DNA Manipulation and Analysis

251 Pages·2023·8.046 MB·English

by  Garry Scarlett

Methods in
Molecular Biology 2633
Garry Scarlett Editor
DNA
Manipulation
and Analysis
M M B
ETHODS IN OLECULAR IO LO GY
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JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
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DNA Manipulation and Analysis
Edited by
Garry Scarlett
Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth, UK
Editor
GarryScarlett
BiophysicsLaboratories
SchoolofBiologicalSciences
UniversityofPortsmouth
Portsmouth,UK
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-3003-7 ISBN978-1-0716-3004-4 (eBook)
https://doi.org/10.1007/978-1-0716-3004-4
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Preface
If there is such a thing as a single secret to the mystery of life, then that secret is probably
DNA.Since1953thathasbeenaverybadlykeptsecret,detaileddescriptionsandimagesof
its structure and function abound in everything from learned articles to pop culture.
However, we now not only know the structure of DNA but can decipher its code and
even modify that code. We have learned to read and write on a molecular and biological
level,thetechnologiesthatallowustoreadandwriteDNAformthefocusofthisbook.This
book is designed for scientists moving into recombinant DNA technologies, covering
techniques that are important for conducting experiments in fields as wide ranging as
developmentaltostructuralbiology.Thechaptersarelaidoutasreadersofthelongrunning
MethodsinMolecularBiologyserieshavecometoexpect,withanaccessibletheorysection
followedbyadetailedmethodandfinallytheever-usefulnotesandtroubleshootingpages.
Noprefaceontheartofgeneengineeringcouldgowithoutmentioningthediscoveryof
the structure of DNA and the subsequent cracking of the code by a series of elegant
experiments in the 1950s and 1960s. However, the key developments that are centrally
relevanttothisbookwerethefirstrecombinantDNAexperimentsofBergand,shortlyafter,
Boyer-Cohen in 1971 and 1972; indeed, some of the principles in those early procedures
haverecognisabledescendantsdescribedinthefollowingchapters.Alandmarkdevelopment
inthefieldinthemid-1970swasthearrivalofSangerdideoxysequencing,replacingdifficult
anddangerouschemicalmethodsfordeterminingtheorderofbasesonapieceaDNA,with
a much easier enzymatic approach. The following decade of the 1980s was dominated by
twomajorbreakthroughs,thepolymerasechainreaction(PCR)andthelessmentionedbut
equally important phosphoramidite chemistry approaches to making short single-stranded
oligonucleotides.Withoutthesedevelopments,manyofthetechniquesinthisbookwould
simplynotbepossible,theyhaveenablednotonlydenovogenerationofDNAfragmentsbut
alsotherapidamplification,siteselectionandtargetedmutationofsequences.
Initially, much of the early recombinant DNA work undertaken was on sequences of
DNAinsertedintoplasmids,replicatingcircularextra-chromosomalDNAfoundinbacteria
that encode useful attributes for the cell. Indeed, plasmids remain the work horses of the
DNAlaboratory,andthefirstfewchaptersdetailavarietyofdifferentmethodsofinserting
DNAsequencesintothem,eachwithspecificstrengthsandweaknesses.Followingonfrom
these,thenextthreechaptersdiscusshowtomanipulateandcreateDNAsequences,which
inconjunctionwiththemolecularcloningmethodsdiscussedintheearlierpartofthebook
provide powerful options in the laboratory for the would-be molecular biologist. The last
decadeofthetwentiethcenturyandthedawnofthenewmillenniumsawincreasinglyrapid
stridesinthecomplexfieldofgenomicmodification,notonlybacterialgenomesbutalsothe
genomes of eukaryotic cells, including those in multicellular organisms. Early attempts at
eukaryoticgenomicmodificationsufferedfromsignificantissueswithtargetingthecorrect
location within the genome, a problem that became worse the bigger the genome to be
manipulated. The first technology that really tackled this was based upon zinc finger
nucleases; this was rapidly replaced by TALENs before the extremely powerful and easy to
use CRISPR/Cas9 system rose to its current dominance. Chapters 9, 10, and 11 discuss
transgenics in the model system Xenopus, with Chaps. 10 and 11 describing methods for
CRISPRtargetedgeneknockoutsandgeneinsertions,respectively.Thebookthenhastwo
v
vi Preface
examplesofthegrowingfieldof‘cell-free’DNAwork,dealingwiththeraisingofaptameric
sequences and high throughput array technologies. Chapters 14, 15 and 16 deal with the
keyunderpinningtechnologiesofallDNAwork,makingoligonucleotidesandreadingthe
sequence. Much of this is now available as outsourced services for starter laboratories, but
in-house provision allows for flexibility and increased speed. The last chapter deals with
ethical considerations. The growing importance of DNA and gene manipulation to wider
societyhasledtoincreasingpublicandgovernmentalscrutiny,notonlyofethicsbutalsothe
risksofrecombinantDNAtechnologies.Manyjurisdictionshavenowintroducedguidelines
andlawsgoverningthemakingandcontainmentofgeneticallymodifiedorganisms,thisisin
additiontolegalimplicationsofusinganimalsinresearch.
Recombinant technologies have developed remarkably since those first experiments in
the early 1970s; the growth of manufactured kits and outsourced services have brought
nucleic acid–based experiments into the range of many laboratories that previously would
havestruggledtosetuptheinfrastructure.Wehopethisbookhelpsprovidetheexpertisefor
scientistsembarkingontheirfirstforaysintothesetypesofprojects.Finally,Iwouldliketo
thank the many chapter authors to who have contributed and provided their time and
expertisetohelpsupportthescientificcommunitywiththeresourcesheldwithinthisbook.
Portsmouth,UK GarryScarlett
Contents
Preface ..................................................................... v
Contributors................................................................. ix
1 ClassicalRecombinantDNACloning ....... ....... ........ ....... ........ 1
AnaMikic´,ArqamAlomari,andDarrenM.Gowers
2 ASequence-andLigation-IndependentCloning(SLIC)Procedure
for theInsertionofGenesintoaPlasmidVector..... ........ ....... ........ 25
RobertA.Holland
3 MolecularCloningUsingInVivoDNAAssembly ........... ....... ........ 33
SandraArroyo-Urea,JakeF.Watson,
andJavierGarcı´a-Nafrı´a
4 AssemblingMultipleFragments:TheGibsonAssembly....... ....... ........ 45
LuisanaAvilan
5 TACloningApproachestoCloningDNAwithDamagedEndsDNA ....... .. 55
CharlotteAyling
6 PCR-BasedAssemblyofGeneSequencesbyThermodynamicallyBalanced
Inside-Out(TBIO)GeneSynthesis ......... ....... ........ ....... ........ 65
TimothyJ.RaganandHelenA.Vincent
7 RandomMutagenesisbyPCR....... ....... ....... ........ ....... ........ 81
F.A.Myers
8 InVitroSiteDirectedMutagenesis.......... ....... ..... ... ...... .... ..... 87
MichaelJ.McClellan
9 XenopusTransgenesisUsingthepGatewaySystem ....... .. .. ....... ........ 97
LiliyaNazlamova
10 CRISPR/Cas9GeneDisruptionStudiesinF XenopusTadpoles:
0
UnderstandingDevelopmentandDiseaseintheFrog........ ....... ........ 111
AnitaAbu-DayaandAnnieGodwin
11 ACRISPR/Cas-BasedMethodforPreciseDNAIntegrationinXenopuslaevis
OocytesFollowedbyIntracytoplasmicSpermInjection(ICSI)
Fertilization........ ....... ........ ....... ....... ........ ... .... .. ...... 131
SianAngelaMartin
12 ALambda-ExonucleaseSELEXMethodforGeneratingAptamers
toBacterialTargets ........ ........ ....... ....... ........ ....... ........ 145
RobertGowlandandDarrenM.Gowers
13 GenerationofFunctional-RNAArraysbyInVitroTranscription
andInSituRNACapturefor theDetectionofRNA-RNAInteractions ....... 163
HelenA.Vincent,CharlotteA.Henderson,DanielaLopesCardoso,
andAnastasiaJ.Callaghan
vii
viii Contents
14 ChemicalSynthesisofOligonucelotideSequences:Phosphoramidite
Chemistry ......... ....... ........ ....... ....... ........ ....... ........ 185
JohnBrazier
15 LowThroughputDirectCycleSequencingofPolymeraseChain
Reaction(PCR)Products........... ....... ....... ........ ....... ........ 195
GeorgeD.ZouganelisandNikolaosTairis
16 NanoporeSequencingforMixedSamples.... ...... .... ..... ....... ........ 213
AngelaH.BeckettandSamuelC.Robson
17 Ethics,Legality,andSafetyforGeneticists .......... ........ ....... ........ 235
SimonE.Kolstoe
Index .......... ........ ....... ........ ....... ....... ........ ....... ........ 247
Contributors
ANITAABU-DAYA • EuropeanXenopusResourceCentre,UniversityofPortsmouth,
Portsmouth,UK
ARQAMALOMARI • DepartmentofBasicSciences,CollegeofAgricultureandForestry,
UniversityofMosul,Mosul,Iraq
SANDRAARROYO-UREA • InstituteforBiocomputationandPhysicsofComplexSystems(BIFI)
andLaboratoriodeMicroscopı´asAvanzadas(LMA),UniversityofZaragoza,
Zaragoza,Spain
LUISANAAVILAN • CentreforEnzymeInnovation,UniversityofPortsmouth,
Portsmouth,UK
CHARLOTTEAYLING • BiophysicsLaboratories,SchoolofBiologicalSciences,
UniversityofPortsmouth,Portsmouth,UK
ANGELAH.BECKETT • CentreforEnzymeInnovation,UniversityofPortsmouth,
Portsmouth,UK
JOHNBRAZIER • ReadingSchoolofPharmacy,UniversityofReading,Reading,UK
ANASTASIAJ.CALLAGHAN • BiophysicsLaboratories,SchoolofBiologicalSciences,Universityof
Portsmouth,Portsmouth,UK
JAVIERGARCI´A-NAFRI´A • InstituteforBiocomputationandPhysicsofComplexSystems(BIFI)
andLaboratoriodeMicroscopı´asAvanzadas(LMA),UniversityofZaragoza,
Zaragoza,Spain
ANNIEGODWIN • EuropeanXenopusResourceCentre,UniversityofPortsmouth,Portsmouth,
UK
DARRENM.GOWERS • BiophysicsLaboratories,SchoolofBiologicalSciences,Universityof
Portsmouth,Portsmouth,UK
ROBERTGOWLAND • DepartmentofBiochemistry,UniversityofCambridge,Cambridge,UK
CHARLOTTEA.HENDERSON • BiophysicsLaboratories,SchoolofBiologicalSciences,University
ofPortsmouth,Portsmouth,UK
ROBERTA.HOLLAND • SyngentaCropProtectionResearch,Berkshire,UK;Centrefor
EnzymeInnovation,UniversityofPortsmouth,Portsmouth,UK
SIMONE.KOLSTOE • ReaderinBioethics,UniversityofPortsmouth,Portsmouth,UK
DANIELALOPESCARDOSO • BiophysicsLaboratories,SchoolofBiologicalSciences,Universityof
Portsmouth,Portsmouth,UK
SIANANGELAMARTIN • EuropeanXenopusResourceCentre(EXRC),Universityof
Portsmouth,Portsmouth,UK
MICHAELJ.MCCLELLAN • LudwigInstituteforCancerResearchLtd,UniversityofOxford,
Oxford,UK
ANAMIKIC´ • BiophysicsLaboratories,SchoolofBiologicalSciences,UniversityofPortsmouth,
Portsmouth,UK
F.A.MYERS • BiophysicsLaboratories,SchoolofBiologicalSciences,UniversityofPortsmouth,
Portsmouth,UK
LILIYANAZLAMOVA • ClinicalandExperimentalSciences,SouthAcademicBlock,
SouthamptonGeneralHospital,Southampton,UK
TIMOTHYJ.RAGAN • LeicesterInstituteofStructuralandChemicalBiology,Departmentof
MolecularandCellularBiology,UniversityofLeicester,Leicester,UK
ix