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Polymerase Chain Reaction: In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.

Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

JoVE 10081

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz

Polymerase chain reaction (PCR) is a technique used to detect microorganisms that are present in soil, water, and atmospheric environments. By amplifying specific sections of DNA, PCR can facilitate the detection and identification of target microorganisms down to the species, strain, and serovar/pathovar level. The technique can also be utilized to characterize entire communities of microorganisms in samples. The culturing of microorganisms in the laboratory using specialized growth media is a long-established technique and remains in use for the detection of microorganisms in environmental samples. Many microbes in the natural environment, while alive, maintain low levels of metabolic activity and/or doubling times and are thus referred to as viable but non-culturable (VBNC) organisms. The use of culture-based techniques alone cannot detect these microbes and, therefore, does not provide a thorough assessment of microbial populations in samples. The use of PCR allows for the detection of culturable microbes, VBNC organisms, and those that are no longer alive or active, as the amplification of genetic sequences does not generally require the pre-enrichment of microorga

 Environmental Microbiology

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

1Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, 2Department of Orthopaedics and Trauma Surgery, University Hospital Bonn, 3Division of EU Cooperation/Microbiology, Paul-Ehrlich-Institute

JoVE 55147


PCR: The Polymerase Chain Reaction

JoVE 5056

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling – cyles of temperature changes at fixed time intervals. Using a thermostable DNA polymerase, PCR can create numerous copies of DNA from DNA building blocks called dinucleoside triphosphates or dNTPs. There are three steps in PCR: denaturation, annealing, and elongation. Denaturation is the first step in the cycle and causes the DNA to melt by disrupting hydrogen bonds between the bases resulting in single-stranded DNA. Annealing lowers the temperature enough to allow the binding of oligonucleotide primers to the DNA template. During the elongation step DNA polymerase will synthesize new double-stranded DNA. This video provides an introduction to the PCR procedure. The basic principles of PCR are described as well as a step-by-step procedure for setting up a generalized PCR reaction. The video shows the necessary components for a PCR reaction, includes instruction for primer design, and provides helpful hints for ensuring successful PCR reactions.

 Basic Methods in Cellular and Molecular Biology

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

1UCLA AIDS Institute, University of California at Los Angeles (UCLA), 2Department of Microbiology, Immunology, & Molecular Genetics, University of California at Los Angeles (UCLA), 3Departments of Biomathematics and Mathematics, University of California at Los Angeles (UCLA), 4Personalized Genomic Medicine Research Center, Division of Strategic Research Groups, Korea Research Institute of Bioscience and Biotechnology, 5Department of Medicine, University of California at Los Angeles (UCLA), 6Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University (OSU)

JoVE 55812


Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

1Department of Microbiology, University of Washington, 2Departments of Medicine and Laboratory Medicine, University of Washington, 3U.S Military HIV Research Program, Walter Reed Army Institute of Research, 4Henry M. Jackson Foundation

JoVE 52610

 Immunology and Infection

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

1Department of Medicine, Weill Cornell Medical College, 2Institute for Computational Biomedicine, Weill Cornell Medical College, 3Department of Physiology and Biophysics, Weill Cornell Medical College, 4Department of Pathology, University of Michigan

JoVE 52246


Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 2NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 3Food Science and Chemical Engineering, Singapore Institute of Technology

JoVE 54371


Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

1Department of Translational Research, Tsurumi University School of Dental Medicine, 2Endowed Department of International Oral Health Science (affiliated with Department of Translational Research), Tsurumi University School of Dental Medicine, 3Cariology and Operative Dentistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University

JoVE 56319


Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

1Center for AIDS Health Disparities Research, Meharry Medical College, 2Department of Biochemistry and Cancer Biology, Meharry Medical College, 3School of Graduate Studies and Research, Meharry Medical College, 4Department of Microbiology and Immunology, Meharry Medical College, 5Tennessee Center for AIDS Research (TN-CFAR), Meharry Medical College

JoVE 54581

 Immunology and Infection

Testing For Genetically Modified Foods

JoVE 10044

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Genetic modification of foods has been a controversial issue due to debated concerns over health and environmental safety. This experiment demonstrates technical understanding of how food DNA is genetically identified, allowing for educated decision making about the safety and potential dangers of using genetically modified organisms (GMOs) in food supplies. Polymerase Chain Reaction (PCR) is used to amplify food DNA to test for the presence of genetically modified DNA in food products. Presence of specific DNA bands is detected by using gel electrophoresis to pull extracted food DNA through a 3% agarose gel, a concentration dense enough to separate the bands of DNA containing the genetically modified DNA. Several controls are used in the electrophoresis procedure to ensure DNA is successfully extracted from test foods (plant primer), and to provide known examples of both genetically modified DNA (purchased genetically modified DNA) and non-genetically modified DNA (purchased certified non-GMO food control).

 Environmental Science

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-Derived Cells

1Chang Gung University, 2Department of Periodontics, Chang Gung Memorial Hospital, 3College of Medicine, California Northstate University, 4Department of Nephrology and Clinical Poison Center, Chang Gung Memorial Hospital, 5Kidney Research Center, Chang Gung Memorial Hospital, 6Center for Tissue Engineering, Chang Gung Memorial Hospital, 7Department of Periodontics, Chang Gung Memorial Hospital, 8School of Dental Technology, College of Oral Medicine, Taipei Medical University

Video Coming Soon

JoVE 57166

 JoVE In-Press

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

1SS Annunziata University Hospital, Unit of Clinical Molecular Biology and Predictive Medicine, University of Chieti, 2Department of Medical, Oral and Biotechnological Sciences, University of Chieti, 3Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 4CNR - Institute of Molecular Genetics, Section of Chieti

Video Coming Soon

JoVE 57289

 JoVE In-Press

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

1Africa Centre for Health and Population Studies, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa, 2Unit D11, Jembi Health Systems, 3Academic Medical Center, Department of Global Health, Amsterdam Institute for Global Health and Development (AIGHD), University of Amsterdam, 4Division of Infectious Diseases and Geographic Medicine, Centre for AIDS Research, Stanford Medical School

JoVE 51242


Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

1Department of Mechanical Engineering, Carnegie Mellon University, 2Engineering Science and Mechanics Program, Virginia Polytechnic Institute and State University, 3Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 4Department of Bioengineering, University of Pittsburgh

JoVE 55300


Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

1Laboratory of Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 2Laboratory of Functional Device, Institute of Advanced Technology, Universiti Putra Malaysia, 3Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 4La Trobe Institute for Molecular Science, La Trobe University

Video Coming Soon

JoVE 56585

 JoVE In-Press

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

1Department of Research, Hackensack University Medical Center, 2Department of Medical Sciences, Seton Hall University, 3Department of Epidemiology and Population Health, Albert Einstein College of Medicine, 4Department of Nephrology and Hypertension, Hadassah - Hebrew University Medical Center

Video Coming Soon

JoVE 57471

 JoVE In-Press

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