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Methods in Molecular Biology 2633 Garry Scarlett Editor DNA Manipulation and Analysis M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. 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 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. 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, andJavierGarcıá-Nafrıá 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) andLaboratoriodeMicroscopıásAvanzadas(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 JAVIERGARCIÁ-NAFRIÁ • InstituteforBiocomputationandPhysicsofComplexSystems(BIFI) andLaboratoriodeMicroscopıásAvanzadas(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