DOCSLIB.ORG

Blotting Techniques

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Blotting Techniques BLOTTING TECHNIQUES Dr.Ramesh C.K. Associate Professor and Chairman Department of Biotechnology Sahyadri Science College Shimoga - 577 203 Karnataka, India BLOTTING TECHNIQUES Southern Blot Northern blot Western blot It is used to detect It is used to detect It is used to detect the DNA. the RNA. proteins. SOUTHERN BLOTTING A technique for identifying specific sequences of DNA in which DNA fragments are separated by electrophoresis, transferred to a membrane, and identified with a suitable probe. Detects restriction fragments following a restriction enzyme digest of genomic DNA (RFLPs) Named after British biochemist Edwin Southern (alive and publishing!) Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol Biol. 1975 Nov 5;98(3):503-17. History/Background • ‘Southern’ hybridization named after Sir Edwin Southern • Developed in 1975 • One of the most highly cited scientific publications “Used to detect the DNA” • This method Involves: Separation Transfer Hybridization. • This DNA can be: • Single gene • Part of a larger piece of DNA……..viral genome “The key to this method is Hybridization” Hybridization “Process of forming a dsDNA molecule between a ssDNA probe and a ss-target DNA” PRINCIPLE The mixture of molecules is separated. Immobilized on a matrix. Probe addition to the matrix to bind to the molecules. Unbound probes are removed. “The place where the probe is connected corresponds to the location of the immobilized target molecule.” Steps The DNA is digested Fragments Gel electrophoresis Transfer to membrane Probing Autoradiogram I. DNA Purification – Isolate the DNA in question from the rest of the cellular material in the nucleus. – Incubate specimen with detergent to promote cell lysis. – Lysis frees cellular proteins and DNA. SOUTHERN BLOTTING • Proteins are enzymatically degraded by incubation with proteinase. • Organic or non-inorganic extraction removes proteins. • DNA is purified from solution by alcohol precipitation. • Visible DNA fibers are removed and suspended in buffer. SOUTHERN BLOTTING II. DNA Fragmentation – Cut the DNA into different sized pieces. – Use restriction endonucleases (RE) – Bacterial proteins – In vivo, they are involved in DNA metabolism and repair or in bacterial host defense. Step 1. Restriction Enzyme Digestion SOUTHERN BLOTTING • This allows for specific sequences to be identified more readily. • Fragments are now easily separated by gel electrophoresis. SOUTHERN BLOTTING III. Gel Electrophoresis – Sorts the DNA pieces by size – Gels are solid with microscopic pores – Agarose or polyacrimide – Gel is soaked in a buffer which controls the size of the pores – Standards should also be run SOUTHERN BLOTTING • Nucleic acids have a net negative charge and will move from the left to the right. The larger molecules are held up while the smaller ones move faster. This results in a separation by size. Gel Electrophoresis _ + Gel Electrophoresis _ + SOUTHERN BLOTTING • Gels can be stained with ethidium bromide. • This causes DNA to fluoresce under UV light which permits photography of the gel. • You can tell the exact migration of DNA standards and the quality of the RE digestion of the test DNA. Step 4. Transfer DNA to Membrane Goals of Southern Hybridization • Blotting Transfer the DNA from the gel to a solid support. Immobilize DNA onto a permanent substrate . ‘Membrane’ – paper-like matrix nitrocellulose paper – Or nylon usually has a slight positive charge SOUTHERN BLOTTING • DNA is partially depurinated with dilute HCL which promotes higher efficiency transfer by breaking down fragments into smaller pieces. • DNA is then denatured with an alkaline solution such as NAOH. • This causes the double stranded to become single-stranded. DNA Denaturation • Eliminate hydrogen bonds with sodium hydroxide (NaOH) A C T T G A T G A A C T SOUTHERN BLOTTING • DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe. • Transferred by either electroblotting or capillary blotting. Transfer DNA to Membrane • Two methods for transferring DNA to a membrane – capillary – Electrophoretic/electroblotting SOUTHERN BLOTTING • 1) Electrophoresis- takes advantage of the molecules negative charge. SOUTHERN BLOTTING • 2) Capillary blotting-fragments are eluted from the gel and deposited onto the membrane by buffer that is drawn through the gel by capillary action. SOUTHERN BLOTTING • The blot is made permanent by: – Drying at ~80°C – Exposing to UV irradiation Pre-hybridization V. Blocking Buffer binds to areas on the blot not occupied by • target DNA. Blocks the empty sites from being bound during hybridization. Prehybridization buffers contain ‘blocking reagents’ that occupy available binding sites on the membrane Probes Short sequence of nucleotides which can bind to target sequence. Synthetic oligo nucleotides Should be specific, complementary to target sequence Probe Size- 10 -10,000 bases Most common 14 – 40 bases Long Probes –More stable Takes longer time to hybridize Short Probes – Rapid hybridization, SOUTHERN BLOTTING • VI. Preparing the probe – Small piece of DNA used to find another piece of DNA – Must be labeled to be visualized – Usually prepared by making a radioactive copy of a DNA fragment. SOUTHERN BLOTTING • The DNA fragment is labeled by the Random Hexamer Labeling Process: – 1. The template DNA is denatured by boiling. – 2. A mixture of hexamers (6 nucleotides) containing all possible sequences is added and allow to base pair. – 3. DNA polymerase is added with radioactive nucleotides. – 4. The mixture is boiled to separate the strands and is ready for hybridization. SOUTHERN BLOTTING • The Random Hexamer Labeling Process produces a radioactive single-stranded DNA copy of both strands of the template for use as a probe. Probe • How do we detect the probe? – Radioactivity (32P) Probe • How do we detect the probe? – Digoxigenin (DIG) U SOUTHERN BLOTTING • VII. Hybridization – The labeled probe is added to the blocked membrane in buffer and incubated for several hours to allow the probe molecules to find their targets. Hybridization Hybridization SOUTHERN BLOTTING • VIII. Washing – Excess probe will have bound nonspecifically to the membrane despite the blocking reagents. – Blot is incubated with wash buffers containing NaCl and detergent to wash away excess probe and reduce background. Washes Anti-DIG Anti-DIG Washes Step 11. CSPD Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2´-(5'-chloro)tricyclo [3.3.1.13,7]decan}-4-yl)phenyl phosphate Step 12. Detection • DIG-labeled probes emitting minute amounts of light • Digoxigenin (DIG), which is used to label the probe, is widely used for immune detection. This plant steroid molecule is highly antigenic. Anti- digoxigenin can be used to very specifically detect the digoxigenin on a hybridised blot. • To be able to visualise the (polyclonal) DIG antibodies when they are bound to the digoxigenin, they have been covalently linked to the enzyme alkaline phosphatase (AP). • (chemiluminescence) • 32P-labeled probes emitting β- particles Step 12. Detection • DIG-labeled probes emitting minute amounts of light (chemiluminescence) • 32P-labeled probes emitting ß-particles • Autoradiography film can detect this radiation Making A Southern Blot 4 Probe Hybridization Addition of blocking reagent Probe addition After washing Membrane with Parts of the bound DNA membrane not already covered Probe covers the with DNA now membrane, but only binds to Probe only remains bind blocking annealed to reagent complimentary DNA complimentary DNA Making A Southern Blot 5 Autorad Development Membrane with probe bound to complimentary DNA X-ray film is placed over the membrane and left until radiation Fragments from the probe has complimentary to the exposed the film probe appear as bands on the autorad USES • Identify mutations, deletions, and gene rearrangements • Used in prognosis of cancer and in prenatal diagnosis of genetic diseases • Leukemias • Diagnosis of HIV-1 and infectious disease USES • Every person has repeated sequences of base pairs which are called Variable Number Tandem Repeats (VNTRs) • To find a particular VNTR we use a radioactive version of the one in question. • This pattern is known as a DNA fingerprint. USES • Applications of DNA fingerprinting include: – Paternity and Maternity Testing – Criminal Identification and Forensics – Personal Identification NORTHERN BLOTTING James Alwine, David Kemp, and George Stark at Stanford University in 1979. In this technique RNA is separated by gel electrophoresis. Southern’s technique has been of enormous value, but it was thought that it could not be applied directly to the blot-transfer of RNAs separated by gel electrophoresis, because RNA was found not to bind to nitrocellulose. Alwine et al. (1979) therefore found a procedure in which RNA bands are blot-transferred from the gel on to chemically reactive paper, where they are bound covalently.. 61 Conti… . The reactive paper is prepared by diazotization of aminobenzyloxymethyl paper (creating diazo benzyloxy methyl (DBM) paper). It is prepared from Whattman 540 paper by a series of uncomplicated reactions. Once covalently bound, the RNA is available for hybridization with radiolabelled DNA probes. As before, hybridizing bands are located by autoradiography. 62 Conti… • Subsequently it was found that RNA bands can indeed be blotted on to nitrocellulose membranes under appropriate conditions (Thomas 1980) and suitable nylon membranes have been developed. • Because of the convenience
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]
  • 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]
  • HUMAN GENE MAPPING WORKSHOPS C.1973–C.1991
    HUMAN GENE MAPPING WORKSHOPS c.1973–c.1991 The transcript of a Witness Seminar held by the History of Modern Biomedicine Research Group, Queen Mary University of London, on 25 March 2014 Edited by E M Jones and E M Tansey Volume 54 2015 ©The Trustee of the Wellcome Trust, London, 2015 First published by Queen Mary University of London, 2015 The History of Modern Biomedicine Research Group is funded by the Wellcome Trust, which is a registered charity, no. 210183. ISBN 978 1 91019 5031 All volumes are freely available online at www.histmodbiomed.org Please cite as: Jones E M, Tansey E M. (eds) (2015) Human Gene Mapping Workshops c.1973–c.1991. Wellcome Witnesses to Contemporary Medicine, vol. 54. 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 and ancillary guides xi Introduction Professor Peter Goodfellow xiii Transcript Edited by E M Jones and E M Tansey 1 Appendix 1 Photographs of participants at HGM1, Yale; ‘New Haven Conference 1973: First International Workshop on Human Gene Mapping’ 90 Appendix 2 Photograph of (EMBO) workshop on ‘Cell Hybridization and Somatic Cell Genetics’, 1973 96 Biographical notes 99 References 109 Index 129 Witness Seminars: Meetings and publications 141 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. The meeting is recorded, transcribed, and edited for publication.
    [Show full text]
  • Blotting Techniques M.W
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector RESEARCH TECHNIQUES MADE SIMPLE North, South, or East? Blotting Techniques M.W. Nicholas1 and Kelly Nelson2 Journal of Investigative Dermatology (2013) 133, e10; doi:10.1038/jid.2013.216 One of the cornerstones of modern molecular biology, blotting is a powerful and sensitive technique for identifying WHAT BLOTTING DOES the presence of specific biomolecules within a sample. • Blotting allows specific and sensitive detection of a Subtypes of blotting are differentiated by the target protein (western) or specific DNA or RNA sequence molecule that is being sought. The first of these tech­ (Southern, northern) within a large sample isolate. niques developed was the Southern blot, named for Dr. • Targets are first separated by size/charge via gel Edwin Southern, who developed it to detect specific DNA electrophoresis and then identified using a very sequences (Southern, 1975). Subsequently, the method was sensitive probe. modified to detect other targets. The nomenclature of these • Variations of these techniques can detect post­ techniques was built around Dr. Southern’s name, resulting translational modifications and DNA­bound proteins. in the terms northern blot (for detection of RNA), western blot (for detection of protein), eastern blot (for detection • Western blotting may also be used to detect a of posttranslationally modified proteins), and south­ circulating antibody in a patient sample or confirm western blot (for detection of DNA binding proteins). Most an antibody’s specificity. researchers consider the eastern blot and the southwestern blot variations of western blots rather than distinct entities.
    [Show full text]
  • Oxalate Oxidase Quantification and Relative Mrna Expression in Transgenic American Chestnut (Castanea Dentata)
    SUNY College of Environmental Science and Forestry Digital Commons @ ESF Dissertations and Theses Spring 4-27-2020 Oxalate Oxidase Quantification and Relative Mrna Expression in Transgenic American Chestnut (Castanea Dentata) Dakota Matthews SUNY College of Environmental Science and Forestry, [email protected] Follow this and additional works at: https://digitalcommons.esf.edu/etds Part of the Forest Biology Commons Recommended Citation Matthews, Dakota, "Oxalate Oxidase Quantification and Relative Mrna Expression in Transgenic American Chestnut (Castanea Dentata)" (2020). Dissertations and Theses. 175. https://digitalcommons.esf.edu/etds/175 This Open Access Thesis is brought to you for free and open access by Digital Commons @ ESF. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of Digital Commons @ ESF. For more information, please contact [email protected], [email protected]. OXALATE OXIDASE QUANTIFICATION AND RELATIVE MRNA EXPRESSION IN TRANSGENIC AMERICAN CHESTNUT (CASTANEA DENTATA) by Dakota Matthews A thesis submitted in partial fulfillment of the requirements for the Masters of Science Degree State University of New York College of Environmental Science and Forestry Syracuse New York May 2020 Approved by: Dr. William A. Powell, Major Professor Dr. Stephen Shaw, Examining Committee Dr. Melissa K. Fierke, Department Chair S. Scott Shannon, Dean, The Graduate School i Acknowledgements I am extremely grateful to have been able to work on the American chestnut research and restoration project here at SUNY-ESF. This project would not have been possible without the support from many individuals. I would first like to thank my major professor Dr. William A. Powell, who has taught me so much and been extremely patient with me.
    [Show full text]
  • Molecular Techniques in Radiobiology Introduction The
    Introduction • The birth of molecular biology could be ascribed to the discovery of the structure of DNA by Watson Molecular Techniques in and Francis Crick in 1953 Radiobiology • In the late 1940s and early 1950s, Linus Pauling realized that as amino acids were folded into proteins, they formed three-dimensional structures, and that function was related to the structure th Chapter 16 (6 edition) • The whole concept emerged that the sequence of bases, which coded for a protein, ultimately Eric J. Hall., Amato Giaccia, determined function leading the way to breaking the genetic code Radiobiology for the Radiologist The structure of DNA The structure of DNA • DNA - deoxyribonucleic acid is a polymer with the • DNA molecule has many monomer units of nucleotides deoxyribo-nucleotides (bases) linked in a chain- • Each nucleotide consists of a 5-carbon sugar like arrangement (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group • Bases are held by hydrogen bonds and are • There are four different types of nucleotides found in paired complimentary: DNA, differing only in – adenine with thymine the nitrogenous base: – cytosine with guanine A is for adenine • Each half constitutes a G is for guanine template for C is for cytosine reconstruction of the T is for thymine other half RNA and DNA Transcription and translation • RNA – ribonucleic acid, has ribose sugar molecule The flow of genetic information from DNA to protein (gene instead of deoxyribose expression) requires a series of steps: • In the cell RNA is
    [Show full text]
  • Science and the Sustainable Intensification of Global Agriculture
    Reaping the benefits Science and the sustainable intensification of global agriculture October 2009 Cover image: From an illustration of a push-pull system for pest control, courtesy of The Gatsby Charitable Foundation. The Quiet Revolution: Push-Pull Technology and the African Farmer. Gatsby Charitable Foundation 2005. Reaping the benefi ts: science and the sustainable intensifi cation of global agriculture RS Policy document 11/09 Issued: October 2009 RS1608 ISBN: 978-0-85403-784-1 © The Royal Society, 2009 Requests to reproduce all or part of this document should be submitted to: The Royal Society Science Policy 6–9 Carlton House Terrace London SW1Y 5AG Tel +44 (0)20 7451 2500 Email [email protected] Web royalsociety.org Design by Franziska Hinz, Royal Society, London Copyedited and Typeset by Techset Composition Limited Reaping the benefi ts: science and the sustainable intensifi cation of global agriculture Contents Foreword v Membership of working group vii Summary ix 1 Introduction 1 1.1 An urgent challenge 1 1.2 Trends in food crop production 2 1.3 Science in context 5 1.4 The need for sustainable intensifi cation 6 1.5 Agricultural sustainability 7 1.6 Agriculture and sustainable economic development 7 1.7 Other major studies 8 1.8 Further UK work 9 1.9 About this report 9 1.10 Conduct of the study 10 2 Constraints on future food crop production 11 2.1 Climate change 11 2.2 Water 11 2.3 Temperature 12 2.4 Ozone 13 2.5 Soil factors 13 2.6 Crop nutrition 15 2.7 Pests, diseases and weed competition 16 2.8 Energy and greenhouse
    [Show full text]
  • Royal Medals 2012 Congratulations to Professor Peter Higgs FRS
    July 2012 Royal Medals 2012 Her Majesty The Queen has approved the Council’s recommendation that the RSE’s Royal Medals for 2012 should be awarded to: Professor David Milne OBE FREng FRSE (left) for his outstanding contribution to business and commerce in Scotland, and to Professor Sir Edwin Southern FRS HonFRSE (centre) for his outstanding contribution to molecular biology. We are delighted that HRH The Duke of Edinburgh will present the Medals at the RSE during the early afternoon of 26 September 2012. The Duke will also present the 2012 IEEE/RSE/ Wolfson James Clerk Maxwell Award to Professor Gerhard Sessler (right), Darmstadt University of Technology. Fellows are most welcome to attend. There will be a tea with HRH thereafter. To apply for admission tickets, please email: [email protected] or contact the Events Team on 0131 240 2780. There is no charge, but only those with formal admission cards will be able to attend. If you wish to be accompanied by your spouse/partner or a personal guest, please indicate this when you apply for tickets, which are being issued on a first-come, first-served basis. Congratulations to Professor Peter Higgs FRS FRSE The RSE wishes to congratulate Professor Peter Higgs FRS FRSE and the team at CERN on the recent announcement that they have discovered a new particle consistent with the Higgs boson. Ballot for Council and Office Bearers Ballot forms were issued to all Fellows at the beginning of July for the election of RSE Council and Office Bearers. Fellows are reminded to return their ballot forms, in the envelope provided with the form, no later than Wednesday 15 August 2012.
    [Show full text]
  • Communicating Biochemistry: Meetings and Events
    © The Authors. Volume compilation © 2011 Portland Press Limited Chapter 3 Communicating Biochemistry: Meetings and Events Ian Dransfield and Brian Beechey Scientific conferences organized by the Biochemical Society represent a key facet of activity throughout the Society’s history and remain central to the present mission of promoting the advancement of molecular biosciences. Importantly, scientific conferences are an important means of communicating research findings, establishing collaborations and, critically, a means of cementing the community of biochemical scientists together. However, in the past 25 years, we have seen major changes to the way in which science is communicated and also in the way that scientists interact and establish collabo- rations. For example, the ability to show videos, “fly through” molecular structures or show time-lapse or real-time movies of molecular events within cells has had a very positive impact on conveying difficult concepts in presentations. However, increased pressures on researchers to obtain/maintain funding can mean that there is a general reluctance to present novel, unpublished data. In addition, the development of email and electronic access to scientific journals has dramatically altered the potential for communi- cation and accessibility of information, perhaps reducing the necessity of attending meetings to make new contacts and to hear exciting new science. The Biochemical Society has responded to these challenges by progressive development of the meetings format to better match the
    [Show full text]
  • The Mapping of Chromosome 16
    The M:apping of Chrolnosome, A Norman A. 16Carl E. Hil( u The Mapping of Chromosome 16 Setting the Stage mapping techniques had been developed of lambda-phage clones, each containing and were being applied to the genomes DNA from one of the twenty-four hu- Bc}th the molecular and the physical of some of the favorite organisms of man chromosomes (see “Libraries from technology for constructing physical molecular biologists. Flow-sorted Chromosomes”), Those maps of complex genomes have devel- Cassandra Smith and Charles Cantor chromosome-specific libraries were oped at a blistering pace over the past had used pulsed-field gel electrophoresis designed as a source of probes to five years, due largely to the initiation to order the very large restriction frag- find polymorphic DNA markers for of the Human Genome Project. These ments produced by cutting the E. coli constructing genetic-linkage maps (see technologies include thecloning of very genome with two rare-cutting restriction “Modern Linkage Mapping”) and as large DNA fragments, electrophoretic enzymes. The resulting long-range a source of clones’ for rapid isolation separation of million-base-sized DNA restriction map of E. coli demonstrated of genes using cDNAs, or coding-region fragments, and sequence-based mapping that pulsed-field gel electrophoresis is probes, to pick out the appropriate clones using the polymerase chain reaction a way to study the long-range order from the libraries. Deaven and his group (PCFL) to identify unique sequences of landmarks on the DNA of human were also constructing larger-insert along the genome. The latter provides a chromosomes.
    [Show full text]
  • Techniques of Molecular Cell Biology
    Journal of Cell Science & Therapy Editorial Techniques of Molecular Cell Biology Iran Mazadeh * Department of Molecular, Cell and Developmental Biology, Los Angeles, CA 90095, USA MOLECULAR CLONING MACROMOLECULE BLOTTING AND Perhaps the most essential procedures of atomic science to PROBING contemplate protein work is sub-atomic cloning. In this method, The terms northern, western and eastern blotching are gotten DNA coding for a protein of interest is cloned utilizing from what at first was a sub-atomic science joke that played on polymerase chain response (PCR), or potentially limitation the term Southern smudging, after the strategy depicted by chemicals into a plasmid (articulation vector). A vector has 3 Edwin Southern for the hybridisation of smeared DNA. Patricia unmistakable highlights: a beginning of replication, a various Thomas, engineer of the RNA blotch which then, at that point cloning site (MCS), and a particular marker typically anti- got known as the northern smear, really didn't utilize the term. microbial opposition. Found upstream of the various cloning site are the advertiser locales and the record start site which manage the statement of cloned quality. This plasmid can be Southern blotting embedded into either bacterial or creature cells. Bringing DNA Named after its innovator, researcher Edwin Southern, the into bacterial cells should be possible by change by means of Southern blotch is a technique for examining for the presence of take-up of exposed DNA, formation through cell-cell contact or a particular DNA succession inside a DNA test. DNA tests by transduction by means of viral vector. Bringing DNA into previously or after limitation compound (limitation eukaryotic cells, like creature cells, by physical or compound endonuclease) absorption are isolated by gel electrophoresis and methods is called transfection.
    [Show full text]