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Methods in Molecular Biology (Clifton,... 2024In the last few decades, molecular techniques and genetic modification have been used in genotype and phenotype studies of S. suis. Genomic modification of S. suis...
In the last few decades, molecular techniques and genetic modification have been used in genotype and phenotype studies of S. suis. Genomic modification of S. suis requires DNA acquisition and its stable insertion into the chromosome by allelic exchange. In this chapter, we described two techniques for the preparation of genomic constructs (cloning and overlapping extension PCR) and for DNA uptake (electroporation and transformation). The protocols are accompanied with examples. All described protocols were successful on our hands with the reference S. suis strain P1/7.
Topics: Electroporation; Cloning, Molecular; Polymerase Chain Reaction
PubMed: 38884909
DOI: 10.1007/978-1-0716-3898-9_4 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Feb 2017Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated... (Review)
Review
Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.
Topics: DNA; DNA Copy Number Variations; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction
PubMed: 28956373
DOI: 10.13345/j.cjb.160269 -
Molecular Diagnosis & Therapy Jun 2017Somatic mutations bear great promise for use as biomarkers for personalized medicine, but are often present only in low abundance in biological material and are... (Review)
Review
Somatic mutations bear great promise for use as biomarkers for personalized medicine, but are often present only in low abundance in biological material and are therefore difficult to detect. Many assays for mutation analysis in cancer-related genes (hotspots) have been developed to improve diagnosis, prognosis, prediction of drug resistance, and monitoring of the response to treatment. Two major approaches have been developed: mutation-specific amplification methods and methods that enrich and detect mutations without prior knowledge on the exact location and identity of the mutation. CO-amplification at Lower Denaturation temperature Polymerase Chain Reaction (COLD-PCR) methods such as full-, fast-, ice- (improved and complete enrichment), enhanced-ice, and temperature-tolerant COLD-PCR make use of a critical temperature in the polymerase chain reaction to selectively denature wild-type-mutant heteroduplexes, allowing the enrichment of rare mutations. Mutations can subsequently be identified using a variety of laboratory technologies such as high-resolution melting, digital polymerase chain reaction, pyrosequencing, Sanger sequencing, or next-generation sequencing. COLD-PCR methods are sensitive, specific, and accurate if appropriately optimized and have a short time to results. A large variety of clinical samples (tumor DNA, circulating cell-free DNA, circulating cell-free fetal DNA, and circulating tumor cells) have been studied using COLD-PCR in many different applications including the detection of genetic changes in cancer and infectious diseases, non-invasive prenatal diagnosis, detection of microorganisms, or DNA methylation analysis. In this review, we describe in detail the different COLD-PCR approaches, highlighting their specificities, advantages, and inconveniences and demonstrating their use in different fields of biological and biomedical research.
Topics: DNA Methylation; Drug Resistance, Neoplasm; Female; High-Throughput Nucleotide Sequencing; Humans; Multiplex Polymerase Chain Reaction; Mutation; Polymerase Chain Reaction; Precision Medicine; Pregnancy; Prenatal Diagnosis
PubMed: 28101802
DOI: 10.1007/s40291-016-0254-8 -
Cold Spring Harbor Protocols May 2016When screening a large number of individual Schizosaccharomyces pombe strains by polymerase chain reaction (PCR), a rapid "colony PCR" approach may be used. Numerous...
When screening a large number of individual Schizosaccharomyces pombe strains by polymerase chain reaction (PCR), a rapid "colony PCR" approach may be used. Numerous colony PCR protocols are available, and fundamental to them all is that the colony must be fresh (grown overnight) and that as few cells as possible are used. In this protocol, we present three reliable methods for preparing S. pombe cells for colony PCR.
Topics: DNA, Fungal; Polymerase Chain Reaction; Schizosaccharomyces
PubMed: 27140919
DOI: 10.1101/pdb.prot090993 -
Biotechnology and Bioengineering Sep 2018We demonstrate the integration of DNA amplification and detection functionalities developed on a lab-on-a-chip microdevice utilizing solid-phase polymerase chain...
We demonstrate the integration of DNA amplification and detection functionalities developed on a lab-on-a-chip microdevice utilizing solid-phase polymerase chain reaction (SP-PCR) for point-of-need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding to contain microchambers as reservoirs for performing SP-PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine-modified forward primers. During SP-PCR, the immobilized forward primers and freely diffusing fluorescence-labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection. The SP-PCR microdevice was used for the direct identifications of two widely detected foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus, and an alga causing harmful algal blooms annually in South Korea, Cochlodinium polykrikoides. The SP-PCR microdevice would be versatilely applied in PON testing as a universal platform for the fast identification of foodborne pathogens and environmentally threatening biogenic targets.
Topics: Dinoflagellida; Lab-On-A-Chip Devices; Microbiological Techniques; Molecular Diagnostic Techniques; Point-of-Care Systems; Polymerase Chain Reaction; Salmonella; Staphylococcus aureus; Time Factors
PubMed: 29777597
DOI: 10.1002/bit.26734 -
Analytica Chimica Acta Apr 2020Polymerase chain reaction (PCR) is an extremely important tool for molecular diagnosis, as it can specifically amplify nucleic acid templates for sensitive detection. As... (Review)
Review
Polymerase chain reaction (PCR) is an extremely important tool for molecular diagnosis, as it can specifically amplify nucleic acid templates for sensitive detection. As another division of PCR, free convective PCR was invented in 2001, which can be performed in a capillary tube pseudo-isothermally within a significantly short time. Convective PCR thermal cycling is implemented by inducing thermal convection inside the capillary tube, which stratifies the reaction into spatially separate and stable melting, annealing, and extension zones created by the temperature gradient. Convective PCR is a promising tool that can be used for nucleic acid diagnosis as a point-of-care test (POCT) due to the significantly simplified heating strategy, reduced cost, and shortened detection time without sacrificing sensitivity and accuracy. Here, we review the history of free convective PCR from its invention to development and its commercial applications.
Topics: Convection; Heating; Polymerase Chain Reaction
PubMed: 32222239
DOI: 10.1016/j.aca.2020.01.069 -
Bioanalysis Dec 2021Polymerase chain reaction (PCR) is widely used in various fields of laboratory testing, ranging from forensic, molecular biology, medical and diagnostic applications to...
Polymerase chain reaction (PCR) is widely used in various fields of laboratory testing, ranging from forensic, molecular biology, medical and diagnostic applications to a wide array of basic research purposes. COVID-19 infection testing has brought the three-letter PCR abbreviation into the vocabulary of billions of people, making it likely the most well-known laboratory test worldwide. With new modalities and translational medicine gaining importance in pharmaceutical research and development, PCR or more specifically, quantitative PCR (qPCR) is now becoming a standard tool in the (regulated) bioanalytical laboratory, driving the bioanalytical community to define best practices for method development, characterization and validation. In absence of specific guidance from health authorities, qPCR may be vulnerable to scope creep from pharmacokinetics (PK) assay validation as defined in bioanalytical method validation guidance/guidelines. In this manuscript, the European Bioanalysis Forum builds a rationale for applying context of use principles when defining requirements for qPCR assay performance and validation criteria.
Topics: Biological Assay; Europe; Humans; Polymerase Chain Reaction; Research Design
PubMed: 34708666
DOI: 10.4155/bio-2021-0218 -
Pediatric and Developmental Pathology :... 2015Children's healthcare has evolved over the years, and the pediatric laboratory has contributed to the clinical understanding of childhood disease through the application... (Review)
Review
Children's healthcare has evolved over the years, and the pediatric laboratory has contributed to the clinical understanding of childhood disease through the application of new technology and knowledge. This article highlights the evolution of PCR technology to aid in the diagnosis of pediatric infections, from the discovery of the PCR, through the subsequent years when the clinical need exceeded the capability of the technology, until the current day, when application of the PCR is becoming commonplace.
Topics: Bacterial Infections; DNA, Bacterial; DNA, Viral; Diffusion of Innovation; Equipment Design; History, 20th Century; History, 21st Century; Humans; Molecular Diagnostic Techniques; Pathology, Molecular; Pediatrics; Polymerase Chain Reaction; Predictive Value of Tests; Reproducibility of Results; Virus Diseases
PubMed: 26701384
DOI: 10.2350/15-05-1643-OA.1 -
Fa Yi Xue Za Zhi Oct 2017Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a convenient and highly efficient method for the detection of mRNA in tissues or... (Review)
Review
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a convenient and highly efficient method for the detection of mRNA in tissues or body fluid samples. It has the characteristics of easy operation, high sensitivity and specificity, etc. With a wide application in medicine, biology and other fields, RT-qPCR technique has made some progresses in the research field of forensic pathology. This paper reviews the application value of RT-qPCR in the study of forensic pathology and current situation, as well as the research progress at home and abroad reviews. It also summarizes the notes of samples extraction, RT-qPCR experiments and data processing, which aims to provide reference for the forensic research and its application.
Topics: Forensic Genetics; Forensic Pathology; Humans; RNA, Messenger; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 29275561
DOI: 10.3969/j.issn.1004-5619.2017.05.017 -
Expert Review of Molecular Diagnostics Feb 2017Molecular diagnostics is a key component of laboratory medicine. Here, the authors review key triggers of ever-increasing automation in nucleic acid amplification... (Review)
Review
Molecular diagnostics is a key component of laboratory medicine. Here, the authors review key triggers of ever-increasing automation in nucleic acid amplification testing (NAAT) with a focus on specific automated Polymerase Chain Reaction (PCR) testing and platforms such as the recently launched cobas® 6800 and cobas® 8800 Systems. The benefits of such automation for different stakeholders including patients, clinicians, laboratory personnel, hospital administrators, payers, and manufacturers are described. Areas Covered: The authors describe how molecular diagnostics has achieved total laboratory automation over time, rivaling clinical chemistry to significantly improve testing efficiency. Finally, the authors discuss how advances in automation decrease the development time for new tests enabling clinicians to more readily provide test results. Expert Commentary: The advancements described enable complete diagnostic solutions whereby specific test results can be combined with relevant patient data sets to allow healthcare providers to deliver comprehensive clinical recommendations in multiple fields ranging from infectious disease to outbreak management and blood safety solutions.
Topics: Automation, Laboratory; Humans; Infections; Molecular Diagnostic Techniques; Polymerase Chain Reaction
PubMed: 28043179
DOI: 10.1080/14737159.2017.1275962