by 615 publications

References 37 publications

Human Papillomavirus Detection by Whole-Genome Next-Generation Sequencing: Importance of Validation and Quality Assurance Procedures

Human Papillomavirus Detection by Whole-Genome Next-Generation Sequencing: Importance of Validation and Quality Assurance Procedures

Towards a synthetic cell cycle

Variety of Nucleotide Polymerase Mutants Aiming to Synthesize Modified RNA

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