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Oligonucleotide Probes: Synthetic or natural oligonucleotides used in hybridization studies in order to identify and study specific nucleic acid fragments, e.g., DNA segments near or within a specific gene locus or gene. The probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the probe include the radioisotope labels 32P and 125I and the chemical label biotin.

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

1Departments of Pathology and Infectious Diseases and Microbiology, University of Pittsburgh, 2University of Pittsburgh Cancer Institute, 3Department of Environmental and Occupational Health, University of Pittsburgh, 4Departments of Psychiatry, Psychology, Behavioral & Community Health Sciences, University of Pittsburgh

JoVE 56001


 Cancer Research

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

1Department of Entomology, Volcani Center, 2Institute of Plant Sciences and Genetics in Agriculture, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, 3Department of Applied Sciences, Institute for Adriatic Crops and Karst Reclamation, 4The Institute of Plant Sciences, Volcani Center

JoVE 51030


 Immunology and Infection

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz

JoVE 2967


 Medicine

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

1Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital, 2Medical Scientist Training Program, University of Cincinnati, 3Immunology Graduate Program, University of Cincinnati, 4Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital

JoVE 54093


 Biology

Expression Profiling with Microarrays

JoVE 5547

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and nucleic acids derived from samples. Using these arrays, scientists can simultaneously evaluate the expression of thousands of genes. In addition, the expression profiles of different cells or tissue types can be compared, allowing researchers to deduce how the expression of different genes change during biological processes, and thus gain insight into how the genes may function in pathways or networks.Here, JoVE explains the principles behind microarrays. This is followed by a general protocol for performing a microarray experiment, and a brief introduction to analyzing microarray data. We end on a discussion of how scientists are currently using microarrays, for example to compare gene expression between different cell types derived from cancerous and non-cancerous tissues, to study important biological problems.


 Genetics

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

An Overview of Genetics and Disease

JoVE 5543

Many human diseases are associated with mutations or variations in genetic sequences. Some of these genetic variants are heritable, passed down from generation to generation, while others arise sporadically during an organism’s life and cause diseases such as cancer. Researchers are trying to identify and characterize these genetic alterations in the hopes of improving diagnosis and therapeutic options for patients.In this video, we will examine the history of genetic disease research, and explore key questions asked by medical geneticists. Various tools used to identify the genetic basis of diseases are then discussed, including genotyping techniques and genome-wide association studies (GWAS). Finally, several current examples of medical genetics research are presented.


 Genetics

SNP Genotyping

JoVE 5544

Single nucleotide polymorphisms, or SNPs, are the most common form of genetic variation in humans. These differences at individual bases in the DNA often do not directly affect gene expression, but in many cases can still be useful for locating disease-associated genes or for diagnosing patients. Numerous methodologies have been established to identify, or “genotype”, SNPs.JoVE’s introduction to SNP Genotyping begins by discussing what SNPs are and how they can be used to identify disease-associated genes. Several common SNP genotyping methods are then examined, including direct hybridization, PCR-based methods, fragment analysis, and sequencing. Finally, we present several examples of how these techniques are applied to genetic research today.


 Genetics

Cytogenetics

JoVE 5545

Cytogenetics is the field of study devoted to chromosomes, and involves the direct observation of a cell’s chromosomal number and structure, together known as its karyotype. Many chromosomal abnormalities are associated with disease. Each chromosome in a karyotype can be stained with a variety of dyes to give unique banding patterns. More recent techniques, including comparative genomic hybridization and fluorescence in situ hybridization (FISH), allow for detecting specific chromosomal features or abnormalities.This video will begin by examining the principles of these classical and modern cytogenetics techniques. This is followed by an examination of a general protocol for performing FISH. Finally, several examples of how karyotyping can be applied to various medical applications are presented.


 Genetics

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