Gene Cloning DNA Analysis An Introduction.pdf

1、9781405181730_1_pre.qxd 1/11/10 10:23 Page ivGENECLONINGAND DNAANALYSIS9781405181730_1_pre.qxd 1/11/10 10:23 Page i9781405181730_1_pre.qxd 1/11/10 10:23 Page iiGENECLONINGAND DNAANALYSISAn IntroductionT.A.BROWNFaculty of Life SciencesUniversity of ManchesterManchesterSixth EditionA John Wiley&Sons,L

2、td.,Publication9781405181730_1_pre.qxd 1/11/10 10:23 Page iiiThis edition first published 2010,2010,2006 by T.A.BrownFirst,second and third editions published by Chapman&Hall 1986,1990,1995Fourth and fifth editions published by Blackwell Publishing Ltd 2001,2006Blackwell Publishing was acquired by J

3、ohn Wiley&Sons in February 2007.Blackwells publishing program has been merged with Wileys global Scientific,Technical and Medical business to form Wiley-Blackwell.Registered office:John Wiley&Sons Ltd,The Atrium,Southern Gate,Chichester,West Sussex,PO19 8SQ,UKEditorial offices:9600 Garsington Road,O

4、xford,OX4 2DQ,UKThe Atrium,Southern Gate,Chichester,West Sussex,PO19 8SQ,UK111 River Street,Hoboken,NJ 07030-5774,USAFor details of our global editorial offices,for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our web

5、site at right of the author to be identified as the author of this work has been asserted in accordance with the Copyright,Designs and Patents Act 1988.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,electronic

6、,mechanical,photocopying,recording or otherwise,except as permitted by the UK Copyright,Designs and Patents Act 1988,without the prior permission of the publisher.Wiley also publishes its books in a variety of electronic formats.Some content that appears in print may not be available in electronic b

7、ooks.Designations used by companies to distinguish their products are often claimed as trademarks.All brand names and product names used in this book are trade names,service marks,trademarks or registered trademarks of their respective owners.The publisher is not associated with any productor vendor

8、 mentioned in this book.This publication is designed to provide accurate and authoritativeinformation in regard to the subject matter covered.It is sold on the understanding that thepublisher is not engaged in rendering professional services.If professional advice or other expertassistance is requir

9、ed,the services of a competent professional should be sought.Library of Congress Cataloguing-in-Publication DataBrown,T.A.(Terence A.)Gene cloning and DNA analysis:an introduction/T.A.Brown.6th ed.p.cm.ISBN 978-1-4051-8173-0(pbk.:alk.paper)ISBN 978-1-4443-3407-4(hbk.:alk.paper)1.Molecular cloning.2.

10、Nucleotide sequence.3.DNAAnalysis.I.Title.QH442.2.B76 2010572.8633dc222009038739ISBN:9781405181730(paperback)and 9781444334074(hardback)A catalog record for this book is available from the British Library.Set in 10/12pt Classical Garamond by Graphicraft Limited,Hong KongPrinted in Malaysia1201097814

11、05181730_1_pre.qxd 1/11/10 10:23 Page ivPreface to the Sixth Edition xviPart I The Basic Principles of Gene Cloning and DNA Analysis11Why Gene Cloning and DNA Analysis are Important32Vectors for Gene Cloning:Plasmids and Bacteriophages133Purification of DNA from Living Cells254Manipulation of Purifi

12、ed DNA 455Introduction of DNA into Living Cells726Cloning Vectors for E.coli887Cloning Vectors for Eukaryotes1058How to Obtain a Clone of a Specific Gene1269The Polymerase Chain Reaction147Part II The Applications of Gene Cloning and DNA Analysis in Research16310Sequencing Genes and Genomes16511Stud

13、ying Gene Expression and Function18512Studying Genomes207Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology22313Production of Protein from Cloned Genes22514Gene Cloning and DNA Analysis in Medicine24515Gene Cloning and DNA Analysis in Agriculture26416Gene Cloning and DNA Ana

14、lysis in Forensic Science and Archaeology282Glossary298Index312Companion website available at CONTENTSBrief Contents9781405181730_1_pre.qxd 1/11/10 10:23 Page v9781405181730_1_pre.qxd 1/11/10 10:23 Page viPreface to the Sixth EditionxviPart I The Basic Principles of Gene Cloning and DNA Analysis11 W

15、hy Gene Cloning and DNA Analysis are Important31.1The early development of genetics31.2The advent of gene cloning and the polymerase chain reaction41.3What is gene cloning?51.4What is PCR?61.5Why gene cloning and PCR are so important71.5.1Obtaining a pure sample of a gene by cloning71.5.2PCR can als

16、o be used to purify a gene91.6How to find your way through this book112 Vectors for Gene Cloning:Plasmids andBacteriophages132.1Plasmids132.1.1Size and copy number152.1.2Conjugation and compatibility162.1.3Plasmid classification162.1.4Plasmids in organisms other than bacteria172.2Bacteriophages172.2

17、.1The phage infection cycle182.2.2Lysogenic phages19Gene organization in the 2 DNA molecule19The linear and circular forms of 2 DNA19M13a filamentous phage222.2.3Viruses as cloning vectors for other organisms24viiCONTENTSContents9781405181730_1_pre.qxd 1/11/10 10:23 Page vii3 Purification of DNA fro

18、m Living Cells253.1Preparation of total cell DNA253.1.1Growing and harvesting a bacterial culture263.1.2Preparation of a cell extract283.1.3Purification of DNA from a cell extract29Removing contaminants by organic extraction and enzymedigestion29Using ion-exchange chromatography to purify DNA from a

19、 cellextract303.1.4Concentration of DNA samples303.1.5Measurement of DNA concentration313.1.6Other methods for the preparation of total cell DNA323.2Preparation of plasmid DNA333.2.1Separation on the basis of size353.2.2Separation on the basis of conformation36Alkaline denaturation36Ethidium bromide

20、caesium chloride density gradientcentrifugation363.2.3Plasmid amplification393.3Preparation of bacteriophage DNA393.3.1Growth of cultures to obtain a high 2 titer403.3.2Preparation of non-lysogenic 2 phages403.3.3Collection of phages from an infected culture423.3.4Purification of DNA from 2 phage pa

21、rticles423.3.5Purification of M13 DNA causes few problems434 Manipulation of Purified DNA454.1The range of DNA manipulative enzymes464.1.1Nucleases464.1.2Ligases474.1.3Polymerases484.1.4DNA modifying enzymes494.2Enzymes for cutting DNArestriction endonucleases504.2.1The discovery and function of res

22、triction endonucleases514.2.2Type II restriction endonucleases cut DNA at specific nucleotidesequences524.2.3Blunt ends and sticky ends534.2.4The frequency of recognition sequences in a DNA molecule534.2.5Performing a restriction digest in the laboratory544.2.6Analysing the result of restriction end

23、onuclease cleavage56Separation of molecules by gel electrophoresis57Visualizing DNA molecules in an agarose gel584.2.7Estimation of the sizes of DNA molecules584.2.8Mapping the positions of different restriction sites in a DNAmolecule594.2.9Special gel electrophoresis methods for separating largermo

24、lecules60Contentsviii9781405181730_1_pre.qxd 1/11/10 10:23 Page viii4.3Ligationjoining DNA molecules together634.3.1The mode of action of DNA ligase634.3.2Sticky ends increase the efficiency of ligation644.3.3Putting sticky ends onto a blunt-ended molecule64Linkers64Adaptors65Producing sticky ends b

25、y homopolymer tailing674.3.4Blunt end ligation with a DNA topoisomerase695 Introduction of DNA into Living Cells725.1Transformationthe uptake of DNA by bacterial cells745.1.1Not all species of bacteria are equally efficient at DNA uptake745.1.2Preparation of competent E.coli cells755.1.3Selection fo

26、r transformed cells755.2Identification of recombinants765.2.1Recombinant selection with pBR322insertional inactivation of an antibiotic resistance gene775.2.2Insertional inactivation does not always involve antibioticresistance795.3Introduction of phage DNA into bacterial cells815.3.1Transfection815

27、.3.2In vitro packaging of 2 cloning vectors815.3.3Phage infection is visualized as plaques on an agar medium815.4Identification of recombinant phages835.4.1Insertional inactivation of a lacZ gene carried by the phagevector835.4.2Insertional inactivation of the 2 cI gene835.4.3Selection using the Spi

28、 phenotype835.4.4Selection on the basis of 2 genome size845.5Introduction of DNA into non-bacterial cells855.5.1Transformation of individual cells855.5.2Transformation of whole organisms856 Cloning Vectors for E.coli886.1Cloning vectors based on E.coli plasmids896.1.1The nomenclature of plasmid clon

29、ing vectors896.1.2The useful properties of pBR322896.1.3The pedigree of pBR322906.1.4More sophisticated E.coli plasmid cloning vectors90pUC8a Lac selection plasmid92pGEM3Zin vitro transcription of cloned DNA936.2Cloning vectors based on M13 bacteriophage946.2.1How to construct a phage cloning vector

30、946.2.2Hybrid plasmidM13 vectors96Contentsix9781405181730_1_pre.qxd 1/11/10 10:23 Page ix6.3Cloning vectors based on 8 bacteriophage976.3.1Segments of the 2 genome can be deleted without impairingviability986.3.2Natural selection can be used to isolate modified 2 that lackcertain restriction sites98

31、6.3.3Insertion and replacement vectors98Insertion vectors99Replacement vectors1006.3.4Cloning experiments with 2 insertion or replacement vectors1006.3.5Long DNA fragments can be cloned using a cosmid1016.48 and other high-capacity vectors enable genomic libraries to beconstructed1026.5Vectors for o

32、ther bacteria1047 Cloning Vectors for Eukaryotes1057.1Vectors for yeast and other fungi1057.1.1Selectable markers for the 2 3m plasmid1067.1.2Vectors based on the 2 3m plasmidyeast episomal plasmids1067.1.3A YEp may insert into yeast chromosomal DNA1077.1.4Other types of yeast cloning vector1087.1.5

33、Artificial chromosomes can be used to clone long pieces of DNA in yeast110The structure and use of a YAC vector110Applications for YAC vectors1117.1.6Vectors for other yeasts and fungi1127.2Cloning vectors for higher plants1127.2.1Agrobacterium tumefaciensnatures smallest genetic engineer113Using th

34、e Ti plasmid to introduce new genes into a plant cell113Production of transformed plants with the Ti plasmid115The Ri plasmid117Limitations of cloning with Agrobacterium plasmids1177.2.2Cloning genes in plants by direct gene transfer118Direct gene transfer into the nucleus118Transfer of genes into t

35、he chloroplast genome1197.2.3Attempts to use plant viruses as cloning vectors119Caulimovirus vectors120Geminivirus vectors1207.3Cloning vectors for animals1207.3.1Cloning vectors for insects121P elements as cloning vectors for Drosophila121Cloning vectors based on insect viruses1227.3.2Cloning in ma

36、mmals122Viruses as cloning vectors for mammals123Gene cloning without a vector124Contentsx9781405181730_1_pre.qxd 1/11/10 10:23 Page x8 How to Obtain a Clone of a Specific Gene1268.1The problem of selection1268.1.1There are two basic strategies for obtaining the clone you want1278.2Direct selection1

37、288.2.1Marker rescue extends the scope of direct selection1298.2.2The scope and limitations of marker rescue1308.3Identification of a clone from a gene library1318.3.1Gene libraries1318.3.2Not all genes are expressed at the same time1318.3.3mRNA can be cloned as complementary DNA1338.4Methods for cl

38、one identification1338.4.1Complementary nucleic acid strands hybridize to each other1338.4.2Colony and plaque hybridization probing133Labeling with a radioactive marker136Non-radioactive labeling1378.4.3Examples of the practical use of hybridization probing137Abundancy probing to analyse a cDNA libr

39、ary137Oligonucleotide probes for genes whose translation productshave been characterized138Heterologous probing allows related genes to be identified141Southern hybridization enables a specific restriction fragmentcontaining a gene to be identified1428.4.4Identification methods based on detection of

40、 the translationproduct of the cloned gene144Antibodies are required for immunological detection methods144Using a purified antibody to detect protein in recombinantcolonies145The problem of gene expression1469 The Polymerase Chain Reaction1479.1The polymerase chain reaction in outline1479.2PCR in m

41、ore detail1499.2.1Designing the oligonucleotide primers for a PCR1499.2.2Working out the correct temperatures to use1529.3After the PCR:studying PCR products1539.3.1Gel electrophoresis of PCR products1549.3.2Cloning PCR products1549.3.3Problems with the error rate of Taq polymerase1579.4Real-time PC

42、R enables the amount of starting material to be quantified1589.4.1Carrying out a quantitative PCR experiment1599.4.2Real-time PCR can also quantify RNA160Contentsxi9781405181730_1_pre.qxd 1/11/10 10:23 Page xiPart II The Applications of Gene Cloning and DNA Analysis in Research16310 Sequencing Genes

43、 and Genomes16510.1 The methodology for DNA sequencing16510.1.1 Chain termination DNA sequencing166Chain termination sequencing in outline166Not all DNA polymerases can be used for sequencing168Chain termination sequencing requires a single-stranded DNAtemplate169The primer determines the region of

44、the template DNA that willbe sequenced16910.1.2 Pyrosequencing171Pyrosequencing involves detection of pulses ofchemiluminescence171Massively parallel pyrosequencing17110.2 How to sequence a genome17310.2.1 The shotgun approach to genome sequencing174The Haemophilus influenzae genome sequencing proje

45、ct174Problems with shotgun sequencing17610.2.2 The clone contig approach177Clone contig assembly by chromosome walking177Rapid methods for clone contig assembly178Clone contig assembly by sequence tagged site content analysis17910.2.3 Using a map to aid sequence assembly180Genetic maps180Physical ma

46、ps181The importance of a map in sequence assembly18311 Studying Gene Expression and Function18511.1 Studying the RNA transcript of a gene18611.1.1 Detecting the presence of a transcript and determining itsnucleotide sequence18611.1.2 Transcript mapping by hybridization between gene and RNA18811.1.3

47、Transcript analysis by primer extension19011.1.4 Transcript analysis by PCR19111.2 Studying the regulation of gene expression19211.2.1 Identifying protein binding sites on a DNA molecule193Gel retardation of DNAprotein complexes193Footprinting with DNase I194Modification interference assays19411.2.2

48、 Identifying control sequences by deletion analysis197Reporter genes197Carrying out a deletion analysis198Contentsxii9781405181730_1_pre.qxd 1/11/10 10:23 Page xii11.3 Identifying and studying the translation product of a cloned gene19911.3.1 HRT and HART can identify the translation product of a cl

49、onedgene19911.3.2 Analysis of proteins by in vitro mutagenesis200Different types of in vitro mutagenesis techniques202Using an oligonucleotide to create a point mutation in a cloned gene203Other methods of creating a point mutation in a cloned gene204The potential of in vitro mutagenesis20512 Studyi

50、ng Genomes20712.1 Genome annotation20712.1.1 Identifying the genes in a genome sequence208Searching for open reading frames208Simple ORF scans are less effective at locating genes ineukaryotic genomes209Gene location is aided by homology searching210Comparing the sequences of related genomes21112.1.