Polymorphism, Single Nucleotide:
A single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population.
JoVE General
Pyrosequencing: A Simple Method for Accurate Genotyping
Department of Internal Medicine, Washington University in St. Louis
Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.
JoVE Immunology and Infection
Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Department of Microbiology, Immunology, & Cancer Biology, University of Virginia Health System
We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.
JoVE Immunology and Infection
Facilitating the Analysis of Immunological Data with Visual Analytic Techniques
1Department of Paediatrics, Division of Infectious and Immunological Diseases, Child and Family Research Institute, University of British Columbia, 2Department of Computer Science, University of British Columbia, 3Department of Psychology, University of British Columbia
Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.
JoVE General
Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes
1Experimental and Clinical Research Center (ECRC), Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC), 2Medical Department, Division of Cardiology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, 3Medical Department, Division of Cardiology and Angiology, Campus Mitte, Charité - Universitätsmedizin Berlin
Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.
JoVE Neuroscience
Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain
Max Planck Institute of Psychiatry
A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.
JoVE Bioengineering
Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules
1Department of Chemistry and Biochemistry, University Of California Santa Barbara, 2Department of Chemistry and Biochemistry, Program in BioMolecular Science and Engineering, University Of California Santa Barbara
"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.
JoVE General
A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation
1Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Emory University School of Medicine and Children's Health Care of Atlanta, 2Division of Rheumatology, Department of Pediatrics, Emory University School of Medicine and Children's Health Care of Atlanta
We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.
JoVE Immunology and Infection
Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites
1National Institute of Allergy and Infectious Diseases, National Institutes of Health, 2School of Life Science, Xiamen University
Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.
JoVE General
An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations
Wellcome Trust Centre for Human Genetics, University of Oxford
Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.
JoVE Clinical and Translational Medicine
FISH for Pre-implantation Genetic Diagnosis
This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.
JoVE General
RNA Secondary Structure Prediction Using High-throughput SHAPE
High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.
JoVE Immunology and Infection
A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Institute for Bioscience and Biotechnology Research, University of Maryland
A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.
JoVE General
Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA
Department of Medicine, University of Toledo Health Science Campus
We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.
JoVE General
Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.
JoVE General
Genotyping of Plant and Animal Samples without Prior DNA Purification
Thermo Scientific Molecular Biology Products, Thermo Fisher Scientific
The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.
JoVE General
Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm
Department of Biology, Saint Louis University
Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.
JoVE Bioengineering
Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)
1Department of Electrical and Computer Engineering, Boston University, 2Department of Biomedical Engineering, Boston University, 3Center for Advanced Genomics Technology, Boston University, 4Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 5Department of Microbiology, Boston University School of Medicine, 6CNR (National Research Council), Istituto di Chimica del Riconoscimento Molecolare
Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.
JoVE General
Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting
1Department of Chemistry, Hunter College, 2Department of Biochemistry, Albert Einstein College of Medicine
This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.
JoVE Immunology and Infection
Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides
1Department of Medical Microbiology and Immunology, University of California, Davis, 2Département Génétique et Développement, Institut Cochin, Université Paris Descartes
Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.
JoVE General
Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique
1Department of Molecular Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, 2Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw
DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.
JoVE Immunology and Infection
Examination of the Telomere G-overhang Structure in Trypanosoma brucei
Biological, Geo. & Env. Sciences, Cleveland State University
Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.
JoVE Clinical and Translational Medicine
A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe
1Fondazione Banca Degli Occhi del Veneto O.N.L.U.S., 2Telethon Institute for Genetics & Medicine (T.I.G.E.M.)
The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.
JoVE General
An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase
Department of Biochemistry, University of Toronto
The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.
JoVE General
Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes
Department of Biochemistry and Biomedical Sciences, McMaster University
Crystal structure of protein–DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.
JoVE Neuroscience
Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice
1Department of Cell and Developmental Biology, Vanderbilt University, 2Department of Neurology, Vanderbilt University
This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.
JoVE General
A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals
We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.
JoVE General
DNA Methylation: Bisulphite Modification and Analysis
1Epigenetics Group, Cancer Research Program, Garvan Institute of Medical Research, 2St Vincent's Clinical School, University of NSW
The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.
JoVE General
Substrate Generation for Endonucleases of CRISPR/Cas Systems
Prokaryotic Small RNA Biology, Max-Planck-Institute for Terrestrial Microbiology
CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.
JoVE Clinical and Translational Medicine
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
1Centre of Excellence in Neuromics, CHUM Research Center and the Department of Medicine, Universite de Montreal, 2Center of Excellence in Neuromics, CHU Sainte Justine and CHUM Notre-Dame Research Centers, Universite de Montreal, 3Department of Medicine, Universite de Montreal
Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.
JoVE Immunology and Infection
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Viral Populations and Pathogenesis lab and CNRS 3015, Institut Pasteur
The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.
JoVE Applied Physics
Angle-resolved Photoemission Spectroscopy At Ultra-low Temperatures
1Institute for Solid State Research, IFW-Dresden, 2Institute of Metal Physics of National Academy of Sciences of Ukraine, 3Diamond Light Source LTD, 4Department of Physics, University of Johannesburg, 5CNR-SPIN, and Dipartimento di Fisica "E. R. Caianiello", Università di Salerno, 6Institute of Physics of Complex Matter, École Polytechnique Fédérale de Lausanne
The overall goal of this method is to determine the low-energy electronic structure of solids at ultra-low temperatures using Angle-Resolved Photoemission Spectroscopy with synchrotron radiation.
JoVE General
Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates
1Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, 2Department of Surgery, University of Toronto, 3Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill
The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.
JoVE General
Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR
The Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University
An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.
JoVE General
Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)
1School of Biological Sciences, Nanyang Technological University, Singapore - NTU, 2Singapore-MIT Alliance for Reserach and Technology (SMART)
Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.
JoVE General
A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA
1Department of Molecular and Cellular Biology, Brown University, 2Center for Computational Molecular Biology, Brown University
Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.
JoVE General
RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs
Department of Biological Sciences, Union College
By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.
JoVE Clinical and Translational Medicine
Single Port Donor Nephrectomy
1Surgery, Weill Cornell Medical College of Cornell University, 2Urology, Weill Cornell Medical College of Cornell University
Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.
JoVE General
Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
1Department of Biological Sciences, University of Alabama Huntsville, 2Department of Biology, Stanford University
A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.
JoVE Immunology and Infection
An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
Department of Population Health, University of Georgia
We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.
JoVE General
Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California
Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.
JoVE General
Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast
Department of Plant Sciences, Tel Aviv University
In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.
JoVE General
Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions
1Department of Biochemistry, University of Toronto, 2Department of Molecular Genetics, University of Toronto, 3Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), University of Toronto
MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.
JoVE General
Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
1The Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Graduate Program in Quantitative and Computational Biology, Princeton University, 3Department of Molecular Biology, Princeton University
This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.
JoVE General
Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School
This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.
JoVE General
Intravitreous Injection for Establishing Ocular Diseases Model
The University of Hong Kong - HKU
Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.
JoVE Neuroscience
Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice
1Department of Neurology, University of Connecticut Health Center, 2Department of Neurology, School of Medicine, University of Pennsylvania, 3Department of Neurosurgery, Hartford Hospital, 4Department of Neurosurgery, School of Medicine, University of Pennsylvania
The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.
JoVE General
Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping
1Horticulture Department, University of Wisconsin-Madison, 2Department of Zoology, Oregon State University
The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.
JoVE General
Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass
Department of Pathology, University of Utah School of Medicine
We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.
JoVE Immunology and Infection
Avian Influenza Surveillance with FTA Cards: Field Methods, Biosafety, and Transportation Issues Solved
1Resource Ecology Group, Wageningen University, 2Section for Zoonotic Ecology and Epidemiology, School of Natural Sciences, Linnaeus University, 3Centre for Wildlife Ecology, Simon Fraser University
A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.
JoVE General
Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation
1Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, 2A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, 3Department of Biochemistry, National University of Singapore, Singapore
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.
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