The Nucleic Acid Protocols Handbook

997 Pages·2000·18.83 MB·English

by  Ian Garner (auth.)

THE NUCLEIC ACID PROTOCOLS HANDBOOK
The
Nucleic Acid
Protocols
Handbook
Edited by
Ralph Rapley
University ofHertfordshire
Hatfield
UK
*-
Humana Press Totowa, New Jersey
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Thenucleicacidprotocolshandbook 1editedbyRalphRapley.
p. em.
Includesindex.
ISBN0-89603-459-3(alk. paper)(hardcover),ISBN0-89603-841-6(paper)
I.Nucleicacids~Laboratorymanuals. 2.Polymerasechainreaction~Laboratorymanu
als. 3.Genemapping~Laboratorymanuals. 4.Moleculargenetics~Laboratorymanuals.
I. Rapley, Ralph.
QP620.N7987 1999
572.8---dc21 98-43385
CIP
Preface
There can be no doubt that some ofthe most spectacularadvances made in science
over the past few decades have been in the isolation, analysis, and manipulation of
nucleicacids.Thishasledtoamuchgreaterunderstandingofmechanismsandprocesses
across many fields of bioscience, such as biochemistry, microbiology, physiology,
pharmacology, andthemedicalsciencestonameafew. Ithasalsoledto the growthof
the biotechnology industry, which seeks to develop andcommercialize many ofthese
importantprocesses and methods. Much ofthis has come about because ofthe devel
opment of numerous molecular biology and genetic manipulation techniques. The
discovery ofrestriction enzymes and the development ofcloning vectors in the early
1970sopenedthedoortowaysofisolatingandmanipulatingnucleicacidsthathadnever
been thought possible. Gene probe labeling and hybridization were developed and
refinedtoprovidepowerfulmethodsofanalysis.These-togetherwiththedevelopment
of DNA sequencing methods, protein engineering techniques, and PeR-have all
continuedtocontributesubstantiallytotheunderstandingofbiologicalprocessesatthe
molecularlevel. TheprotocolsfortheseimportantmethodsarethefocusofTheNucleic
AcidProtocolsHandbook, whose aim is to provide acomprehensive setoftechniques
inonevolumethatwillenablethe isolation,analysis,andmanipulationofnucleicacids
to be readily undertaken.
TheNucleicAcidProtocolsHandbookisdividedinto 10parts;withineachthereare
approximately10chapters.Thefirstfourpartsfollowoneanotherlogically:nucleicacid
extraction(PartI),basicseparationandanalysisofDNA(II), throughprobedesignand
labeling(III),andRNAanalysistechniques(IV).Thefollowingthreesectionsdealwith
genelibraryconstruction andscreening(V),DNAsequencing(VI),andthepolymerase
chainreaction (VII). PartVIII deals with the analysis ofgenes, mutations, and protein
interactionsandisfollowedbyPartIX,onmutagenesis,transcription,andtranslationin
vitro. This is followed finally by Part X, on gene localization and mapping in situ.
In compiling this volume a number oftechniques have been drawn and updated from
versionsappearinginearliervolumesofHumanaPress'Methods inMolecularBiology
series. These highly successful books have provided numerous laboratories with the
techniques needed to undertake modern laboratory molecular biology success
fully. Assuch,theirformathasbeenfollowedinTheNucleicAcidProtocolsHandbook.
Thusashortintroductiontothebasictheoryofthetechniqueisfollowed byacomplete
listing ofall materials and reagents needed before a particular protocol is presented.
Step-by-stepinstructionsarethenprovidedintheMethodssection.Inaddition,Notesare
citedthroughout the Methods andappearatthe end ofthe chapter, providing valuable
andhighlyusefulinformationnotfoundintraditionalscientificliterature.Thisessential
v
vi Preface
infonnation in many casesmay mean the difference betweenthe success or failure of
aparticulartechniqueandisoneofthe recognizedkeypointsofthe HumanaMethods
in MolecularBiology series.
Itisinevitablethatadegreeofoverlapoccursbetweensomeofthechapters. Indeed,
the use ofthepolymerase chainreaction is now sowidespreadthat it is akey element
ofmany ofthe protocols. These have beencrossreferencedwhere possible, although
mostoftheprotocolsare self-containedandcanbeattemptedwithoutthe need to read
further chapters. For those new or unfamiliar to laboratory molecular biology, the
compilation ofprotocols in The Nucleic AcidProtocols Handbook also provides the
abilitytoattemptprotocolsconfidently.Theintentwasnottolistallprotocolsinmolec
ular biology (within one volume, this is an impossible task), and certainly more
advancedprotocolsmaybefound inanumberofexcellenttexts includingmanyinthe
HumanaMethods in MolecularBiologyseries.Itwas, however,theaimtoprovidethe
mostcommonly usedprotocols andalternatives in onevolume atalevel accessible to
most laboratories, which we believe has been achieved. In such a large compilation,
muchcreditmustgototheauthors,whohavedevotedvaluabletimeandefforttowrite
and update these protocols; to Prof. John M. Walker, the series editor, for his helpful
adviceandguidance;andtothestaffatHumanaPressfortheirsubstantialeffortsinthe
production ofthe volume.
Ralph Rapley
Contents
Preface v
Contributors xv
PART I NUCLEIC ACID EXTRACTION
Isolation of High-Molecular-Weight DNA from Animal Cells 3
Ian Garner
2 Isolation of mRNA by Affinity Chromatography 9
Sian BryantandDavid L. Manning
3 Isolation and Purification of DNA from Plants 13
Justin StaceyandPeter G. Isaac
4 Purification of Uncontaminated, Intact Plant RNA 17
Shu-Hua Cheng, Brandon D. Moore, andJeffreyR. Seemann
5 An Improved Method to Isolate Mitochondrial RNA
from Green Plant Tissue 23
Fei Ye andRal'Reski
6 Isolating Chromosomal DNA from Bacteria 29
Elisabeth ChachatyandPatrickSaulnier
7 Bacterial DNA Extraction for Polymerase Chain Reaction
and Pulsed-Field Gel Electrophoresis 33
Elisabeth ChachatyandPatrickSaulnier
8 Isolation of Fungal Nucleic Acids 37
SurapareddySreenivasaprasad
9 Total RNA Isolation from Bacteria 47
John Heptinstall
10 Simultaneous RNA and DNA Extraction
from Biopsy Material, Culture Cells, Plants, and Bacteria 53
Udo Dobbeling
11 Spectrophotometric Analysis of Nucleic Acids 57
John HeptinstallandRalph Rapley
PART II BASIC SEPARATION AND ANALYSIS OF DNA
12 Restriction Endonuclease Digestion of DNA 63
Duncan R. Smith
13 Agarose Gel Electrophoresis of Nucleic Acids 67
D. Ross Williams andRalph Rapley
vii
viii Contents
14 Preparation of RNA Dot Blots 71
RachelHodge
15 Native Polyacrylamide Gel Electrophoresis 73
Adrian J. Harwood
16 Southern Blotting of Agarose Gels by Capillary Transfer 77
Ralph RapleyandJane Davenport-Jones
17 Pulsed-Field Gel Electrophoresis 81
John Maule
18 HPLC of DNA and PCR Products 105
Elena D. Katz
PART III PROBE DESIGN, SYNTHESIS, AND LABELING
19 End-Labeling of DNA Fragments 117
Adrian J. Harwood
20 Nick Translation and Random Hexamer Labeling of DNA 123
Jane Davenport-Jones
21 Generation of Labeled Probes by Polymerase Chain Reaction 127
Y. M. Dennis LoandShu F. An
22 Nonradioactive Oligonucleotide Probe Labeling 135
Sue FowlerandIan Durrant
23 Preparation of Direct, Enzyme-Labeled DNA Probes 145
Ian Durrantand TimothyStone
24 Random Prime Labeling of DNA Probes
with Fluorescein-Tagged Nucleotides 149
Bronwen M. Harvey, Claire B. Wheeler, andMartin W. Cunningham
25 Hybridization and Detection of Fluorescein-Labeled DNA Probes
Using Chemiluminescence 153
Claire B. Wheeler, BronwenM. Harvey, andMartin W. Cunningham
26 Hybridization of Enzyme-Labeled Probes
and Detection by Chemiluminescence 157
TimothyStoneandIan Durrant
27 Hybridization and Competition Hybridization of Southern Blots 163
Rosemary Kelsell
28 Autoradiography and Fluorography 169
Eric Quemeneur
PART IV RNA ANALYSIS TECHNIQUES
29 Formaldehyde Gel Electrophoresis of Total RNA 177
Sian BryantandDavid L. Manning
30 RNA Probes for the Analysis of Gene Expression 181
Dominique Belin
31 Primer Extension Analysis of mRNA 195
Maggie Walmsley, Mark Leonard, andRogerPatient
Contents ix
32 S1 Mapping Using Single-Stranded DNA Probes 201
Stephane VivilleandRoberto Mantovani
33 Measurements of Rate of Transcription in Isolated Nuclei
by Nuclear"Run-Off" Assay 207
RaiAjitK. Srivastava andGustavSchonfeld
34 One-Tube RT-PCR with Sequence-Specific RT Primers 213
Ulrich PfefferandPaola Ferro
35 Characterization of RNA Using Continuous RT-PCR
Coupled with ELOSA 219
Fram;ois Mallet
36 Quantitative Analysis of RNA Species by Polymerase Chain
Reaction and Solid-Phase Minisequencing 229
AnuSuomalainen andAnn-ChristineSyvanen
37 Nonradioactive Northern Blotting of RNA 239
RainerLow
38 Analysis of RNA by Northern Blotting Using Riboprobes 249
RaiAjit K. Srivastava
PART V GENE LIBRARY CONSTRUCTION AND SCREENING
39 Production of Double-Stranded cDNA for Gene Library Synthesis 261
Jane KirkandSteve Mayall
40 Using Rapid Amplification of cDNA Ends (RACE)
to Obtain Full-Length cDNAs 267
Yue ZhangandMichaelA. Frohman
41 cDNA Library Construction
Using Streptavidin-Paramagnetic Beads and PCR 289
Kris N. Lambertand Valerie M. Williamson
42 Rapid (Ligase-Free) Subcloning
of Polymerase Chain Reaction Products 295
A/an R. Shu/dinerandKeith Tanner
43 Subtraction Hybridization cDNA Libraries 305
Clifford W. Schweinfest, PeterS. Nelson, Michael W. Graber,
Rita I. Demopoulos, and Takis S. Papas
44 Cloning Polymerase Chain Reaction Products
Utilizing the T/A Overhang and a Kit 319
Melissa Lail-Trecker
45 Extraction and Purification of Plasmid DNA 327
Craig Winstanley andRalph Rapley
46 Biotinylated Probes in Colony Hybridization 333
MichaelJ. Haas
47 Cloning Long Polymerase Chain Reaction Products 339
Songrong Ren andJ. Michael Ruppert
48 Cloning DNA Fragments in M13 Vectors 347
David Walsh
x Contents
49 cDNA Library Construction for the Lambda ZAP®-Based Vectors 355
MarjoryA. Snead, Michelle A. Alting-Mees, andJayM. Short
50 Expression and Preparation of Fusion Proteins
from Recombinant )",gt11 Phages 367
Sheng-HeHuangandAmbroseJong
51 Antibody Screening of Bacteriophage ,,-gt11 DNA
Expression Libraries 373
PeterJones
52 Screening cDNA Libraries by Hybridization
with Double-Stranded DNA Probes and Oligonucleotides 381
Caroline A. Austin
53 cDNA Library Screening with the Tetramethylammonium Chloride (TMAC)
Technique Using Highly Degenerate Oligoonucleotide Probes 389
BentHonore andPederMadsen
54 Screening Recombinant Libraries by Polymerase Chain Reaction 397
Michael W. King
55 Construction and Screening of Cosmid Libraries 405
Jens Hanke andJorg D. Hoheisel
56 Generation of Large Insert YAC Libraries 415
Zoia Larin, AnthonyP. Monaco, andHans Lehrach
57 YAC Library Storage and Transport 425
John E. Collins, Sheila Hassock, andIan Dunham
58 YAC Library Screening: Preparation ofHybridization Filters
andPolymerase Chain Reaction Pools 431
Charlotte G. Cole, John E. Collins, andIan Dunham
59 YAC Library Screening: Hybridization
andPCR-BasedScreening Protocols 437
Charlotte G. Cole, John E. Collins, andIan Dunham
60 Phage-Display Libraries of Murine
and Human Antibody'Fab Fragments 449
Jan Engberg, Lene K. Johansen, Michelle Westengaard-Hildinge,
Erik S. Riise, andBjarne Albrechtsen
PART VI DNA SEQUENCING
61 Preparation and Analysis of DNA Sequencing Gels 481
BimalD. M. Theophilus
62 DNA Sequencing of Plasmids 489
George Murphy
63 Sequencing DNA Fragments Cloned into M13
and Phagemid Vectors 493
Neil Brewis
64 Direct cDNA Sequencing Using Sequential
Linear/Asymmetric Polymerase Chain Reaction 499
IvorJ. Mason