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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.
 Science Education: Essentials of Environmental Microbiology

Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

JoVE Science Education

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

 Science Education: Basic Methods in Cellular and Molecular Biology

PCR: The Polymerase Chain Reaction

JoVE Science Education

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.

 JoVE Immunology and Infection

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

 JoVE Biology

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

 JoVE Genetics

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

 Science Education: Essentials of Environmental Science

Testing For Genetically Modified Foods

JoVE Science Education

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).

 JoVE Medicine

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

 JoVE 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

 JoVE Immunology and Infection

High-throughput Detection Method for Influenza Virus

1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, 2Department of Microbiology, Mount Sinai School of Medicine, 3Laboratory of Molecular Genetics, Blood Research Institute, 4City of Milwaukee Health Department Laboratory, 5Division of Hematology-Oncology/BMT, Children's Hospital of Wisconsin, Medical College of Wisconsin, 6Division of Hematology and Oncology, Dept Medicine, Medical College of Wisconsin


JoVE 3623

 Science Education: Essentials of Environmental Microbiology

Quantifying Environmental Microorganisms and Viruses Using qPCR

JoVE Science Education

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

Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, is a widely-used molecular technique for enumerating microorganisms in the environment. Prior to this approach, quantifying microorganisms was limited largely to classical culture-based techniques. However, the culturing of microbes from environmental samples can be particularly challenging, and it is generally held that as few as 1 to 10% of the microorganisms present within environmental samples are detectable using these techniques. The advent of qPCR in environmental microbiology research has therefore advanced the field greatly by allowing for more accurate determination of concentrations of microorganisms such as disease-causing pathogens in environmental samples. However, an important limitation of qPCR as an applied microbiological technique is that living, viable populations cannot be differentiated from inactive or non-living populations. This video demonstrates the use of qPCR to detect pepper mild mottle virus from an environmental water sample.

 JoVE Immunology and Infection

Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries

1The Recombinant Antibody Network, 2The Banting and Best Department of Medical Research, University of Toronto, 3Antibiome Center, University of California, San Francisco at Mission Bay, 4Department of Biochemistry and Molecular Biology, The University of Chicago


JoVE 51492

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