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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.

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 …

 Basic Methods in Cellular and Molecular Biology

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

1Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department, Institut Curie, PSL Research University, 2CNRS UMR144, Institut Curie, PSL Research University, 3Department of Medical Oncology, Institut Curie, PSL Research University, 4University Versailles Saint-Quentin-en-Yvelines, 5University Paris Descartes, 6Inserm U830, Institut Curie, PSL Research University

JoVE 58051

 Genetics

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

 Medicine

Enzyme Activity- Concept

JoVE 10585

Biological Catalysts

All living organisms continuously perform numerous biochemical reactions to sustain their presence. Most of these reactions require an input of energy to start, which is called the activation energy. Catalysts are chemicals that lower the activation energy. Even though catalysts facilitate a chemical reaction, they are not consumed by it. This means a catalyst …

 Lab Bio

PCR

JoVE 10819

The polymerase chain reaction, or PCR, is a widely used technique for copying segments of DNA. Due to exponential amplification, PCR can produce millions or billions of DNA copies within just a few hours. In a PCR reaction, a heat-resistant DNA polymerase enzyme amplifies the original DNA through a series of temperature changes inside an automated machine called a thermocycler.

Kary Mullis developed PCR in 1983, for which he was awarded the 1993 Nobel Prize in Chemistry. Being a relatively fast, inexpensive, and precise way of copying a DNA sequence, PCR became an invaluable tool for numerous applications, including molecular cloning, gene mutagenesis, pathogen detection, gene expression analysis, DNA quantitation and sequencing, and genetic disease diagnosis. PCR mimics the natural DNA replication process that occurs in cells. The reaction mixture includes a template DNA sequence to be copied, a pair of short DNA molecules called primers, free DNA building blocks called deoxynucleotide triphosphates (dNTPs), and a specialized DNA polymerase enzyme. PCR involves a series of steps at high temperatures, requiring a DNA polymerase enzyme that is functional at such temperatures. The most commonly used DNA polymerase is Taq polymerase, named after Thermus aquaticus, the bacterium from which the polymerase was initially isolated. DNA

 Core: Biotechnology

Proofreading

JoVE 10790

Synthesis of new DNA molecules starts when DNA polymerase links nucleotides together in a sequence that is complementary to the template DNA strand. DNA polymerase has a higher affinity for the correct base to ensure fidelity in DNA replication. The DNA polymerase furthermore proofreads during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand. Genomic DNA is synthesized in the 5’ to 3’ direction. Each cell contains a number of DNA polymerases that play different roles in synthesizing and correcting mistakes in DNA; DNA polymerase delta and epsilon possess proofreading ability when replicating nuclear DNA. These polymerases “read” each base after it is added to the new strand. If the newly-added base is incorrect, the polymerase reverses direction (moving from 3’ to 5’) and uses an exonucleolytic domain to cut off the incorrect base. Subsequently, it is replaced with the correct base. Proofreading is important for preventing mutations from occurring in newly-synthesized DNA, but what happens when the proofreading mechanism fails? When a mutation alters the exonuclease domain of DNA polymerase, it loses the ability to remove incorrect nucleotides. In consequence, mutations can accumulate rapidly throughout the genome. This type of mutation has been linked to

 Core: DNA Structure and Function

16S rRNA Sequencing: A PCR-based Technique to Identify Bacterial Species

JoVE 10510

Source: Ewa Bukowska-Faniband1, Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden


Planet Earth is a habitat for millions of bacterial species, each of which has specific characteristics. Identification of bacterial species is…

 Microbiology

Biodiversity and Human Values

JoVE 10952

Human civilization relies on biodiversity in many ways. Sudden changes in species biodiversity result in environmental changes that can modify weather patterns and therefore human civilizations.

Humans are dependent on agriculture, which developed when ancestral humans found species that made suitable foods. At least 11,000 years ago, humans started to select plant and animal species to be cultivated on farms. Going back for thousands of years, humans have been artificially selecting species for food, building materials, textiles, and medicine. That progress is ongoing. Human ingenuity continues to benefit from studying the natural world and either directly using or modifying materials and compounds for industrial use. Maintaining the current level of biodiversity will make it substantially more likely that discoveries can be made. For example, in 1969, Thomas D. Brock and Hudson Freeze were studying the Lower Geyser Basin of Yellowstone National Park and discovered a strain of bacteria that is surprisingly heat-tolerant. From this bacteria, an enzyme called Taq polymerase was isolated. This enzyme allows researchers to perform polymerase chain reaction (PCR), which underlies most biotechnological advancements that revolutionized the production of pharmaceuticals, food, and consumer goods among others. For some, discoveries that benefit

 Core: Biological Diversity

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 …

 Environmental Science

Quantifying Environmental Microorganisms and Viruses Using qPCR

JoVE 10186

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…

 Environmental Microbiology

RNA Analysis of Environmental Samples Using RT-PCR

JoVE 10104

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


Reverse transcription-polymerase chain reaction (RT-PCR) involves the same process as conventional PCR — cycling temperature to amplify nucleic acids. However, while conventional PCR only amplifies…

 Environmental Microbiology

Molecular Cloning

JoVE 5074

Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant DNA fragments in host organisms. The DNA fragment, which may be a gene, can be isolated from a prokaryotic or eukaryotic specimen. Following isolation of the fragment of interest, or insert, both the vector and insert must be cut with…

 Basic Methods in Cellular and Molecular Biology

Optical Biosensing

JoVE 5795

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable signal such as fluorescence. Or these sensors can be label-free, and use the changes in optical properties, such as refractive index, to sense for the binding of the target molecule. This video introduces both label and…

 Bioengineering

An Introduction to Aging and Regeneration

JoVE 5337

Tissues are maintained through a balance of cellular aging and regeneration. Aging refers to the gradual loss of cellular function, and regeneration is the repair of damaged tissue generally mediated by preexisting adult or somatic stem cells. Scientists are interested in understanding the biological mechanisms behind these two complex processes. By doing so, researchers may be able to use…

 Developmental Biology

Restriction Enzyme Digests

JoVE 5070

Restriction enzymes or endonucleases recognize and cut DNA at a specific sequence. These enzymes occur naturally in bacteria as a defense against bacteriophages - viruses that infect bacteria. Bacterial restriction enzymes cut the invading bacteriophage DNA while leaving the bacterial genomic DNA unharmed due to addition of methyl groups.


This video explains the basic principles …

 Basic Methods in Cellular and Molecular Biology

Mouse Genotyping

JoVE 5160

Even though the human genome was mapped over 10 years ago, scientists are still far from understanding the function of every human gene! One way to evaluate how a gene functions is to disrupt the sequence encoding it and then evaluate the impact of this change (the phenotype) on the animal’s biology. This approach is commonly used in the mouse (Mus musculus), since it shares a…

 Biology II

Blood Withdrawal I

JoVE 10246

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN


Blood collection is a common requirement for research studies that involve mice and rats. The method of blood withdrawal in mice and rats is dependent upon the volume of blood needed, the frequency of the sampling, the health status of the …

 Lab Animal Research

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

1UMR 1173 Institut national de la santé et de la recherche médicale (INSERM), Université de Versailles St. Quentin, 2UR892 Institut national de la recherche agronomique (INRA), Unité de virologie et immunologie moléculaires, 3Assistance Publique–Hôpitaux de Paris (AP-HP), Hôpital Ambroise Paré, Laboratoire de Microbiologie

JoVE 59218

 Immunology and Infection

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

1Molecular Genetics of Chronic Inflammation and Allergic Disease, Max-Delbrück Center for Molecular Medicine, 2Murdoch Childrens Research Institute, 3Department of Paediatrics, University of Melbourne, 4Centre for Social and Early Emotional Development, Faculty of Health, Deakin University, 5Department of Paediatrics, University of Western Australia

JoVE 57601

 Genetics

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

 Immunology and Infection

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

JoVE 57471

 Genetics

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

1Center for Pathobiochemistry and Genetics, Institute of Medical Chemistry and Pathobiochemistry, Medical University Vienna, 2School of Medical Engineering and Applied Social Sciences, University of Applied Sciences Upper Austria

JoVE 59573

 Biochemistry

Probiotic Studies in Neonatal Mice Using Gavage

1Department of Experimental Medicine, University of British Columbia, 2Department of Pediatrics, University of British Columbia, 3Department of Epidemiology and Pediatrics, University of Nebraska Medical Centre, 4Animal Care Services, University of British Columbia

JoVE 59074

 Immunology and Infection

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

1Laboratory of Viruses Contaminants of Water and Food (VIRCONT), Department of Genetics, Microbiology and Statistics, Section of Microbiology, Virology and Biotechnology, University of Barcelona, 2Grupo de Investigación Biodiversidad, Medio Ambiente y Salud (BIOMAS), Facultad de Ingenierías y Ciencias Agropecuarias (FICA), Ingeniería en Biotecnología, Universidad de las Américas, 3Municipal Laboratory - Waters of Mataró, 4GenIUL

JoVE 59463

 Immunology and Infection
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