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1、GenomicSequencingofSARS-CoV17716GenomicSequencingoftheSevereAcuteRespiratorySyndrome-CoronavirusStephenS.C.Chim,RossaW.K.Chiu,andY.M.DennisLoSummaryThepolymerasechainreaction(PCR),whichcanexponentiallyreplicateatargetDNAsequence,hasformedthebasisforthesensitiveanddirectexamin

2、ationofclinicalsamplesforevidenceofinfection.Duringtheepidemicofsevereacuterespiratorysyn-drome(SARS)in2003,PCRnotonlyofferedarapidwaytodiagnoseSARS-coronavirus(SARS-CoV)infection,butalsomadethemolecularanalysisofitsgenomicsequencepossible.SequencevariationswereobservedintheS

3、AR-CoVobtainedfromdifferentpatientsinthisepidemic.Theseuniqueviralgeneticsignaturescanbeappliedasapow-erfulmoleculartoolintracingtherouteoftransmissionandinstudyingthegenomeevolutionofSARS-CoV.ToextractthiswealthofinformationfromthelimitedprimaryclinicalspecimensofSARSpatient

4、s,wewerepresentedwiththechallengeofefficientlyamplifyingfragmentsoftheSARS-CoVgenomeforanalysis.Inthischapter,wewilldiscusshowwemanagedtoaccomplishthistaskwithouroptimizedprotocolsonreverse-transcription,nestedPCRamplification,andDNAcyclesequencing.Wewillalsodis-cussthesequen

5、cevariationsthattypifiedsomestrainsofSARS-CoVinthedifferentphasesduringthisepidemic.PCRamplificationoftheviralsequenceandgenomicsequencingofthesecriticalsequencevariationsofre-emergingSARS-CoVstrainswouldgiveusquickinsightsintothevirus.KeyWords:SARScoronavirus;viralRNAextract

6、ion;reverse-transcriptionPCR;sequencing;genomicsequencevariation.1.IntroductionSevereacuterespiratorysyndrome-coronavirus(SARS-CoV),theetiologicagentofSARS(1–3),isavirusthatwasunknowntousbeforetheSARSepidemic.Theconcertedeffortsofresearchershavepromptlyelucidateditsgeneticcod

7、e.ThegenomeofSARS-CoVisa29,727-nucleotide,polyadenyl-atedRNA.Thegenomicorganizationistypicalofcoronaviruses,havingthecharacteristicgeneorder(5'-polymerase[Orf1ab],spike[S],envelope[E],mem-From:MethodsinMolecularBiology,vol.336:ClinicalApplicationsofPCREditedby:Y.M.D.Lo,R.W.K.

8、Chiu,andK.C.A.Chan?HumanaPressInc.,Totowa,NJ177178Chim,Chiu,andLobra