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Reverse Transcriptase Polymerase Chain Reaction: A variation of the Pcr technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard Pcr protocols.
 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 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 Cancer Research

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

1Department of Surgery and Translational Medicine (DCMT), University of Florence, 2Neurofarba Department, University of Florence, 3Department of Traumatology and General Orthopedics, Azienda Ospedaliera Universitaria Careggi

JoVE 53884

 Science Education: Essentials of Environmental Microbiology

RNA Analysis of Environmental Samples Using RT-PCR

JoVE Science Education

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 deoxyribonucleic acids (DNA), RT-PCR enables the amplification of ribonucleic acids (RNA) through the formation of complementary DNA (cDNA). This enables RNA-based organisms found within the environment to be analyzed utilizing methods and technologies that are designed for DNA. Many viruses found in the environment use RNA as their genetic material. Several RNA-based viral pathogens, such as Norovirus, and indicator organisms, such as pepper mild mottle virus (PMMoV), do not have culture-based detection methods for quantification. In order to detect for the presence of these RNA viruses in environmental samples from soil, water, agriculture, etc., molecular assays rely on RT-PCR to convert RNA into DNA. Without RT-PCR, microbiologists would not be able to assay and research numerous RNA-based viruses that pose risks to human and environmental health. RT-PCR can also be employed as a tool to measure microbial activity in the env

 JoVE Developmental Biology

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

1Department of Nanobiomedical Sciences and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, 2Insititute of Tissue Regeneration Engineering, Dankook University, 3Department of Biological Sciences, Ajou University, 4Department of Pharmacy, Sahmyook University, 5Department of Animal Biotechnology, Sahmyook University

JoVE 53696

 JoVE Bioengineering

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

1Florey Neuroscience Institutes and Centre for Neuroscience, University of Melbourne, 2Fluid Dynamics Group, CSIRO Materials Science and Engineering, 3Swinburne University of Technology, Faculty of Engineering and Industrial Sciences

JoVE 3144

 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

 JoVE Immunology and Infection

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

1Institute of Virology, University of Cologne, 2Max Planck Institute for Informatics, 3Institute for Immune genetics, 4Department of Gastroenterology, Hepatology and Infectiology, University of Duesseldorf, 5Department of Dermatology, University of Essen, 6Department of Internal Medicine, University of Cologne, 7Augustinerinnen Hospital

JoVE 3264

 JoVE Immunology and Infection

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

1USGS Western Ecological Research Center, 2Wildlife Health Center, University of California, Davis, 3Department of Population Health and Reproduction, University of California, Davis, 4Department of Veterinary and Biomedical Sciences, University of Minnesota, 5Science Applications International Corporation

JoVE 2829

 Science Education: Basic Methods in Cellular and Molecular Biology

Molecular Cloning

JoVE Science Education

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 restriction enzymes and purified. The purified pieces are joined together though a technique called ligation. The enzyme that catalyzes the ligation reaction is known as ligase. This video explains the major methods that are combined, in tandem, to comprise the overall molecular cloning procedure. Critical aspects of molecular cloning are discussed, such as the need for molecular cloning strategy and how to keep track of transformed bacterial colonies. Verification steps, such as checking purified plasmid for the presence of insert with restrictions digests and sequencing are also mentioned.

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