DOCSLIB.ORG

Techniques Used to Test Native DNA Downloaded from by Guest on 29 September 2021

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Techniques Used to Test Native DNA Downloaded from by Guest on 29 September 2021 CE UPDATE —MOLECULAR BIOLOGY II James Wisecarver, MD, PhD Techniques Used To Test Native DNA Downloaded from https://academic.oup.com/labmed/article/28/2/121/2503736 by guest on 29 September 2021 The first article of this series described the key structural features of DNA and how it is extracted ABSTRACT Several techniques commonly are used to test from cells for study. After a DNA sequence has native high-molecular-weight DNA obtained from cells and been extracted from cells and purified, the genetic tissues, including the Southern blot, dot and slot blot proce­ information contained within the sequence can be dures, and in situ hybridization of tissue sections with examined through a variety of techniques. This article discusses the techniques commonly oligonucleotide probes. These techniques work well when used to test native high-molecular-weight DNA there is an adequate amount of fresh tissue or cells available to obtained from cells and tissues, including provide a source for the intact DNA. These techniques involve Southern blot, dot blot, and in situ hybridization. separating, or denaturing, the double-stranded DNA into individual strands, and then applying a marked nucleotide Southern Blot The technique known as Southern blot analysis is probe and allowing it to hybridize to a complementary DNA used widely for analyzing the size of certain DNA sequence that may be present. These techniques are used to fragments. The sequence of interest is prepared determine whether a particular DNA sequence is present. by digesting intact DNA collected from a patient's This is the second article in a three-part series on DNA. Other articles discuss the struc­ C cells with a restriction endonuclease enzyme. 0 tural properties of DNA, how it is extracted from cells for study, some of the basic tools '£ Following complete digestion, a collection of II used to gain useful clinical information, and DNA amplification techniques. On comple­ o double-stranded DNA fragments remains, rang­ 'E tion of this series, readers will be able to describe the composition of DNA, how the 3 ing in size from a few hundred to several thou­ strands are arranged, how to extract DNA from tissues prior to testing, and how DNA £ £ sand base pairs. fragments are prepared by enzyme digestion, separated using gel electrophoresis, and o These double-stranded fragments can be sepa­ then isolated for further study. 0 rated by size using a technique known as agarose gel electrophoresis. This technique involves plac­ After the DNA fragments have been separated, From the ing the fragmented DNA into wells in a slab of the double-stranded fragments are denatured Department of Pathology and agarose gel and submersing the gel in a buffer into single strands by soaking the gel in sodium Microbiology, © chamber in the electrophoresis apparatus. hydroxide followed by neutralization. These DNA University of c 0 Electric voltage is applied, causing the negatively pieces then are transferred onto a piece of elec­ Nebraska Medical ti charged nucleic acid fragments to migrate toward trostatically charged paper or filter membrane. In Center, Omaha. ( the positively charged region, or anode, of the gel the original description of this technique, the slab Reprint requests apparatus. The gel matrix acts as a sieve, allowing of gel was placed in a tray of buffer, and the filter to Dr Wisecarver, the smaller fragments to move through the gel material was placed on top of the agarose gel. Department of Pathology and matrix easily while the progressively larger frag­ Absorbent paper toweling was placed on top of Microbiology, ments have more difficulty migrating through the the filter material allowing the buffer to work University of gel. These DNA fragments are invisible to the upward through the gel and the filter membrane Nebraska Medical naked eye but can be visualized if the gel is into the absorbent toweling (Fig 1). The migra­ Center, 600 S 42nd stained with a dye such as ethidium bromide, tion of buffer also transfers the DNA fragments St, Omaha, NE 68198-3135. which binds to the DNA. After staining, the gel from the gel onto the membrane surface. Most can be placed on an ultraviolet light source. laboratories use vacuum- or air pressure-blotting Orange fluorescence indicates the presence of the devices that greatly shorten the time necessary to DNA within the lanes of the gel. transfer the DNA onto the membrane. The single-stranded DNA fragments that have been Downloaded from https://academic.oup.com/labmed/article-abstract/28/2/121/2503736 by guest on 28 May 2018 FEBRUARY 1997 VOLUME 28, NUMBER 2 LABORATORY MEDICINE 121 transferred are immobilized permanently onto sequence. The appropriate conditions for each the membrane surface by brief exposure to ultra­ procedure must be determined. When using a violet light. commercially prepared kit, care should be taken The next step is to determine the location and during probing and washing steps to adhere to size of the particular DNA sequence of interest. the times and temperatures outlined in the An oligonucleotide probe with a base sequence instructions to obtain accurate results. that is complementary to the sequence of interest Following these wash steps, the membrane is prepared containing a label to permit detection. should be exposed to x-ray film for a period of One simple technique for labeling probes is to hours to days, depending on the strength of the Test Your incorporate radioactive nucleotides as the probe is signal emitted from the probe. If radioactive Knowledge assembled. The location of the radioactive probe probes are used, no further steps are needed. Downloaded from https://academic.oup.com/labmed/article/28/2/121/2503736 by guest on 29 September 2021 Look for the CE can be detected by exposing the membrane to x- When using enzyme-labeled probes such as alka­ Update exam on ray film. More recently, nonisotopic techniques line phosphatase, however, a substrate must be Molecular Biology have been developed to label probes with enzymes added to the membrane surface before exposure to (702) in the March such as alkaline phosphatase. A suitable enzyme issue of Laboratory the film. This substrate, when cleaved by the alka­ Medicine. Participants substrate can be used to detect the presence and line phosphatase enzyme, emits photons of light. will earn 3 CMLE location of the bound probe. These light photons act similarly to the particles credit hours. The membrane blot is placed in a salt solution released from decay of a radioactively labeled containing the labeled probe at a temperature probe and alter the silver present in the x-ray film. that will permit the probe to hybridize, or bind, Development of the x-ray film following exposure to its complementary DNA fragment bound to to the membrane surface yields an image that will the membrane. During the incubation period, disclose the location of the DNA fragments con­ Fig 1. In the which often is several hours, the probe eventually taining the sequence of interest (Fig 2). Southern blot proce­ dure, DNA fragments will find the complementary base pair sequence To determine the size of this fragment, a size that have been pre­ on the membrane and bind to this target DNA. marker is placed in the gel and is transferred to pared by cutting Following hybridization, the solution containing the membrane. By comparing the size of fragments native DNA with a the labeled probe is removed, and the membrane detected by the probe with the size marker, it is restriction enzyme is washed several times with a salt-buffer solution possible to estimate the number of bases present in are separated in to remove any unbound probe from its surface. agarose gel through the fragment to which the probe hybridized. electrophoresis. The By adjusting the salt concentration and the Most laboratories use a 1-kilobase DNA ladder in fragments are trans­ temperature, one can carefully remove the non- which the sizing bands are approximately 1,000 ferred onto a nylon specifically bound probe, being careful not to dis­ bases apart. membrane by plac­ rupt the bonds between the probe and the ing the gel in a intended target sequence. This careful considera­ buffer bath and plac­ Northern and Western Blots ing the membrane tion of the ionic strength and temperature used The Southern blot was named for its inventor, Dr on top of the gel. during the wash steps often is referred to as the Edwin Southern. Other investigators began to use Paper toweling is "stringency" of the procedure. If the temperature this technique to separate and identify RNA frag­ placed on top of the is too low or the ionic strength is too high, the ments and proteins. Investigators using this tech­ membrane, drawing probe might stick to the membrane in a nonspe­ the buffer upward nique to isolate messenger RNA (mRNA) dubbed from the sponge cific fashion. If the temperature is too high, the it northern blotting, as opposed to Southern blot­ located in the pan, probe might separate from the intended target ting, which examines DNA fragments. Not to be through the gel and outdone, protein chemists membrane, and into used the term western the toweling. As this The Southern Blot Procedure occurs, the DNA blotting to describe a tech­ fragments leave the nique in which proteins gel and are deposited Buffer wicks up through are separated electro- on the membrane membrane into paper toweling phoretically, transferred surface, producing a Paper towels Filter membrane to membranes, and iden­ "blot." After this ^4, A transfer is complete, tified through the use of the fragments are labeled antibodies specific immobilized on the for the protein of interest. membrane surface, Northern blot analysis of and a labeled probe mRNA is more technically is added to localize Gel containing Buffer in tray Sponge soaked in demanding than similar the DNA fragments separated DNA buffer supporting gel containing the fragments analyses of DNA.
Load more

Recommended publications
  • Biochemistry and the Genomic Revolution 1.1
    Dedication About the authors Preface Tools and Techniques Clinical Applications Molecular Evolution Supplements Supporting Biochemistry, Fifth Edition Acknowledgments I. The Molecular Design of Life 1. Prelude: Biochemistry and the Genomic Revolution 1.1. DNA Illustrates the Relation between Form and Function 1.2. Biochemical Unity Underlies Biological Diversity 1.3. Chemical Bonds in Biochemistry 1.4. Biochemistry and Human Biology Appendix: Depicting Molecular Structures 2. Biochemical Evolution 2.1. Key Organic Molecules Are Used by Living Systems 2.2. Evolution Requires Reproduction, Variation, and Selective Pressure 2.3. Energy Transformations Are Necessary to Sustain Living Systems 2.4. Cells Can Respond to Changes in Their Environments Summary Problems Selected Readings 3. Protein Structure and Function 3.1. Proteins Are Built from a Repertoire of 20 Amino Acids 3.2. Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains 3.3. Secondary Structure: Polypeptide Chains Can Fold Into Regular Structures Such as the Alpha Helix, the Beta Sheet, and Turns and Loops 3.4. Tertiary Structure: Water-Soluble Proteins Fold Into Compact Structures with Nonpolar Cores 3.5. Quaternary Structure: Polypeptide Chains Can Assemble Into Multisubunit Structures 3.6. The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure Summary Appendix: Acid-Base Concepts Problems Selected Readings 4. Exploring Proteins 4.1. The Purification of Proteins Is an Essential First Step in Understanding Their Function 4.2. Amino Acid Sequences Can Be Determined by Automated Edman Degradation 4.3. Immunology Provides Important Techniques with Which to Investigate Proteins 4.4. Peptides Can Be Synthesized by Automated Solid-Phase Methods 4.5.
    [Show full text]
  • SOP Template Northern Blotting with P-32
    Standard Operating Procedure Procedure Northern Blotting using 32P Department Location SOP Prepared By: Section 1: Purpose Northern blotting is a standard method for the detection and quantification of RNA from a cell. This is done by isolating and purifying RNA and using a radioactively-labeled DNA or RNA probe to hybridize to and detect the RNA. Using this technique, temporal and spatial location of RNA expression can be found8. Unlike RT-PCR (real-time PCR), which quantifies the amount of RNA or DNA at various times, Northern blotting can be used not only to quantify but also determine the size of the RNA. It is also useful to perform Northern blotting when studying transfer RNA (tRNA), which appears as the two lowest bands on a gel1. The probes used in Northern blotting do not have to be radioactive, but radioactive probes still have the greatest sensitivity. Quantifying mRNA of tRNA can provide insight into gene expression in cells that are exposed to different environments. Section 2: Personal Protective Equipment and Survey Equipment PPE: • Nitrile Gloves • Lab coat or lab gown • Proper enclosed shoes • Safety glasses Other Equipment: • Geiger counter • Personal chest dosimeter Section 3: Radioactive Material 32P, specific type varies according to what procedure is followed for probe design Supplier: Perkin Elmer Starting activity: 10 mCi/mL Typical use quantities: no more than 10uCi per experiment • Radioactive material (RAM) benchtop exposure time: ~10-20 minutes Activity used per experiment: _______________ RAM handling time: _______________ Frequency of experiment: _______________ Section 4: Potential Hazards • 32P is a high-energy beta emitter and has a half-life of 14.29 days.
    [Show full text]
  • Glycoprotein Detection with the Odyssey Infrared Imaging System
    Glycoprotein Detection with the Odyssey ® Infrared Imaging System Julie A. Champoux, Kristi L.H. Ambroz, Joseph B. Hwang, William M. Volcheck, and Amy R. Schutz-Geshwender LI-COR Biosciences, Lincoln, NE 68504 INTRODUCTION bination of lectins can also be used to dissect the Glycosylation is one of the most common and carbohydrate composition of a target protein. important events in post-translational modifica- For more information about IRDye products tion, with over half of all proteins believed to be used in this study, go to http://www.licor.com/bio/ glycosylated.1 Cellular glycoconjugates play reagents/irdyes.jsp. important roles in many biological processes and have been implicated in cancer development, MATERIALS AND METHODS retrovirus infection and other diseases. Carbohy- Sensitivity of biotinylated Con A / IRDye drate-binding proteins known as lectins bind to 800CW-streptavidin and IRDye 800CW-Con A specific oligosaccharides, and can serve as markers Two-fold serial dilutions of α2-macroglobulin, to identify certain cell types or cellular compo- glucose oxidase and RNAse B (Sigma Cat# nents. Lectins have very high binding specificity M6159, 49178 and NEB Cat# P7817S were and have been used to characterize and purify mixed and separated on 4-12% polyacrylamide oligosaccharides.2 This application note describes Tris-glycine gels (Novex/Invitrogen). Samples the use of lectins to detect glycoproteins in West- were electrophoretically transferred onto nitro- ern blot format using the Odyssey® Infrared cellulose membrane (Osmonics). For all blots in Imaging System. this study, blocking was carried out with Odyssey Concanavalin A (Con A) is a carbohydrate- Blocking Buffer (LI-COR, Part # 927-40000) for 1 h binding protein that binds specifically to the at room temperature; washing was performed in most commonly occurring sugars: α-D-mannose, PBST (PBS + 0.1% Tween®-20) for 4 x 5 min, fol- α-D-glucose and, with lower affinity, α-N-acetyl- lowed by a 5 min rinse in PBS before imaging.
    [Show full text]
  • Blotting Techniques Blotting Is the Technique in Which Nucleic Acids Or
    Blotting techniques Blotting is the technique in which nucleic acids or proteins are immobilized onto a solid support generally nylon or nitrocellulose membranes. Blotting of nucleic acid is the central technique for hybridization studies. Nucleic acid labeling and hybridization on membranes have formed the basis for a range of experimental techniques involving understanding of gene expression, organization, etc. Identifying and measuring specific proteins in complex biological mixtures, such as blood, have long been important goals in scientific and diagnostic practice. More recently the identification of abnormal genes in genomic DNA has become increasingly important in clinical research and genetic counseling. Blotting techniques are used to identify unique proteins and nucleic acid sequences. They have been developed to be highly specific and sensitive and have become important tools in both molecular biology and clinical research. General principle The blotting methods are fairly simple and usually consist of four separate steps: electrophoretic separation of protein or of nucleic acid fragments in the sample; transfer to and immobilization on paper support; binding of analytical probe to target molecule on paper; and visualization of bound probe. Molecules in a sample are first separated by electrophoresis and then transferred on to an easily handled support medium or membrane. This immobilizes the protein or DNA fragments, provides a faithful replica of the original separation, and facilitates subsequent biochemical analysis. After being transferred to the support medium the immobilized protein or nucleic acid fragment is localized by the use of probes, such as antibodies or DNA, that specifically bind to the molecule of interest. Finally, the position of the probe that is bound to the immobilized target molecule is visualized usually by autoradiography.
    [Show full text]
  • Near-Infrared Fluorescent Northern Blot
    Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press METHOD Near-infrared fluorescent northern blot BRET R. MILLER,1,4 TIANQI WEI,1,4 CHRISTOPHER J. FIELDS,1,2 PEIKE SHENG,1,2 and MINGYI XIE1,2,3 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA 2UF Health Cancer Center, University of Florida, Gainesville, Florida 32610, USA 3UF Genetics Institute, University of Florida, Gainesville, Florida 32610, USA ABSTRACT Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present al- ternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of dif- ferent RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.
    [Show full text]
  • NORTHERN BLOT SEM.I Dr.Ramesh Pathak the Northern Blot Is Also
    NORTHERN BLOT SEM.I Dr.Ramesh Pathak The Northern blot is also called RNA blot. It is a technique used in molecular biology to study gene expression by detection of RNA in a sample. The northern blot technique developed by James Alwine, David Kemp and Jeorge Stark at Stanford University with contribution from Gerhard Heinrich in 1977.Northern blotting takes its name from its similarity to the first blotting technique, the southern blotting named after biologist Edwin Southern. Procedure The blotting process starts with extraction of total RNA from a homogenized tissue sample or cells on agarose gel containing formaldehyde which is a denaturing agent for the RNA to limit secondary structure. Eukaryotic mRNA can then be isolated through the use of oligo(dT) cellulose chromatography to isolate only those RNAs with a poly A tail. RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the probes are unable to enter matrix, the RNA samples, now separated by size, are transferred to nylon membrane through a capillary or vacuum blotting system. A nylon membrane with a positive charge, is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. The transfer buffer used for the blotting usually contain formamide because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating the need for high temperature, which could cause RNA degradation. Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat.
    [Show full text]
  • Southern Blotting Teacher’S Guidebook
    PR139 G-Biosciences ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected] A Geno Technology, Inc. (USA) brand name Southern Blotting Teacher’s Guidebook (Cat. # BE-315) think proteins! think G-Biosciences www.GBiosciences.com MATERIALS INCLUDED ....................................................................................................... 3 SPECIAL HANDLING INSTRUCTIONS ................................................................................... 3 ADDITIONAL EQUIPMENT REQUIRED ................................................................................ 3 TIME REQUIRED ................................................................................................................. 3 AIMS .................................................................................................................................. 4 BACKGROUND ................................................................................................................... 4 TEACHER’S PRE EXPERIMENT SET UP ................................................................................ 5 PREPARATION OF AGAROSE GEL ................................................................................... 5 PREPARE THE DNA LADDER ........................................................................................... 5 PREPARE DILUTE DNA STAINING SOLUTION .................................................................. 5 MATERIALS FOR DEMONSTRATION ................................................................................... 6 DEMONSTRATION
    [Show full text]
  • Gene Expression Detection Methods RNA
    Gene expression detection methods RNA Dr. Ingrid Müller - AG von Laer 1 TRANSCRIPTIONAL CONTROL REGULATES DIFFERENTIATION Four different human cells - same genes, different structures and functions due to differential gene expression 2 THE CENTRAL DOGMA • Flow of Information: DNA Replication Transcription Translation A B Cells in all living organisms are continually activating or deactivating genes through gene expression, which contain the information required for producing proteins through proteins synthesis. When a particular protein is required by the cell, the gene coding for that protein is activated. The first stage in producing a protein involves the production of an RNA copy of the gene's DNA sequence. This RNA copy is the messenger RNA. The amount of mRNA produced correlates with the amount of protein eventually synthesised and measuring the amount of a particular mRNA produced by a given cell or tissue is often easier than measuring the amount of the final protein. Levels in gene activation may vary between cancerogenic and healthy cells. 3 MOLECULAR METHODS TOOL BOX I. Analysis II. Overexpression III. Inhibition •Western Blot •(adding proteins to in •pharmacological inhibitors, •Proteomics vitro reactions) •dominant-negative Protein •immuno- proteins, histochemisty • protein depletion using •protein chimera antibodies •Electrophoresis •microinjection •RNAi & •Northern Blot •siRNA •RNAse protection •Morpholinos RNA assays •Microarrays •RT-PCR •RNA in-situ •Reporter genes •(microinjection) •Knock-out •Electroporation, •Integrational mutagenesis DNA (gene) •lipofection, •Classic genetics •transgenics 4 RNA METHODS: ELECTROPHORESIS 5 PURIFICATION OF MESSENGER RNA USING OLIGO DT COLUMNS Total cellular RNA; apply at room temperature to Break open anneal polyA tail to oligo(dT) cells in the presence of RNAse Oligo(dT) inhibitors attached to AAAA.
    [Show full text]
  • Bioanalytical Chemistry 4. Gel Electrophoresis
    73 Bioanalytical chemistry 4. Gel Electrophoresis Required reading: Sections 9.1, 9.2.3, 9.2.4, 9.5.1, 10.1 to 10.7, 11.1 to 11.5, and 15.5 of Mikkelsen and Cortón, Bioanalytical Chemistry Some objectives for this section ⇒ You will know what DNA agarose gel electrophoresis is ⇒ You will know what the difference between normal and pulsed field electrophoresis of DNA ⇒ You will know what SDS-PAGE is. ⇒ You will understand how the basis for molecular basis for size-based separation of proteins by SDS- PAGE. ⇒ You will know what IEF is. ⇒ You will know how SDS-PAGE and IEF can be combined in 2-dimensional gel electroophoresis ⇒ You will know what a Western Blot is. ⇒ You will appreciate how these techniques can be used in the analysis of DNA and proteins. Primary Source Material • Chapter 4 and 6 of Biochemistry: Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert (NCBI bookshelf). • Chapter 3 and 7 of Molecular Cell Biology 4th ed. (Ch. 9, 5th ed.): Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. (NCBI bookshelf). • Chapter 12 of Introduction to Genetic Analysis Anthony: J.F. Griffiths, Jeffrey H. Miller, David T. Suzuki, Richard C. Lewontin, William M. Gelbart (NCBI bookshelf). • Some animations are from http://www.wiley-vch.de/books/info/3-527-30300-6/. • http://www.piercenet.com/ Electrophoresis 74 The velocity of migration (v) of a molecule in an electric field depends on the electric field strength (E), the net charge on the protein (z), and the frictional coefficient (f).
    [Show full text]
  • Clinical Molecular Genetics in the Uk C.1975–C.2000
    CLINICAL MOLECULAR GENETICS IN THE UK c.1975–c.2000 The transcript of a Witness Seminar held by the History of Modern Biomedicine Research Group, Queen Mary, University of London, on 5 February 2013 Edited by E M Jones and E M Tansey Volume 48 2014 ©The Trustee of the Wellcome Trust, London, 2014 First published by Queen Mary, University of London, 2014 The History of Modern Biomedicine Research Group is funded by the Wellcome Trust, which is a registered charity, no. 210183. ISBN 978 0 90223 888 6 All volumes are freely available online at www.history.qmul.ac.uk/research/modbiomed/ wellcome_witnesses/ Please cite as: Jones E M, Tansey E M. (eds) (2014) Clinical Molecular Genetics in the UK c.1975–c.2000. Wellcome Witnesses to Contemporary Medicine, vol. 48. London: Queen Mary, University of London. CONTENTS What is a Witness Seminar? v Acknowledgements E M Tansey and E M Jones vii Illustrations and credits ix Abbreviations xi Ancillary guides xiii Introduction Professor Bob Williamson xv Transcript Edited by E M Jones and E M Tansey 1 Appendix 1 Photograph, with key, of delegates attending The Molecular Biology of Thalassaemia conference in Kolimbari, Crete, 1978 88 Appendix 2 Extracts from the University of Leiden postgraduate course Restriction Fragment Length Polymorphisms and Human Genetics, 1982 91 Appendix 3 Archival material of the Clinical Molecular Genetics Society 95 Biographical notes 101 References 113 Index 131 Witness Seminars: Meetings and Publications 143 WHAT IS A WITNESS SEMINAR? The Witness Seminar is a specialized form of oral history, where several individuals associated with a particular set of circumstances or events are invited to meet together to discuss, debate, and agree or disagree about their memories.
    [Show full text]
  • Journal of Biomedical Discovery and Collaboration
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE Journal of Biomedical Discovery and provided by PubMed Central Collaboration BioMed Central Case Study Open Access The emergence and diffusion of DNA microarray technology Tim Lenoir*† and Eric Giannella† Address: Jenkins Collaboratory for New Technologies in Society, Duke University, John Hope Franklin Center, 2204 Erwin Road, Durham, North Carolina 27708-0402, USA Email: Tim Lenoir* - [email protected]; Eric Giannella - [email protected] * Corresponding author †Equal contributors Published: 22 August 2006 Received: 09 August 2006 Accepted: 22 August 2006 Journal of Biomedical Discovery and Collaboration 2006, 1:11 doi:10.1186/1747-5333-1-11 This article is available from: http://www.j-biomed-discovery.com/content/1/1/11 © 2006 Lenoir and Giannella; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The network model of innovation widely adopted among researchers in the economics of science and technology posits relatively porous boundaries between firms and academic research programs and a bi-directional flow of inventions, personnel, and tacit knowledge between sites of university and industry innovation. Moreover, the model suggests that these bi-directional flows should be considered as mutual stimulation of research and invention in both industry and academe, operating as a positive feedback loop. One side of this bi-directional flow – namely; the flow of inventions into industry through the licensing of university-based technologies – has been well studied; but the reverse phenomenon of the stimulation of university research through the absorption of new directions emanating from industry has yet to be investigated in much detail.
    [Show full text]
  • Protein Blotting Guide
    Electrophoresis and Blotting Protein Blotting Guide BEGIN Protein Blotting Guide Theory and Products Part 1 Theory and Products 5 Chapter 5 Detection and Imaging 29 Total Protein Detection 31 Transfer Buffer Formulations 58 5 Chapter 1 Overview of Protein Blotting Anionic Dyes 31 Towbin Buffer 58 Towbin Buffer with SDS 58 Transfer 6 Fluorescent Protein Stains 31 Stain-Free Technology 32 Bjerrum Schafer-Nielsen Buffer 58 Detection 6 Colloidal Gold 32 Bjerrum Schafer-Nielsen Buffer with SDS 58 CAPS Buffer 58 General Considerations and Workflow 6 Immunodetection 32 Dunn Carbonate Buffer 58 Immunodetection Workflow 33 0.7% Acetic Acid 58 Chapter 2 Methods and Instrumentation 9 Blocking 33 Protein Blotting Methods 10 Antibody Incubations 33 Detection Buffer Formulations 58 Electrophoretic Transfer 10 Washes 33 General Detection Buffers 58 Tank Blotting 10 Antibody Selection and Dilution 34 Total Protein Staining Buffers and Solutions 59 Semi-Dry Blotting 11 Primary Antibodies 34 Substrate Buffers and Solutions 60 Microfiltration (Dot Blotting) Species-Specific Secondary Antibodies 34 Stripping Buffer 60 Antibody-Specific Ligands 34 Blotting Systems and Power Supplies 12 Detection Methods 35 Tank Blotting Cells 12 Colorimetric Detection 36 Part 3 Troubleshooting 63 Mini Trans-Blot® Cell and Criterion™ Blotter 12 Premixed and Individual Colorimetric Substrates 38 Transfer 64 Trans-Blot® Cell 12 Immun-Blot® Assay Kits 38 Electrophoretic Transfer 64 Trans-Blot® Plus Cell 13 Immun-Blot Amplified AP Kit 38 Microfiltration 65 Semi-Dry Blotting Cells
    [Show full text]