AnalyticalBiochemistryxxx(2008)xxx–xxxwww.elsevier.com/locate/yabio
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Identificationofryanodinereceptor1single-nucleotidepolymorphismsbyhigh-resolutionmeltingusingtheLightCycler480System
HilbertGrievink,KathrynMStowell*
Institute of Molecular Biosciences, Massey University, Palmerston North 11-222, New Zealand
Received7October2007
Abstract
High-resolutionmelting(HRM)allowssingle-nucleotidepolymorphism(SNP)detection/typingusinginexpensivegenericheterodu-plex-detectingdouble-strandedDNA(dsDNA)bindingdyes.UntilrecentlyHRMhasbeenapost-PCRprocess.WiththeLightCycler480System,however,theentiremutationscreeningprocess,includingpost-PCRanalysis,canbeperformedusingasingleinstrument.HRMassaysweredevelopedtoallowscreeningoftheryanodinereceptorgene(RYR1)forpotentialmutationscausingmalignanthyperthermia(MH)and/orcentralcoredisease(CCD)usingtheLightCycler480System.Theassayswerevalidatedusingengineeredplasmidsand/orgenomicDNAsamplesthatareeitherhomozygouswildtypeorheterozygousforoneofthreeSNPsthatleadtotheRyR1aminoacidsubstitutionsT4826I,H4833Y,and/orR4861H.TheHRManalyseswereconductedusingtwodiVerentheteroduplex-detectingdsDNAbindingdyes:LightCycler480HRMdyeandLCGreenPlus.HeterozygoussamplesforeachoftheHRMassayswerereadilydistinguishedfromhomozygoussampleswithbothdyes.Byusingengineeredplasmids,itwasshownthatevenhomozygoussequencevariationscanbeidentifiedbyusingeithersmallampliconsortheadditionofexogenousDNAafterPCR.Thus,theLightCycler480Systemprovidesanovel,integrated,real-timePCR/HRMplatformthatallowshighthroughput,inexpensiveSNPdetection,andgenotypingbasedonhigh-resolutionampliconmelting.©2007ElsevierInc.Allrightsreserved.
Keywords: High-resolutionmelting;SNPidentification;LightCycler480;Ryanodinereceptor1;Malignanthyperthermia;Centralcoredisease
Genetictestinghasanimportantroleinmanydiagnosticlaboratoriesandcanprovidedramaticprognosticandclini-calbenefits.Manygenetictestsareavailabletodetectand/ortypesingle-nucleotidepolymorphisms(SNPs).1Mostofthese techniques, however, require an additional separa-tionstepthatmakesthemlessfavorableforhigh-through-put assays. Examples of such methods are single-strandconformation polymorphism [1], denaturing gradient gelelectrophoresis [2], restriction endonuclease analysis, andDNAsequencing.Homogeneous,closed-tubemethodsforSNPdetection/typing thatdonotrequireseparationstepsare available and are based on either allele-specific PCR
003-2697/$-seefrontmatter©2007ElsevierInc.Allrightsreserved.oi:10.1016/j.ab.2007.11.019
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* Correspondingauthor.Fax:+64063505688.E-mail address: [email protected](K.M.Stowell).
1 Abbreviations used: SNP, single-nucleotide polymorphism; HRM,igh-resolution melting; dsDNA, double-stranded DNA; Tm, meltingemperature; MH, malignant hyperthermia; CCD, central core disease;YR1,ryanodinereceptorgene;cDNA,complementaryDNA.
usingSYBRGreenI[3,4]orexpensivefluorescentlylabeledprobes[5,6]orprimers[7].SNPgenotypingbasedonallele-specificPCRrequiresthreeprimers, twoofwhichneedtobeallelespecific.Thus,diVerentmutationsrequirediVerentallele-specificprimers.WhenusinglabeledprobesforSNPdetection/typing,onlySNPsthatlieundertheprobecanbedetected. Consequently, multiple relatively costly probesareneededtocoverallpotentialSNPs.Inaddition,theuseofprobesoftenrequiresextensiveoptimization.Thesecon-ditions limit theusefulnessof thesemethodsforscreeningpurposes.IfPCRisperformedwitha59-labeledprimerasdescribed by Gundry and coworkers [7], high-resolutionampliconmeltingallowsgenotypingandmutationscanningwithoutprobes.However,thismethodrequiresatleastoneexpensivelabeledoligonucleotide.
High-resolution melting (HRM) was introduced as ahomogeneousclosed-tubesystemthatallowsmutationscan-ning and genotyping without the need for costly labeled
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Table1Primersequences,primerconcentrations,andampliconsizes
Target Primers(59–39) Primerconcentrations(lM)
Ampliconsize(bp)
4826 ACTTCTTCTTTGCTGCC 0.3 77GGTGACAGAGGACAGGAT 0.3
4833 TCTCCTGGACATCGCC 0.3 78CACACCTGTTTCCCATTG 0.3
4861 CCGTGGTGGCCTTCAA 0.2 81GGTTCATCCTCATCCTCG 0.2
4861 GGTGGTCGTCTACCTGT 0.2 61GGTTCATCCTCATCCTCG 0.2
oligonucleotides. It reliesonanewgenerationofgenericheteroduplex-detecting double-stranded DNA (dsDNA)bindingdyes.Heteroduplexproductsareidentifiedbythepresence of a second low-temperature melting transition[8].TheLightCycler480HRMdyeisarecentlyintroducedmember of this new family. Unlike SYBR Green I, thegenericheteroduplex-detectingdsDNAdyescanbeusedatsaturatingconcentrationswithout inhibitingoradverselyaVectingthePCR.Thereasonwhythisnewfamilyofdyescandetectheteroduplexes,whereasSYBRGreenIcannot,isnotentirelyclear,butdyeredistributionduringmeltingisthoughttobeonereason[8].
TheLightCycler480Systemprovidesauniqueformatinwhichtheentireexperiment, includingreal-timeandpost-PCRanalysis, canbedoneonone instrument ina96-or384-wellformatandcanbecompletedwithin1h.DiVerentsequencevariantscanbeidentifiedbasedondiVerences inmelting curves using the LightCycler 480 Gene ScanningSoftware.Heterozygoussamplesarebestdistinguishedfromhomozygous samples by an altered shape in the meltingcurve.ThesediVerencesarebestvisualizedusingdiVerenceplotsbecauseslightdiVerencesincurveshapeandmeltingtemperature(Tm)becomeobvious.Amoredetaileddescrip-tioncanbefoundelsewhere[8].DiVerenthomozygoussam-ples,ontheotherhand,arebestdistinguishedbyachangeinTm.Smallerampliconshavebeenfoundtoimprovediscrim-inationbetweengenotypes[7].
In this study, inexpensive and high-throughput HRMassaysweredevelopedandanalyzedusingtheLightCycler480Systemtoallowscreeningofthegenethatencodestheryanodinereceptorskeletalmusclecalciumreleasechannel(RyR1)formutationsassociatedwithmalignanthyperther-mia (MH, MIM no. 145600) and/or central core disease(CCD,MIMno.117000).Thecodingregionoftheryano-dinereceptorgene(RYR1,MIMno.180901,NM_000540)is more than 15,000bp in size; thus, there is a constantsearch formoredistinctive, faster,andcheaperscreeningmethodologies. Both MH and CCD are associated withdefectsintheRYR1geneonchromosome19q13.1,whichistheprimarylocusofMHinhumans(MHS1)[9].Untilrecently, approximately 50% of MH had been linked tothis locus [10].Preliminaryanalysesbasedonsequencingthe entire RYR1 complementary DNA (cDNA) suggestthat the linkage to the MHS1 locus might be as high as70% [9]. Genomic DNA samples of known RYR1 geno-types with either the wild-type sequence or a mutationassociatedwithMHand/orCCDwereusedtovalidatetheHRM assays. The SNPs investigated in this study led totheRyR1aminoacidsubstitutionsT4826I(linkedtoMH),H4833Y(linkedtoMH),and/orR4861H(linkedtoCCD).Nearly all mutations associated with MH and/or CCDoccurintheheterozygousstate.Nevertheless,homozygousmissensemutationshavebeenreportedonrareoccasions[11,12].Therefore,fourdiVerenthomozygousRYR1geno-typeswerestudiedusingengineeredplasmidstoshowthateven homozygous sequence variations can be identifiedusingHRMontheLightCycler480System.HRManaly-
Pleasecitethisarticleinpressas:H.Grievink,.K.M.Stowell,Ident...,Anal.Biochem.(2007),doi:10.1016/j.ab.2007.11.019
seswereconductedwithboththeLightCycler480HRMdyeandLCGreenPlus.
Materials and methods
DNA samples
Human genomic DNA was prepared from wholeblood samples using the Wizard Genomic DNA Kit(Promega) or the MagNA Pure LC DNA Isolation KitI(Roche)accordingtothemanufacturer’sstandardpro-tocol. Informed consent was obtained from participat-ing subjects, and the study was carried out after ethicalapprovalwasobtained from theWhanganui–Manawatuhumanethicscommittee.TovalidatetheHRMassays,3homozygouswild-typeand3heterozygousmutantgeno-micDNAsamplesofknowngenotypeswerescreenedfortheR4861HRYR1mutation.ForeachoftheT4826IandH4833YRYR1mutations,10homozygouswild-typeand10heterozygousgenomicDNAsamplesofknowngeno-type were screened. Engineered plasmids were createdby cloning wild-type genomic DNA flanking the RYR14861 wild-type sequence into the vector pGEM–T Easy(Promega).SNPs representingC,T,orA sequencevari-antsatthedefinedpositionwereintroducedusingQuik-Change site-directedmutagenesis (Stratagene)accordingtothemanufacturer’sstandardprotocol.Theengineeredplasmidswereusedtoaddressthepossibilityofdiscrimi-nationbetweendiVerenttypesofhomozygotes.DNAcon-centrationsweredeterminedbyA260.
PCR and HRM conditions
Primers were designed using the LightCycler ProbeDesign Software 2.0. Primer sequences used in PCR arelisted in Table 1. Amplicon lengths were kept relativelyshort(61–81bp)toimprovediscriminationbetweengeno-types.Real-timePCRcyclingandHRManalysisoftheengi-neeredplasmidsandgenomicDNAsampleswerecarriedoutontheLightCycler480System(Roche).ExperimentswereconductedwithboththeLightCycler480HRMdye(Roche)andLCGreenPlus(ITBiochem).
ThereactionmixtureforHRMusingtheLightCycler480HRMdyeconsistedof0.2to0.3lMofeachprimer,
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1£LightCycler 480 HRM dye, and 3mM MgCl2. ThereactionmixtureforHRMusingLCGreenPlusconsistedof0.2to0.3lMofeachprimer,1£LC480ProbeMasterMix, and 1£LCGreen Plus. DNA templates were usedatapproximately104copies forengineeredplasmidcon-structsorat10to150ngforgenomicDNAsamples.
Assayswerecarriedoutina96-wellformatin10-llvol-umesandwereperformedusingthefollowingtouchdownPCRcyclingandHRMconditions.ThePCRwasinitiatedwitha10-minholdat95°C,followedby40cyclesof95°Cfor10s,atouchdowncyclingstep(decreasing0.5°C/cycle)annealingrangingfrom62to56°Cfor10s,and72°Cfor4s.Afteramplification,thesampleswereheatedto95°Cfor1minandthencooledto40°Cfor1mintoencourageheteroduplexformation.HRMcurvedatawereobtainedby melting over the desired range (76–92°C unlessotherwisestated)atarateof25acquisitionsper1°C.
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Fig.1.DiVerenceplotsofHRManalysesofthe486181-bpamplicons.Here3wereanalyzedusingtheLightCycler480HRMdye(A)orLCGreenPlus(B).
Results
Ampliconmeltinganalysesinthepresenceofthehetero-duplexdetecting thedsDNAbindingdyeLightCycler480HRMdyeorLCGreenPluswereusedtodetectSNPsusingtheLightCycler480System.Ampliconswere61to81bpinlengthtoallowdefinitivediscriminationandidentificationofhomozygoussequencevariations.Fig.1showsthediVer-enceplotsproducedbytheHRManalysis,whichfollowedthe real-timePCRamplificationof81-bpamplicons fromgenomicDNAflanking the4861positionusingeither theLightCycler480HRMdyeorLCGreenPlus.HRManal-ysis with either dye allows clear discrimination betweenthehomozygousandheterozygousgenomicDNAsamplesbased on diVerences in melting curve shapes. All sampleswereofknowngenotypesandweregroupedcorrectlybytheLightCycler480GeneScanningSoftware.
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heterozygoussamples(dottedlines)and3homozygoussamples(solidlines)
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Two other HRM assays were designed and allowedscreening of the RYR1 gene for the T4826I and H4833YRYR1mutations.Eachoftheassayswasvalidatedbyscreen-ing10homozygouswild-typeand10heterozygousgenomicDNAsamplesofknowngenotypesfortheSNPscausingtheT4826IandH4833Yaminoacidsubstitutions.Unambigu-ousdiVerenceswerevisibleintheshapesofthemeltingcurvesforheteroduplexesandhomoduplexes.ThediVerenceplotsshowninFigs.2and3clearlyseparatehomozygousgeno-micDNAsamplesfromheterozygousonesforthe4826and4833HRMassays,respectively.Allsamplesweregroupedcorrectlyby theLightCycler480GeneScanningSoftwarewith both the LightCycler 480 HRM dye and LCGreenPlus.Bothhomozygousandheterozygoussamplesanalyzedfor the4833SNPbyHRMusingLCGreenPlus showanincreaseinvariabilitybetweenmeltingcurves(Fig.3B).TheHRMassayperformedwiththeLightCycler480HRMdye
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Fig.2.DiVerenceplotsofHRManalysesofthe482677-bpamplicons.Here10wereanalyzedusingtheLightCycler480HRMdye(A)orLCGreenPlus(B).
shows no such variability (Fig. 3A). The real-time PCR,whichprecedes theHRManalysis, revealed thatalthoughtheamplificationcurvesofbothassayslooksimilarandupto standard, thecrossingpointsof the reactionsusing theLC480ProbeMasterMixwithLCGreenPlusweredelayedbyatleastthreecyclescomparedwiththeLightCycler480HRMdye.Thistrendcouldbedetectedinallexperiments.Inaddition,theLightCycler480HRMdyegeneratesafluo-rescencesignalthatisatleasteighttimesstrongerthanthatwithLCGreenPlus.
Engineered plasmids were used to study homozygotediscrimination.Fourplasmids(identicalexceptforaG,C,T,orAatthespecifiedposition)containingthesequenceflankingthe4861SNPwereusedalonetosimulatehomo-zygous genotypes or in binary combinations to simulateheterozygousgenotypes.HRManalysesof81-and61-bpampliconswereconductedtodeterminetheeVectofampli-
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heterozygoussamples(dottedlines)and10homozygoussamples(solidlines)
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cpt4zphdnaTmttHtm
Fig.3.DiVerenceplotsofHRManalysesofthe483378-bpamplicons.Here10heterozygoussamples(dottedlines)and10homozygoussamples(solidlines)wereanalyzedusingtheLightCycler480HRMdye(A)orLCGreenPlus(B).
on length on genotype diVerentiation. The diVerencelot in the HRM assay for the 81-bp amplicon causinghe4861SNPusingtheLC480HRMdyeisshowninFig.. Heterozygotes were easily distinguished from homo-ygotesbasedonshapeof themeltingcurves.DiVerencelotanalysisalsoallowsdiscriminationbetweendiVerenteterozygotes. Homozygote discrimination is based oniVerences in Tm. These diVerences are best detected byormalized melting curves without temperature shiftingnd not by the temperature-shifted diVerence curves [7].hus,forthedetectionofhomozygotevariants,ampliconelting data should be analyzed both with and without
emperatureshifting.AsshowninFig.4B,nodiVerentia-ion is possible between homozygous A and T based onRM analysis of the 81-bp amplicon. The Tm values of
hehomozygousAandTvariantsdiVerbyonlyapproxi-ately0.1°C(Fig.4B).
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Completegenotypingofallthe4861SNPsin81-bpampli-conswithHRMwaspossiblebyaddingexogenouswild-typeDNA amplicons (in a 1:1 ratio) to unknown homozygoussamples. If unknown samples are wild type, their meltingcurvesdonotchangeaftertheadditionofexogenouswild-typeDNAamplicons.Iftheunknownsamplesarehomozy-gousmutants,heteroduplexesareproducedandsamplescanbecorrectlyidentifiedashomozygousmutant.Fig.5showstheresultofadding81-bpampliconscontainingtheflankingwild-type 4861 sequence to the homozygous samples. Het-eroduplexes were formed when homozygous mutants werepresent.TheshapesofthemeltingcurvesthatweregeneratedbytheadditionofexogenousDNAtohomozygousmutantscorrelatedwiththoseoftheoriginalheterozygotesand,there-fore,allowedSNPgenotyping.
HRM analyses using smaller 61-bp amplicons alloweddiscriminationbetweendiVerentheterozygousanddiVerent
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Fig.4.HRManalysisofpossibleSNPgenotypesatthe4861positionusingtheLightCycler480HRMdye(81-bpamplicons).Here2samplesofeachgenotypewereanalyzedandincludedfourhomozygotes(solidlines)andthreeheterozygotes(dottedlines).(A)DiVerenceplotoftheHRManalyses.(B)NormalizedHRMcurvesofthewild-typesamples.Tmvaluesofhomozygotevariants:82.96and82.94°CforG/G,82.67and82.60°CforC/C,82.22and82.28°CforT/T,and82.34and82.37°CforA/A.
homozygous samples without the addition of exogenousDNA. Heterozygous SNP variants were readily identifiedusingdiVerenceplots(Fig.6A).HomozygousSNPvariantsatthe4861positionwereidentifiedusingnon-temperature-shifted normalization curves (Fig. 6B). The Tm diVerencebetween the homozygous A and T variants was approxi-mately0.2°CandprovedtobesuYcientfordiscriminationbetweenthetwo.Occasionally,homozygoteSNPidentifica-tionmayalsobepossiblebyusingdiVerenceplots(Fig.6A).Becausethesesmaller(61-bp)ampliconshavelowerTmval-ues,themeltingrangewasadjustedto69to92°C.
Discussion
HRMhasbeen introducedasahomogeneousclosed-tubepost-PCRmethodforgenotypingandmutationscan-
Pleasecitethisarticleinpressas:H.Grievink,.K.M.Stowell,Ident...,Anal.Biochem.(2007),doi:10.1016/j.ab.2007.11.019
ningthatdoesnotneedcostlylabeledoligonucleotides[8].Instead,itreliesonnewgenerationgenericheteroduplex-detectingdsDNAbindingdyes.Usingthisnewtechnique,SNPshavebeengenotypedinproductsaslargeas544bp[7].HRMSNPdetectionand/orgenotyping,however, isstrongly sequencedependent,andoften shortampliconsand/or unlabeled oligonucleotide probes are necessaryor preferred [13–15]. This study focused on usingHRManalysisof relatively smallamplicons forSNPdetectionand identification without the use of unlabeled probes.Byusingonlytwostandardunlabeledprimers,therobust-nessoftheassayincreasessignificantlybecauseoptimiza-tiontypicallyisnotneeded.Hence,allassaysdescribedinthisarticle couldbe conductedusing identicalPCRandHRM conditions, making it ideal for high-throughputscreeningpurposes.Inaddition,theLightCycler480Sys-
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Fig.5.HRManalysisofpossibleSNPgenotypesatthe4861positionbyaddingwild-typeDNAampliconsusingtheLightCycler480HRMdye(81-bpamplicons).Unknownhomozygousgenotypes(blacksolidlines)weremixedwithwild-typeampliconsafterPCR,creatingheterozygotes(G/T,G/C,andG/A,thickgraylines)thathavemeltingcurvessimilartothoseoftheoriginalheterozygotes(blackdottedlines).
temprovidesauniqueformatinwhichtheentireexperi-ment,includingreal-timePCRandpost-PCRHRManaly-sis,canbedoneina96-or384-wellformatandcompletedwithin1h.
Inthisstudy,HRMassaysweredevelopedandanalyzedusingtheLightCycler480System.Theassaysscreened61-to 81-bp RYR1 amplicons for mutations associated withMH(T4826IandH4833Y)and/orCCD(R4861H).HRManalyseswereconductedusingtwodiVerentheteroduplex-detectingdsDNAbindingdyes:LightCycler480HRMdyeandLCGreenPlus.
Whenthepurposeoftheanalysisistoscanforhetero-zygotes,usingnormalizedandtemperature-shifteddiVer-ence plots is a convenient way of viewing HRM databecauseslightdiVerencesincurveshapebecomeobvious.Allassaysthatweredevelopedinthisstudyallowedunam-biguousdiscriminationbetweenheterozygousandhomo-zygoussamples.TheuseoftheLightCycler480HRMdyehassomeadvantagesovertheuseoftheLC480ProbeMas-terMixwithLCGreenPlus.Thefluorescencesignalgen-eratedbytheLightCycler480HRMdye isat leasteighttimesasstrong,andPCRcrossingpointsareloweredbyatleastthreecycles.Thelatterofthetwocanbecrucialforaccuratemutationscanningand/orgenotypingbecauseithasbeensuggested that thevalidityofHRManalysisofsamples with late or poor amplification is questionable[16].Thereal-timePCRprecedingtheHRManalysis,there-fore,canprovideausefulqualitycontrolmeasure.Thus,thelatePCRcrossingpointsarelikelytobethecauseoftheincreaseinvariabilitybetweenthemeltingplotsshowninFigs.3Aand3B.
Engineeredplasmids,whichcontainthegenomicDNAsequenceflankingthe4861SNP,wereusedtoshowthatall fourpossiblehomozygousgenotypesatoneposition
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couldreadilybedistinguishedfromeachotherbyHRManalysis.ThisisanimportantelementforRYR1screeningforpossibleMHand/orCCDmutationsbecausehomozy-gousmissensemutationshavebeenreportedonrareocca-sions [11,12]. First, diVerentiation between genotypes of81-bpampliconswaspossiblebyspikingunknownsam-pleswithexogenousDNAafterPCR(Fig.5).Spikingsam-plesafterPCRhastheadvantagethatonlyhomozygoussamplesneedtoberetestedbecauseheterozygoussamplescanalreadybeidentifiedbasedondiVerenceplotanalysis.In addition, this technique eliminates strict monitoringof DNA concentrations and diVerences in amplificationeYcienciesbetweensamplesandspikebecauseexogenousDNA is added after the PCR. Second, SNP genotypingwithout the addition of exogenous DNA was possibleby using 61-bp amplicons that maximize diVerences inTmand,therefore,improvediscriminationbetweengeno-types(Fig.6).WhenlookingatdiVerencesinTm,however,one should acknowledge the possible eVects that ionicstrength,productconcentrations,anddiVerencesinPCRamplificationscanhaveontheTmbetweendiVerentsam-ples[7].
Studies with genomic DNA samples and engineeredplasmidssuggestthatbothSNPdetectionandgenotypingofallpossiblebasecombinationsatonepositionbyHRManalysisofrelativelysmallamplicons(61–81bp)ispossi-bleusing theLightCycler480System.Dependingon thesequencethatisstudied,HRMassaysonlargerampliconsmight need to be used in conjunction with a sequencingmethod to determine the precise mutation. Nevertheless,HRMisinexpensive,hasthepotentialforhighthroughput,andcangreatlybenefitmutationscreeningandgenotypingof clinical samples for many genetic disorders, includingMHandCCD.
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Fig.6.HRManalysisofpossibleSNPgenotypesatthe4861positionusingtheLightCycler480HRMdye(61-bpamplicons).Here2samplesofeachgenotypewereanalyzedandincludedfourhomozygotes(solidlines)andthreeheterozygotes(dottedlines).(A)DiVerenceplotoftheHRManalyses.(B)NormalizedHRMcurvesofthewild-typesamples.Tmvaluesofhomozygotevariants:80.50and80.39°CforG/G,79.97and79.95°CforC/C,79.37and79.43°CforT/T,and79.58and79.64°CforA/A.
Acknowledgments
We thank Elaine Langton (Wellington Hospital)andNeilPollock (PalmerstonNorthHospital) forsup-plying blood samples for genomic DNA extractions.We thank Anthony Thrush (Roche Diagnostics NewZealand)forprovidingtechnicalsupport.Wealsothankthe Royal Society of New Zealand Marsden Fund forfunding.
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ficationofryanodinereceptor1single-nucleotidepolymorphisms