Essential Molecular Biology: A Practical Approach Volume I (Practical Approach Series) (2nd edition) PDF
Preview Essential Molecular Biology: A Practical Approach Volume I (Practical Approach Series) (2nd edition)
Essential Molecular Biology Volume One The Practical Approach Series Related Practical Approach Series Titles Human Cytogenetics: Malignancy and Genome Mapping Acquired Abnormalities 2 Protein Function 2/e Human Cytogenetics: Constitutional Protein Structure 2/e Analysis* DNA and Protein Sequence Animal Cell Culture 3/e Analysis RNA Viruses Protein Structure Prediction Differential Display Antibody Engineering Mouse Genetics and Transgenics DNA Cloning 4: Mammalian DNA Viruses Systems Gene Targeting 2/e DNA Cloning 3: Complex Protein Phosphorylation 2/e Genomes Crystallization of Nucleic Gene Probes 2 Acids and Proteins Gene Probes 1 DNA Microarray Technology Pulsed Field Gel Post-Translational Modification Electrophoresis Protein Expression Non-isotopic Methods in Molecular Biology Chromosome Structural Analysis PCR 2 Gel Electrophoresis of Proteins 3/e DNA Cloning 2: Expression In Situ Hybridization 2/e Systems Chromatin DNA Cloning 1: Core Techniques Mutation Detection Molecular Genetics of Yeast Molecular Genetic Analysis of RNA Processing Volume II Populations 2/e RNA Processing Volume I PCR 3: PCR In Situ Hybridization PCR 1 Antisense Technology Plant Molecular Biology * indicates a forthcoming title Please see the Practical Approach series website at http://www.oup.co.uk/pas for full contents lists of all Practical Approach titles. Essential Molecular Biology Volume One Second Edition A Practical Approach Edited by T. A. Brown Department of Biomolecular Sciences UMIST, Manchester, U.K. OXFORD UNIVERSITY PRESS OXFORD UNIVERSITY PRESS Great Clarendon Street, Oxford 0X2 6DP Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Athens Auckland Bangkok Bogota Buenos Aires Calcutta Cape Town Chennai Dar es Salaam Delhi Florence Hong Kong Istanbul Karachi Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Paris Sao Paulo Singapore Taipei Tokyo Toronto Warsaw with associated companies in Berlin Ibadan Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries Published in the United States by Oxford University Press Inc., New York © Oxford University Press, 2000 The moral rights of the author have been asserted Database right Oxford University Press (maker) First published 2000 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, or under terms agreed with the appropriate reprographics rights organization. Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above You must not circulate this book in any other binding or cover and you must impose this same condition on any acquirer British Library Cataloguing in Publication Data Data available Library of Congress Cataloguing in Publication Data 1 3 5 7 9 1 0 8 6 4 2 ISBN 0 19 963643 5 (Hbk.) ISBN 0 19 963642 7 (Pbk.) Typeset in Swift by Footnote Graphics, Warminster, Wilts Printed in Great Britain on acid-free paper by The Bath Press, Avon Preface to the first edition There are now a number of molecular biology manuals on the market and the editor of an entirely new one has a duty to explain why his contribution should be needed. My answer is that although there are some excellent handbooks for researchers who already know the basic principles of gene cloning there are very few that cater for the absolute beginner. Unfortunately, everyone is a beginner at some stage in their careers and, even in an established molecular biology lab- oratory, the new research student can spend a substantial amount of time not really understanding what is going on. For the experienced biologist expert in a discipline other than molecular biology, and perhaps without direct access to a tame gene cloner, guidance on how to introduce recombinant DNA techniques into their own research programme can be very difficult to obtain. For several years I have run a basic gene cloning course at UMIST and I have continually been impressed by the number of biochemists, botanists, geneticists, cell biolo- gists, medics and others who want to learn how to clone and study genes. The contributors to Essential Molecular Biology: A Practical Approach were asked to write accounts that combine solid practical information with sufficient back- ground material to ensure that the novice can understand how a technique works, what it achieves, and how to make modifications to suit personal require- ments. Where appropriate, the reader is also given advice on more advanced or specialized techniques. In all cases the authors have responded to the challenge and produced chapters that make concessions to the beginner without jeopard- izing scientific content or practical value. I hope that the result is a handbook that will guide newcomers into molecular biology research. The book is split into two parts. Volume I deals with the fundamental tech- niques needed to carry out DNA cloning experiments. The emphasis is on coming to grips with the necessary practical skills and understanding the back- ground in sufficient detail to be able to adjust to circumstances as the project progresses. In Volume II, procedures for preparing gene libraries and identifying genes are described, together with methods for studying the structure of a cloned gene and the way it is expressed in the cell. It is assumed that the basics from Volume I are now in place, but the procedures are still described in the same down-to-earth fashion, with protocols complemented by background information and troubleshooting hints. PREFACE TO THE FIRST EDITION I must thank a number of people for their help with this book. First, I am grateful to the authors who provided the manuscripts, more or less on time, and were prepared in many cases to make revisions according to my requests. I would especially like to thank Paul Towner for stepping in at the last minute after I had been let down with one chapter. The participants and assistants on the recent UMIST gene cloning courses helped me formulate the contents of the book and my colleague Paul Sims provided valuable advice. The Series Editors and publishers made encouraging noises when the going got tough, and sorted out a number of problems for me. I am also grateful to my research students for giving their opinion on the practical contents of the book. Finally, I would like to thank my wife Ken for proving that even an archaeologist can learn how to clone genes. 1990 T.A.B. VI Preface to the Second Edition The primary objective of Essential Molecular Biology: A Practical Approach, that the book should be accessible and informative to researchers encountering molecu- lar biology techniques for the first time, is as valuable today as it was when the first edition was written 10 years ago. If anything, the need for a basic DNA man- ual has increased as molecular biology techniques have become more and more mainstream in disparate areas of research. Gene cloning, sequencing and PCR are now central to those various research fields grouped together under 'molec- ular life sciences' and an ever-increasing number of new research students enter these fields every year. Equally impressive has been the spread of DNA tech- niques into areas of research such as molecular ecology and biomolecular archaeology, where the power of DNA analysis is contributing to scientific knowledge in disciplines that just a few years ago either did not exist at all or had little use for clones and PCR products. When planning the second edition of Essential Molecular Biology: A Practical Approach I therefore determined from the very start that the objective of the book should remain the same: 'to combine solid practical information with sufficient background material to ensure that the novice can understand how a technique works, what it achieves, and how to make modifications to suit personal requirements'. Since the first edition was published there has been a dramatic expansion in the range and sophistication of molecular biology techniques, even in those basic techniques that are of greatest importance to the newcomer. To address these changes, every chapter has been revised and updated as appropriate, with some chapters completely rewritten. This is particularly true for the more advanced techniques described in Volume II but also applies to the very basic procedures covered in Volume I, the advances over the last 10 years even in- volving such fundamental techniques as DNA purification and agarose gel electrophoresis. Despite these changes, the two volumes are still organized according to the rationale of the first edition, with the procedures for DNA and RNA manipulation (purification, electrophoresis and the construction and cloning of recombinant molecules) in Volume I and those for isolating and study- ing individual genes (preparation and screening of libraries, polymerase chain reactions, DNA sequencing and studying gene expression) in Volume II. VII PREFACE TO THE SECOND EDITION An edited volume is only as good as the chapters it contains and I therefore wish to thank all the contributors for taking such great care to address the objective of Essential Molecular Biology by making their chapters lucid and infor- mative. I also thank Liz Owen and Lisa Blake of Oxford University Press for ensuring that the chapters became a book rather than languishing in my com- puter, and my wife Keri Brown for helping me find the time to organize and assemble these two volumes. 2000 TAB. VIII Contents List of protocols page xv Abbreviations xix 1 Getting started in molecular biology 1 T. A. Brown 1 Introduction 1 2 Practical requirements for molecular biology research 2 Experimental skills 2 Equipment 2 A word on kits 5 3 Health hazards and safety procedures 5 Microbiological safety 5 Radiochemicals 6 Chemical hazards 6 Ultraviolet radiation 7 High-voltage electricity 7 4 Research strategies for molecular biology 8 Gene cloning in outline 8 PCR in outline 10 The choice between cloning and PCR 10 Basic techniques needed for cloning and PCR 12 5 Planning an informative project 16 Will it be possible to identify the correct clone? 16 Will the DNA sequence provide any new information? 17 Will studies of gene expression be informative? 17 Conclusion 18 References 18 2 Microbiological techniques for molecular biology: bacteria and phages 21 Brian W. Bainbridge 1 Introduction: techniques for handling microbes 21 Basic microbiological techniques 21 IX
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