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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

Video Coming Soon

JoVE 55147


 JoVE In-Press

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

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

1Department of Pediatrics, Section of Diabetes and Endocrinology, McNair Medical Institute, Baylor College of Medicine, Texas Children's Hospital, 2Benaroya Research Institute at Virginia Mason, 3Center for Human Immunobiology, Baylor College of Medicine, Texas Children's Hospital, 4Department of Pediatrics, Section of Diabetes and Endocrinology, Baylor College of Medicine, Texas Children's Hospital

JoVE 55379


 Immunology and Infection

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


 Genetics

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


 Biology

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


 Genetics

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

Video Coming Soon

JoVE 56319


 JoVE In-Press

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

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


 Medicine

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


 Bioengineering

Nanomanipulation of Single RNA Molecules by Optical Tweezers

1Nanoscale Engineering Graduate Program, College of Nanoscale Science and Engineering, University at Albany, State University of New York, 2Nanoscale Science Undergraduate Program, College of Nanoscale Science and Engineering, University at Albany, State University of New York, 3Nanobioscience Constellation, College of Nanoscale Science and Engineering, University at Albany, State University of New York, 4The RNA Institute, University at Albany, State University of New York, 5Department of Biological Sciences, University at Albany, State University of New York

JoVE 51542


 Bioengineering

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