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Muscle & Nerve Jun 2020Transgenic animals are widely used for research and for most of them, genotyping is unavoidable. Published protocols may be powerful but may also present disadvantages...
BACKGROUND
Transgenic animals are widely used for research and for most of them, genotyping is unavoidable. Published protocols may be powerful but may also present disadvantages such as their cost or the requirement of additional steps/equipment. Moreover, if more than one strain must be genotyped, several protocols may need to be developed.
METHODS
we adapted the existing amplification-resistant mutation protocol to develop the 1-h universal genotyping protocol (1-HUG), which allows the robust genotyping of genetically modified mice in 1 h from sample isolation to polymerase chain reaction gel running.
RESULTS
This protocol allows the genotyping of different mouse models including mdx mouse, and FLExDUX4 and HSA-MerCreMer alone or in combination. It can be applied to different types of genomic modifications and to sexing.
CONCLUSIONS
The 1-HUG protocol can be used routinely in any laboratory using mouse models for neuromuscular diseases.
Topics: Animals; Genotype; Genotyping Techniques; Mice; Mice, Inbred mdx; Mice, Transgenic; Polymerase Chain Reaction; Species Specificity
PubMed: 32086834
DOI: 10.1002/mus.26841 -
Small (Weinheim An Der Bergstrasse,... Apr 2022Digital PCR (dPCR) surpasses the performance of earlier PCR formats because of highly precise, absolute quantification and other unique merits. A simple thermocycling...
Digital PCR (dPCR) surpasses the performance of earlier PCR formats because of highly precise, absolute quantification and other unique merits. A simple thermocycling approach and durable microcarrier are of great value for dPCR advancement and application. Herein, a near-infrared (NIR) controlled thermocycling approach by embedding magnetic graphene oxide (GO) composite into the agarose microcarriers is developed. The core-shell composite is constructed by sequentially encapsulating GO and silica outside the magnetic nanocores. Benefiting from these additives, the resultant composite agarose gains appealing features as light-driven temperature changing, switchable gel-sol phase transforming, biocompatibility, and magnetic traction. By further emulsifying into droplets via the microfluidics method, the influence of typical parameters including material loading amount, laser intensity, and droplet diameter at various ranges is investigated for assembling microcarriers with different responsiveness. Then a paradigm of the NIR program can be easily tailored for PCR thermocycling. Finally, the feasibility of the approach is verified by detecting statistically diluted Klebsiella pneumoniae DNA samples, from 0.1 to 2 copies per drop. It is anticipated that this method has promising prospects for dPCR-based and other temperature-controlled applications.
Topics: DNA; Microfluidics; Polymerase Chain Reaction; Sepharose
PubMed: 35212452
DOI: 10.1002/smll.202107858 -
International Journal of Molecular... Jul 2023DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an...
DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing DNA polymerase for routine use in laboratories. We developed a method using () that expresses thermostable DNA polymerase directly in the PCR without purification. The gene was transformed into and expressed. After overnight incubation and washing, -expressing DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.
Topics: Taq Polymerase; Escherichia coli; Polymerase Chain Reaction; DNA; DNA Replication
PubMed: 37511160
DOI: 10.3390/ijms241411405 -
Methods in Molecular Biology (Clifton,... 2022DEL technology is dependent on the generation and analysis of large amounts of DNA sequence data. In this chapter, we describe a method to sequence DEL libraries that...
DEL technology is dependent on the generation and analysis of large amounts of DNA sequence data. In this chapter, we describe a method to sequence DEL libraries that uses a customized preparative PCR protocol, along with standard steps for purification, analysis, and sequencing of the amplified library DNA on an Illumina sequencing platform. Compared with standard Illumina sequencing library preparation protocols, in which a PCR reaction is followed by end repair, adenylation, ligation of Illumina adapters, and a second preparative PCR reaction, the customized operations described here provide significantly improved process efficiency and sequencing quality.
Topics: DNA; Gene Library; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 36083556
DOI: 10.1007/978-1-0716-2545-3_21 -
Canadian Journal of Microbiology Jun 2022Since the introduction of the polymerase chain reaction (PCR) technique in 1983, nucleic acid amplification has permeated all fields of biological science, particularly... (Review)
Review
Since the introduction of the polymerase chain reaction (PCR) technique in 1983, nucleic acid amplification has permeated all fields of biological science, particularly clinical research. Despite its importance, PCR has been restricted to specialized centers and its use in laboratories with few resources is limited. In recent decades, there has been a notable increase in the development of new isothermal technologies for molecular diagnosis with the hope of overcoming the traditional limitations of the laboratory. Among these technologies, recombinase polymerase amplification (RPA) has a wide application potential because it does not require thermocyclers and has high sensitivity, specificity, simplicity, and detection speed. This technique has been used for DNA and RNA amplification in various pathogenic organisms such as viruses, bacteria, and parasites. In addition, RPA has been successfully implemented in different detection strategies, making it a promising alternative for performing diagnoses in environments with scarce resources and a high burden of infectious diseases. In this study, we present a review of the use of RPA in clinical settings and its implementation in various research areas.
Topics: Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Recombinases; Sensitivity and Specificity
PubMed: 35394399
DOI: 10.1139/cjm-2021-0329 -
Nature Communications Aug 2023DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases....
DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.
Topics: Polymerase Chain Reaction; DNA; DNA Methylation; Temperature; Oligonucleotides; CpG Islands
PubMed: 37620381
DOI: 10.1038/s41467-023-40873-y -
Cold Spring Harbor Protocols Jan 2024A simple method to determine the genetic sex of a mouse is to amplify DNA from a male-specific gene by polymerase chain reaction (PCR). This protocol is used to detect...
A simple method to determine the genetic sex of a mouse is to amplify DNA from a male-specific gene by polymerase chain reaction (PCR). This protocol is used to detect the Y-chromosome-specific gene in tissue lysates of tail tip or ear punch samples.
Topics: Mice; Male; Animals; Genotype; Y Chromosome; Polymerase Chain Reaction; DNA
PubMed: 37932078
DOI: 10.1101/pdb.prot108062 -
Methods in Molecular Biology (Clifton,... 2022Multiplex quantitative polymerase chain reaction (multiplex qPCR) enables the amplification of more than one target in a single reaction using different reporter dyes...
Multiplex quantitative polymerase chain reaction (multiplex qPCR) enables the amplification of more than one target in a single reaction using different reporter dyes with distinct fluorescent spectra. The number of reporter fluorophores is typically restricted to three or four, depending upon the capability of the real-time PCR platform and software used. Each target is amplified by a different set of primers and a uniquely labeled probe that distinguishes each PCR amplicon. Thus, the levels of several targets of interest can be quantified in real time. By combining several reactions in a single tube, multiplex qPCR reduces the quantity, and cost of reagents needed to screen a sample for multiple targets. Specificity and efficiency are not affected by the inclusion of the three assays in a multiplex reaction.
Topics: DNA Primers; Multiplex Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Viroids
PubMed: 34845695
DOI: 10.1007/978-1-0716-1464-8_16 -
Lab on a Chip Oct 2019The polymerase chain reaction (PCR) is a popular and well-established DNA amplification technique. Technological and engineering advancements in the field of...
The polymerase chain reaction (PCR) is a popular and well-established DNA amplification technique. Technological and engineering advancements in the field of microfluidics have fuelled the progress of polymerase chain reaction (PCR) technology in the last three decades. Advances in microfluidics-based PCR technology have significantly reduced the sample volume and thermal cycling time. Further advances led to novel and accurate techniques such as the digital PCR. However, contamination of PCR samples, lack of reusability of the microfluidic PCR platforms, complexity in instrumentation and operation remain as some of the significant drawbacks of conventional microfluidic PCR platforms. Liquid marbles, the recently emerging microfluidic platform, could potentially resolve these drawbacks. This paper reports the first liquid marble based polymerase chain reaction. We demonstrated an experimental setup for the liquid-marble based PCR with a humidity-controlled chamber and an embedded thermal cycler. A concentrated salt solution was used to control the humidity of the PCR chamber which in turn reduces the evaporation rate of the liquid marble. The successful PCR of microbial source tracking markers for faecal contamination was achieved with the system, indicating potential application in water quality monitoring.
Topics: Humidity; Lipids; Microfluidic Analytical Techniques; Polymerase Chain Reaction
PubMed: 31464317
DOI: 10.1039/c9lc00676a -
Archives of Razi Institute Jul 2021Authentication of animal cell lines in cell banks is one of the most important programs regulated during cell culture and storage. This operation provides a thorough and...
Authentication of animal cell lines in cell banks is one of the most important programs regulated during cell culture and storage. This operation provides a thorough and beneficial document which can be advantageous for the functional use of animal cell lines. Therefore, various procedures are used to prevent misidentified cells, cross-contamination to other cell lines, and mislabeling errors leading to incorrect assessment. These contaminants can result in major financial disadvantages. One of the practical methods in this field is a molecular procedure which can demonstrate more accurate results. In the present study, the BHK-21 (C5) was characterized, and it was tried to determine the identity of BHK-21 (C5) as a continuous cell line by Polymerase chain reaction (PCR) molecular procedure in Iran. The cytochrome c oxidase I (CO1) gene was selected as a prevalent DNA fragment for the authentication of the BHK-21 (C5) cell line, along with six cell lines, including Chinese hamster ovary, Lamb kidney, Razi Bovine Kidney, Medical Research Council cell strain 5, Monkey Green Kidney, and Goat Lymphocyte. After amplification, PCR products were analyzed by agarose gel electrophoresis to ensure their accuracy. The results of characterization were indicated, cell viability was estimated to be about 92%, and a uniform cell culture was obtained. The doubling time and µ ratio equivalent were obtained at 20.5 h and 0.03, respectively. Sterility tests revealed that the cell seed was free of bacterial, mycoplasma, and mycobacterial infections. The results of molecular identification revealed that the identification of this cell line was approved and can be used in studies, diagnosis, production, and quality control of biological products.
Topics: Animals; CHO Cells; Cattle; Cricetinae; Cricetulus; DNA Primers; Iran; Polymerase Chain Reaction; Sheep
PubMed: 34223718
DOI: 10.22092/ari.2020.128637.1419