Browse
Submit Data
Databases
API
Help

Dataset Information

36 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification.

ABSTRACT: In vitro reconstitution of the bacteriophage T4 replication machinery provides a novel system for fast and processive isothermal DNA amplification. We have characterized this system in two formats: (i) in circular nicking endonuclease-dependent amplification (cNDA), the T4 replisome is supplemented with a nicking endonuclease (Nb.BbvCI) and a reverse primer to generate a well-defined uniform double-stranded linear product and to achieve up to 1100-fold linear amplification of a plasmid in 1 h. (ii) The T4 replisome with its primase (gp61) can also support priming and exponential amplification of genomic DNA in primase-based whole-genome amplification (T4 pWGA). Low amplification biases between 4.8 and 9.8 among eight loci for 0.3-10 ng template DNA suggest that this method is indeed suitable for uniform whole-genome amplification. Finally, the utility of the T4 replisome for isothermal DNA amplification is demonstrated in various applications, including incorporation of functional tags for DNA labeling and immobilization; template generation for in vitro transcription/translation and sequencing; and colony screening and DNA quantification.

SUBMITTER: Schaerli Y

PROVIDER: S-EPMC3001092 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Primase-based whole genome amplification.

Project description:In vitro DNA amplification methods, such as polymerase chain reaction (PCR), rely on synthetic oligonucleotide primers for initiation of the reaction. In vivo, primers are synthesized on-template by DNA primase. The bacteriophage T7 gene 4 protein (gp4) has both primase and helicase activities. In this study, we report the development of a primase-based Whole Genome Amplification (pWGA) method, which utilizes gp4 primase to synthesize primers, eliminating the requirement of adding synthetic primers. Typical yield of pWGA from 1 ng to 10 ng of human genomic DNA input is in the microgram range, reaching over a thousand-fold amplification after 1 h of incubation at 37 degrees C. The amplification bias on human genomic DNA is 6.3-fold among 20 loci on different chromosomes. 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. When circular DNA is used as template in pWGA, 10(8)-fold of amplification is observed from as low as 100 copies of input. The high efficiency of pWGA in amplifying circular DNA makes it a potential tool in diagnosis and genotyping of circular human DNA viruses such as human papillomavirus (HPV).

| S-EPMC2490742 | biostudies-literature

Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities.

Project description:Rapid isothermal nucleic acid amplification technologies can enable diagnosis of human pathogens and genetic variations in a simple, inexpensive, user-friendly format. The isothermal exponential amplification reaction (EXPAR) efficiently amplifies short oligonucleotides called triggers in less than 10 min by means of thermostable polymerase and nicking endonuclease activities. We recently demonstrated that this reaction can be coupled with upstream generation of trigger oligonucleotides from a genomic target sequence, and with downstream visual detection using DNA-functionalized gold nanospheres. The utility of EXPAR in clinical diagnostics is, however, limited by a nonspecific background amplification phenomenon, which is further investigated in this report. We found that nonspecific background amplification includes an early phase and a late phase. Observations related to late phase background amplification are in general agreement with literature reports of ab initio DNA synthesis. Early phase background amplification, which limits the sensitivity of EXPAR, differs however from previous reports of nonspecific DNA synthesis. It is observable in the presence of single-stranded oligonucleotides following the EXPAR template design rules and generates the trigger sequence expected for the EXPAR template present in the reaction. It appears to require interaction between the DNA polymerase and the single-stranded EXPAR template. Early phase background amplification can be suppressed or eliminated by physically separating the template and polymerase until the final reaction temperature has been reached, thereby enabling detection of attomolar starting trigger concentrations.

| S-EPMC4097029 | biostudies-literature

Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification.

Project description:CRISPR/Cas9 is a highly efficient genome engineering tool, yet its off-target effects and sequence-dependent cleavage activity across different sgRNAs remain major concerns for its application. Here, we propose a nicking triggered exponential amplification reaction (NTEXPAR), a fast and sensitive in vitro method, to detect the double strand DNA cleaved by down to 10 pM Cas9 with a linear range of 100 pM to 20 nM. With this newly developed amplification method, Cas9 cleavage activity can be quantified in 40 min and the optimal sgRNA design for specific target sequence can be successfully determined. Using the pre-screened sgRNA, we are able to distinguish single nucleotide mismatch in a gene silencing experiment. This fluorescence based isothermal assay provides a versatile tool for the pre-screening of sgRNAs to achieve highly specific and highly efficient CRISPR/Cas9 genome editing.

| S-EPMC6018437 | biostudies-literature

Helicase-dependent isothermal DNA amplification.

Project description:Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.

| S-EPMC1249482 | biostudies-literature

Simultaneous amplification and screening of whole plasmids using the T7 bacteriophage replisome.

Project description:This study describes a novel helicase-mediated isothermal DNA amplification method that exponentially amplifies circular DNAs. The circular helicase-dependent amplification (cHDA) system is based on the T7 replication machinery, which includes the processive T7 helicase, an exonuclease-deficient T7 DNA polymerase (T7 Sequenase) and the T7 Gp2.5 single-stranded DNA-binding (SSB) protein. After the duplex DNA template is unwound by the T7 helicase, specific primers anneal to the separated DNA strands and T7 Sequenase extends the 3' end of each primer by a rolling circle mechanism to amplify not only a region defined by the primers but also continuous concatemers of the template. The cHDA reaction can be carried out at one temperature (25 degrees C) for the entire process and can achieve up to 10 000-fold amplification. Amplification can be performed using purified plasmid DNA or a crude cell lysate and can amplify inserts as large as 10 kb. Following a cHDA reaction, the amplified products can be used directly for sequencing and restriction enzyme digestion without further purification. By utilizing the helicase enzyme, circular DNA samples can be simultaneously screened and amplified at one constant temperature in one easy step.

| S-EPMC1540742 | biostudies-literature

Understanding DNA replication by the bacteriophage T4 replisome.

Project description:The T4 replisome has provided a unique opportunity to investigate the intricacies of DNA replication. We present a comprehensive review of this system focusing on the following: its 8-protein composition, their individual and synergistic activities, and assembly in vitro and in vivo into a replisome capable of coordinated leading/lagging strand DNA synthesis. We conclude with a brief comparison with other replisomes with emphasis on how coordinated DNA replication is achieved.

| S-EPMC5682956 | biostudies-literature

A novel ultrasensitive ECL sensor for DNA detection based on nicking endonuclease-assisted target recycling amplification, rolling circle amplification and hemin/G-quadruplex.

Project description:In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL) detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease)-assisted target recycling amplification (TRA), rolling circle amplification (RCA) and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form "Y" junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O8(2-) system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM) detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.

| S-EPMC4367324 | biostudies-literature

A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome.

Project description:Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.

| S-EPMC7335327 | biostudies-literature

Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease.

Project description:Many bacteriophage and prophage genomes encode an HNH endonuclease (HNHE) next to their cohesive end site and terminase genes. The HNH catalytic domain contains the conserved catalytic residues His-Asn-His and a zinc-binding site [CxxC](2). An additional zinc ribbon (ZR) domain with one to two zinc-binding sites ([CxxxxC], [CxxxxH], [CxxxC], [HxxxH], [CxxC] or [CxxH]) is frequently found at the N-terminus or C-terminus of the HNHE or a ZR domain protein (ZRP) located adjacent to the HNHE. We expressed and purified 10 such HNHEs and characterized their cleavage sites. These HNHEs are site-specific and strand-specific nicking endonucleases (NEase or nickase) with 3- to 7-bp specificities. A minimal HNH nicking domain of 76 amino acid residues was identified from Bacillus phage ? HNHE and subsequently fused to a zinc finger protein to generate a chimeric NEase with a new specificity (12-13 bp). The identification of a large pool of previously unknown natural NEases and engineered NEases provides more 'tools' for DNA manipulation and molecular diagnostics. The small modular HNH nicking domain can be used to generate rare NEases applicable to targeted genome editing. In addition, the engineered ZF nickase is useful for evaluation of off-target sites in vitro before performing cell-based gene modification.

| S-EPMC3592412 | biostudies-literature

Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.

Project description:Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.

| S-EPMC7570347 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data