Table Of ContentSPRINGER
LAB MANUALS
Springer-Verlag Berlin Heidelberg GmbH
R. CURTIS BIRD BRUCE F. SMITH (EDS.)
Genetic Library
Construction and Screening
Advanced Techniques and Applications
With 30 Figures
Springer
DR. R. CURTIS BIRO DR. BRUCE F. SMITH
Department of Pathobiology Scott-Ritchey Research Center
Auburn University Auburn University
Auburn,AL 36849-5519 Auburn, AL 36849
USA USA
e-mail: [email protected] e-mail: [email protected]
Applied for Library of Congress Cataloging-in-Publication Data
Die Oeutsche Bibliothek -CIP-Einheitsaufnahme
Genetic library construction and screening : advanced techniques and
applications / R. Curtis Bird; Bruce F. Smith (ed.). -Berlin; Heidelberg
; New York ; Barcelona; Hong Kong; London; Milan; Paris; Tokyo:
Springer, 2002
(Springer lab manuals)
ISBN 978-3-642-47733-1 ISBN 978-3-642-56408-6 (eBook)
DOI 10.1007/978-3-642-56408-6
This work is subject to copyright. All rights are reserved, whether the whole or part of the material
is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Ouplica
tion of this publication or parts thereof is permitted only under the provisions of the German
Copyright Law of September 9, 1965, in its current version, and permissions for use must always be
obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright
Law.
http://www.springer.de
© Springer-Verlag Berlin Heidelberg 2002
Originally published by Springer-Verlag Berlin Heidelberg New York in 2002
The use of general descriptive names, registered names, trademarks, etc. in this publication does
not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
Product liability: The publisher cannot guarantee the accuracy of any information about dosage
and application thereof contained in this book. In every individual case the user must check such
information by consulting the relevant literature.
Cover design: design & production GmbH, 0-69121 Heidelberg
Typesetting: Kriiner, D-69115 Heidelberg
SPIN 10531590 39/3130YK - 5 4 3 2 1 0- Printedonacidfreepaper
Preface
In recent years,it has seemedasthough classicalcloning strate
giesweretakingsomethingofabackseattonewadvances inthe
development of applied polymerase chain reaction strategies.
The tried and established methods of library construction and
recombinantDNAcloning pioneeredduringthe 1970sandearly
1980sprovided many of the bedrock approaches used to find
cDNAand genomic clones of significance and made possible
accessible DNAsequencing technologies that did not require
exotic or even hazardous chemistry. Today, these technologies
areenjoying arenaissanceastheyfindnewutilityinnovelguises
combinedwith the newer approachesandtechnologiesbasedon
polymerasechain reaction.Although itisunlikelythat anyscien
tist todaywould employ allofthe technologies presentedin this
volume, every scientist working in the disciplines of molecular
biology and genetics shouldbefamiliar with allofthemandwill
likelyneed aworkingknowledge orhaveanapplicationforsome
ofthem.This volume isdesignedto provide an easilyaccessible
introductiontowhat are rapidlybecomingcorestrategiesin the
application of modern recombinant DNAtechnologies. These
approaches should be of value to both experienced scientists
seeking to applythese technologies to theirsystems aswellasto
scientistsand graduatestudents,inthe more formative stages of
development as molecular biologists, wishing to apply these
powerful technologies.
The first section of this volume, encompassing the first five
chapters,addressesdirectlytheapplicationsofpolymerasechain
reactiontoavarietyofproblemsinDNAcloningthat are,orhave
been, extremely challenging using more traditional approaches
and technologies. In truth,what weused to caution in our labo
ratories,werecloning experimentsonlytobeundertakenbysci
entists and students experienced in advanced cloning technol-
VI Preface
ogyareexperimentsnowwellwithinthe grasp ofthose atamore
formative leveloftrainingandexperience.Although still not for
the novice, these technologies are well within the abilities of
graduate students and scientists new to the discipline once a
basic competence in small volume handling, RNase-free tech
niques and the theoretical precepts of recombinant DNAhave
been mastered.These chaptersintroducestudentsandscientists
toexcitingapplicationsofpolymerasechain reactionthatrender
manageableapplicationssuch assimplecDNAcloning and tran
scriptmapping,mutagenesisandeventhe challengingcloning of
very long transcripts. Additionally, this section demonstrates
howtoclonelibrariessuccessfullyfromlimitingamountsoftotal
RNA, an application that was considered extremely difficult
using earlier approaches.
The second section of this volume, encompassing the sixth
and seventh chapters,describes modern approaches to subtrac
tivecloningtechnologiesthatseektoselectonlythose sequences
ofinterestpriortothe cloningstep.Whenoriginallyattemptedin
the 1980s,these pre-screening approaches to target sequence
enrichmentproved extremely challenging and evenbeyond the
abilities ofsome giventhe limitations ofthe technologies avail
able.Nowsubtractiveapproaches,withorwithoutapplicationsof
polymerase chain reaction, can be successfully undertaken by
scientists of many levels of experience in the application of
recombinant DNAtechnology. These improvements have pro
vided amajor advance in our ability to detect and clone impor
tantandrelativelylowabundancesequences.
In the last section, composed of chapters eight through
eleven, a variety of specialized expression cloning and library
screeningstrategies are introduced.Thesebeginwith twovaria
tions on two-hybrid analysis that provide, first, a strategy for
investigation of protein-protein interactions while the second
provides a strategy for investigation of protein-nucleic acid
interactions. Both provide the means to probe extremelysubtle
interactions not amenable to precipitation or chromatographic
approaches requiring much more stable macromolecular inter
actions.The possibilities for investigation of the subtle touch
and-go types ofmacromolecularinteractions often encountered
between signaling proteinsorbetweentranscriptionfactors and
their DNAbind sites are now greatly enhanced,thus expanding
the possibilities forgenediscoveryinthis discipline.
Preface VII
The last two chapters describe the application of alternative
strategiestocommonexperimentalproblems.Thefirstistopro
vide high throughput screening for applications where func
tional screening is not possible yet, where many thousands of
clones must be interrogated in an informative way to identify
neededclones.Thisapproachisparticularlyuseful asitemploys
fluorescent non-radioactive imaging technologies compatible
with many of the modern automated approaches to high
throughput handling of recombinant clones. Lastly,the final
chapterprovides insightintothe realmofalternativeapproaches
to creatingartificialaffinityreagents -artificialantibodiesifyou
will-that canprovide whatisessentiallyaninfinite arrayofpep
tides from which to select ligand-binding complexes.Addition
ally,because these complexes are in reality bacteriophages,they
come with many thousands of sites for secondary binding of
directly or indirectly fluorescently labeled antibodies. This
capacity has the potential to drive detection limits to ranges
undreamtofforantibodiesand toprovideaffinityreagentswith
out the need to considerantigenicity.
The potential of these reagents in the realm of gene discov
ery,diagnosticsaswellastherapeuticsisnothingshortofbreath
taking when the many applications being investigated are con
sidered. However,perhaps the most exciting applications of all
comefrom the investigationofthese technologiesand the devel
opment and exploration of prophylactic treatments for impor
tant diseases and threats.In this realm, these technologies may
soon demonstratetheirmostimportantvalueinthecampaignto
improve humanand animalwell-being and to alleviatesuffering
due to the ravages of disease.This isparticularlytrue in an era
where,inadditiontothenaturaldevelopmentofdisease,humans
can threatento foment newbiological plagues.
Whilewehavebeen absorbedinreading,writingand editing
this exciting project, there aremanypeople that wewouldliketo
acknowledge and thank for their special contributions. In the
development ofavolume such as this,theircontributions are of
the highest import becausewithout their input and encourage
ment this projectwould not havebeen possible. Though no list
can ever be inclusive and complete, the individual authors of
each chapter and our very patient and supportive editor in the
lifescienceseditorialofficeofSpringer-VerlaginHeidelberg,Dr.
[utta Lindenborn, deserve the most credit. Their contributions
VIII Preface
and the qualityofthe text speakforthemselves.Additionally,we
would liketo thankourwives/collaborators (they are both),our
families, and those in our laboratories for their continuing
efforts, support,and encouragement.Their contributions,while
less tangible within the pages of this volume, are no less im
portantintheeffectstheyhaveonthequalityofour efforts.Addi
tionally, we would like to thank the College of Veterinary
Medicine at Auburn University and our faculty colleagues for
continued support, both financial and intellectual, during the
completionofthis projectand forthe richscholarlyenvironment
inwhich wehavespentso manyyears.Our gratitudeto themall
cannotbe adequatelyexpressedaseach contributeshis/herspe
cial flavor to an exotic, intellectually challenging and stimulat
inglydiverseworkingenvironment.Our gratitudeisextendedto
themall.
AuburnUniversity R.Curtis Bird,Ph.D.
December2001 BruceF.Smith,V.M.D.,Ph.D.
Contents
PCRandcDNACloningTechniques
Chapter1
Strategiesfor cDNAClingand MappingRNA Transcripts
PETERS.SILVERSTEIN,SHILPAJ.BUCH,andR.CURTISBIRD 3
Chapter2
Cloning PCRProducts
WEIWENJIANG and BRUCEF.SMITH 21
Chapter3
SiteDirectedDeletion,Insertion,
and SubstitutionUsingPCR
MICHAELMINGFU LING AND BRIANH.ROBINSON 33
Chapter4
LongRT-PCRCloning-
AmplificationofFull-Length Enterovirus Genomes
KATHLEEN SIMPSONand JOHNW.Gow . . . . . . . 55
Chapter5
ConstructionofcDNALibraries from SmallQuantities
ofTotalRNAUsingTemplateSwitching Catalyzed
byM-MLVReverseTranscriptase
YORKY.ZHu,ALEXCHENCHIK,ROGERLI,
FLORENCEY.HSIEH,and PAULD.SIEBERT 69
SubtractiveandDifferentialDisplayTechniques
Chapter6
SubtractiveHybridizationand cDNACloning
R.CURTISBIRD and GINWu . . . . . . . . . . 97
X Contents
Chapter7
DifferentialDisplayto IdentifySteroid-InducedGenes
in EndocrineCells
ROBERTJ.KEMPPAINENandELLENN.BEHREND . . . . .. 127
SpecializedCloningandlibraryScreeningStrategies
Chapter8
TwoHyridcDNACloning
JORGA.SCHENK 145
Chapter9
YeastOneandTwoHyridcDNACloning
BETTYC.B.HUANG andYING Luo . . . . . . . . . . 167
Chapter 10
High-ThroughputLibraryScreening
byFluorescentHybridisationson GriddedMembranes
HUGUES ROEST CROLLIUS,JOHNO'BRIEN,
andHANS LEHRACH 187
Chapter 11
IdentificationofCellTargetingLigands
UsingRandom Peptide-PresentingPhage Libraries
TATIANAI.SAMOYLOVAandBRUCEF.SMITH . . . . . . . . 209
SubjectIndex 233
