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Genes To Cells : Devoted To Molecular &... Jan 2018Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics...
Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS.
Topics: 3' Untranslated Regions; Arabidopsis; Arabidopsis Proteins; Cell-Free System; Cloning, Molecular; DNA Primers; DNA, Plant; Polymerase Chain Reaction; Protein Biosynthesis; RNA Processing, Post-Transcriptional
PubMed: 29235215
DOI: 10.1111/gtc.12547 -
Biochemistry Aug 2009DNA primases are pivotal enzymes in chromosomal DNA replication in all organisms. In this article, we report unique mechanistic characteristics of recombinant DNA...
DNA primases are pivotal enzymes in chromosomal DNA replication in all organisms. In this article, we report unique mechanistic characteristics of recombinant DNA primase from Bacillus anthracis. The mechanism of action of B. anthracis DNA primase (DnaG(BA)) may be described in several distinct steps as follows. Its mechanism of action is initiated when it binds to single-stranded DNA (ssDNA) in the form of a trimer. Although DnaG(BA) binds to different DNA sequences with moderate affinity (as expected of a mobile DNA binding protein), we found that DnaG(BA) bound to the origin of bacteriophage G4 (G4ori) with approximately 8-fold higher affinity. DnaG(BA) was strongly stimulated (>or=75-fold) by its cognate helicase, DnaB(BA), during RNA primer synthesis. With the G4ori ssDNA template, DnaG(BA) formed short (
primers in the absence of DnaB(BA). The presence of DnaB(BA) increased the rate of primer synthesis. The observed stimulation of primer synthesis by cognate DnaB(BA) is thus indicative of a positive effector role for DnaB(BA). By contrast, Escherichia coli DnaB helicase (DnaB(EC)) did not stimulate DnaG(BA) and inhibited primer synthesis to near completion. This observed effect of E. coli DnaB(EC) is indicative of a strong negative effector role for heterologous DnaB(EC). We conclude that DnaG(BA) is capable of interacting with DnaB proteins from both B. anthracis and E. coli; however, between DnaB proteins derived from these two organisms, only the homologous DNA helicase (DnaB(BA)) acted as a positive effector of primer synthesis. Topics: Amino Acid Sequence; Bacillus anthracis; DNA Primase; DNA Primers; DNA Replication; DNA-Binding Proteins; Escherichia coli Proteins; Molecular Sequence Data; Protein Binding; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 19583259
DOI: 10.1021/bi900086z -
BMC Bioinformatics Sep 2014PRISE2 is a new software tool for designing sequence-selective PCR primers and probes. To achieve high level of selectivity, PRISE2 allows the user to specify a...
BACKGROUND
PRISE2 is a new software tool for designing sequence-selective PCR primers and probes. To achieve high level of selectivity, PRISE2 allows the user to specify a collection of target sequences that the primers are supposed to amplify, as well as non-target sequences that should not be amplified. The program emphasizes primer selectivity on the 3' end, which is crucial for selective amplification of conserved sequences such as rRNA genes. In PRISE2, users can specify desired properties of primers, including length, GC content, and others. They can interactively manipulate the list of candidate primers, to choose primer pairs that are best suited for their needs. A similar process is used to add probes to selected primer pairs. More advanced features include, for example, the capability to define a custom mismatch penalty function. PRISE2 is equipped with a graphical, user-friendly interface, and it runs on Windows, Macintosh or Linux machines.
RESULTS
PRISE2 has been tested on two very similar strains of the fungus Dactylella oviparasitica, and it was able to create highly selective primers and probes for each of them, demonstrating the ability to create useful sequence-selective assays.
CONCLUSIONS
PRISE2 is a user-friendly, interactive software package that can be used to design high-quality selective primers for PCR experiments. In addition to choosing primers, users have an option to add a probe to any selected primer pair, enabling design of Taqman and other primer-probe based assays. PRISE2 can also be used to design probes for FISH and other hybridization-based assays.
Topics: Conserved Sequence; DNA Primers; Oligonucleotide Probes; Polymerase Chain Reaction; Software
PubMed: 25252611
DOI: 10.1186/1471-2105-15-317 -
Genetics and Molecular Research : GMR Feb 2016The whole-genome sequencing of coxsackievirus (CV)-A10 does not follow a conventional experimental protocol. To fully understand the genetic variation and evolution of...
The whole-genome sequencing of coxsackievirus (CV)-A10 does not follow a conventional experimental protocol. To fully understand the genetic variation and evolution of CV-A10, complete genome amplification is necessary. Most previous studies have concentrated on partial sequences of the CV-A10 genome, such as the VP1 gene. The few studies that have investigated CV-A10 at the genomic level have reported only two complete genome sequences to GenBank. The basic fault may be attributed to the regional nature of the genetics and evolution of CV-A10 and to the lack of laboratory procedures for obtaining the genomes. In this study, we present a robust "three-step" protocol performed with A105UF/A820, EVP4/A6141, and A4879/A1005R for the full-length genome amplification of CV-A10. The results revealed that the method is able to accurately and reproducibly amplify three fragments with overlaps of the full-length genome of eight CV-A10 strains. Compared with other methods, this assay is both quick and specific. In addition, the three-step protocol could be capable of amplifying the full-length genomes of CV-A10 strains isolated from different countries and regions. The specific three-step protocol may be particularly useful for investigating samples co-infected with CV-A10 and other viruses.
Topics: Chromosome Mapping; DNA Primers; Enterovirus A, Human; Genome, Viral; Genotype; RNA, Viral; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 26909991
DOI: 10.4238/gmr.15017513 -
Fly Oct 2017The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However,...
The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.
Topics: Alleles; Animals; Base Sequence; CRISPR-Cas Systems; Cell Line; DNA Primers; Drosophila; Genetic Engineering; Genome, Insect; Mutation; Sequence Homology; Single-Cell Analysis
PubMed: 28853976
DOI: 10.1080/19336934.2017.1372068 -
BMC Genomics Nov 2021A large number of variants have been employed in various medical applications, such as providing medication instructions, disease susceptibility testing, paternity...
BACKGROUND
A large number of variants have been employed in various medical applications, such as providing medication instructions, disease susceptibility testing, paternity testing, and tumour diagnosis. A high multiplicity PCR will outperform other technologies because of its lower cost, reaction time and sample consumption. To conduct a multiplex PCR with higher than 100 plex multiplicity, primers need to be carefully designed to avoid the formation of secondary structures and nonspecific amplification between primers, templates and products. Thus, a user-friendly, highly automated and highly user-defined web-based multiplex PCR primer design software is needed to minimize the work of primer design and experimental verification.
RESULTS
Ultiplex was developed as a free online multiplex primer design tool with a user-friendly web-based interface ( http://ultiplex.igenebook.cn ). To evaluate the performance of Ultiplex, 294 out of 295 (99.7%) target primers were successfully designed. A total of 275 targets produced qualified primers after primer filtration, and 271 of those targets were successfully clustered into one compatible PCR group and could be covered by 108 primers. The designed primer group stably detected the rs28934573(C > T) mutation at lower than a 0.25% mutation rate in a series of samples with different ratios of HCT-15 and HaCaT cell line DNA.
CONCLUSION
Ultiplex is a web-based multiplex PCR primer tool that has several functions, including batch design and compatibility checking for the exclusion of mutual secondary structures and mutual false alignments across the whole genome. It offers flexible arguments for users to define their own references, primer Tm values, product lengths, plex numbers and tag oligos. With its user-friendly reports and web-based interface, Ultiplex will provide assistance for biological applications and research involving genomic variants.
Topics: DNA Primers; Internet; Multiplex Polymerase Chain Reaction; Software; Workflow
PubMed: 34794394
DOI: 10.1186/s12864-021-08149-1 -
Nucleic Acids Research Feb 2009A simple isothermal nucleic-acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), was developed to detect specific nucleic-acid sequences...
A simple isothermal nucleic-acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60 degrees C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, 'primers' are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG-RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of 'primers' are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (approximately 60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG-RCA to various molecular diagnostic assays.
Topics: DNA Primers; DNA Probes; DNA, Bacterial; DNA, Circular; Nucleic Acid Amplification Techniques; Sequence Analysis, DNA; Temperature; Time Factors
PubMed: 19106144
DOI: 10.1093/nar/gkn1014 -
BMC Bioinformatics Jan 2014MicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the...
BACKGROUND
MicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the reference that is used to validate other methods. Several commercial qPCR assays are available but they often come at a high price and the sequences of the primers are not disclosed. An alternative to commercial assays is to manually design primers but this work is tedious and, hence, not practical for the design of primers for a larger number of targets.
RESULTS
I have developed the software miRprimer for automatic design of primers for the method miR-specific RT-qPCR, which is one of the best performing microRNA qPCR methods available. The algorithm is based on an implementation of the previously published rules for manual design of miR-specific primers with the additional feature of evaluating the propensity of formation of secondary structures and primer dimers. Testing of the primers showed that 76 out of 79 primers (96%) worked for quantification of microRNAs by miR-specific RT-qPCR of mammalian RNA samples. This success rate corresponds to the success rate of manual primer design. Furthermore, primers designed by this method have been distributed to several labs and used successfully in published studies.
CONCLUSIONS
The software miRprimer is an automatic and easy method for design of functional primers for miR-specific RT-qPCR. The application is available as stand-alone software that will work on the MS Windows platform and in a developer version written in the Ruby programming language.
Topics: Algorithms; Computational Biology; DNA Primers; MicroRNAs; RNA Probes; Real-Time Polymerase Chain Reaction; Software
PubMed: 24472427
DOI: 10.1186/1471-2105-15-29 -
BioTechniques Apr 2018Real-time PCR (qPCR) is widely used in the life sciences. For quantifying DNA, a standard curve is required. Common methods for standard development are time consuming,...
Real-time PCR (qPCR) is widely used in the life sciences. For quantifying DNA, a standard curve is required. Common methods for standard development are time consuming, costly, necessitate a specific skill set, and pose a contamination risk. Using a targeted synthetic oligonucleotide, such as a gBlocks Gene Fragment, overcomes these drawbacks and provides researchers an accurate and quick solution to standard development. Here, we demonstrate that using a gBlocks fragment as a standard provides comparable sensitivity, reliability, and assay performance to a purified amplicon standard.
Topics: DNA; DNA Primers; DNA Probes; Oligonucleotides; Real-Time Polymerase Chain Reaction; Reproducibility of Results
PubMed: 29661012
DOI: 10.2144/btn-2018-2000 -
Nature Protocols Mar 2019CRISPR-Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low...
CRISPR-Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 10 cells. Our method harnesses two simple PCR steps, separated by automated electrophoresis and extraction of size-selected DNA amplicons, thus allowing the removal of unexpanded arrays from the sample pool and enabling 1,000-times more sensitive detection of new spacers than alternative PCR protocols. CAPTURE is a straightforward method that requires only 1 d to enable the detection of spacer acquisition in all native CRISPR systems and facilitate studies aimed both at unraveling the mechanism of spacer integration and more sensitive tracing of integration events in natural ecosystems.
Topics: CRISPR-Cas Systems; DNA Primers; Electrophoresis, Agar Gel; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction
PubMed: 30742049
DOI: 10.1038/s41596-018-0123-5