identification of ryanodine receptor 1 single-nucleotide

AnalyticalBiochemistryxxx(2008)xxx–xxxwww.elsevier.com/locate/yabio

0d

ARTICLE IN PRESS

Available online at www.sciencedirect.com

htR

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

Pleasecitethisarticleinpressas:H.Grievink,.K.M.Stowell,Iden...,Anal.Biochem.(2007),doi:10.1016/j.ab.2007.11.019

* Correspondingauthor.Fax:+64063505688.E-mai­l address: k.m.stowell@massey.ac.nz(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

tifi­cationofryanodinereceptor1single-nucleotidepolymorphisms

ARTICLE IN PRESS

2 Identi­fic­ati­on of ryanodi­ne rec­eptor 1 SNPs / H. Gri­evi­nk, K.M. Stowell / Anal. Bi­oc­hem. xxx (2008) xxx–xxx

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,

ifi­cationofryanodinereceptor1single-nucleotidepolymorphisms

ARTICLE IN PRESS

Identi­fic­ati­on of ryanodi­ne rec­eptor 1 SNPs / H. Gri­evi­nk, K.M. Stowell / Anal. Bi­oc­hem. xxx (2008) xxx–xxx 3

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.

Pleasecitethisarticleinpressas:H.Grievink,.K.M.Stowell,Ident...,Anal.Biochem.(2007),doi:10.1016/j.ab.2007.11.019

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.

ifi­cationofryanodinereceptor1single-nucleotidepolymorphisms

heterozygoussamples(dottedlines)and3homozygoussamples(solidlines)

ARTICLE IN PRESS

4 Identi­fic­ati­on of ryanodi­ne rec­eptor 1 SNPs / H. Gri­evi­nk, K.M. Stowell / Anal. Bi­oc­hem. xxx (2008) xxx–xxx

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

Pleasecitethisarticleinpressas:H.Grievink,.K.M.Stowell,Iden...,Anal.Biochem.(2007),doi:10.1016/j.ab.2007.11.019

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-

tifi­cationofryanodinereceptor1single-nucleotidepolymorphisms

heterozygoussamples(dottedlines)and10homozygoussamples(solidlines)

ARTICLE IN PRESS

Identi­fic­ati­on of ryanodi­ne rec­eptor 1 SNPs / H. Gri­evi­nk, K.M. Stowell / Anal. Bi­oc­hem. xxx (2008) xxx–xxx 5

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).

Pleasecitethisarticleinpressas:H.Grievink,.K.M.Stowell,Ident...,Anal.Biochem.(2007),doi:10.1016/j.ab.2007.11.019

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

ifi­cationofryanodinereceptor1single-nucleotidepolymorphisms

ARTICLE IN PRESS

6 Identi­fic­ati­on of ryanodi­ne rec­eptor 1 SNPs / H. Gri­evi­nk, K.M. Stowell / Anal. Bi­oc­hem. xxx (2008) xxx–xxx

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-

ifi­cationofryanodinereceptor1single-nucleotidepolymorphisms

ARTICLE IN PRESS

Identi­fic­ati­on of ryanodi­ne rec­eptor 1 SNPs / H. Gri­evi­nk, K.M. Stowell / Anal. Bi­oc­hem. xxx (2008) xxx–xxx 7

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

Pleasecitethisarticleinpressas:H.Grievink,.K.M.Stowell,Ident...,Anal.Biochem.(2007),doi:10.1016/j.ab.2007.11.019

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.

ifi­cationofryanodinereceptor1single-nucleotidepolymorphisms

ARTICLE IN PRESS

8 Identi­fic­ati­on of ryanodi­ne rec­eptor 1 SNPs / H. Gri­evi­nk, K.M. Stowell / Anal. Bi­oc­hem. xxx (2008) xxx–xxx

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.

Re­f­e­re­nce­s

[1] M.Orita,H.Iwahana,H.Kanazawa,K.Hayashi,T.Sekiya,Detectionof polymorphisms of human DNA by gel electrophoresis as single-strandconformationpolymorphisms,Proc.Natl.Acad.Sci.USA86(1989)2766–2770.

Pleasecitethisarticleinpressas:H.Grievink,.K.M.Stowell,Identifi­...,Anal.Biochem.(2007),doi:10.1016/j.ab.2007.11.019

[2] S.G.Fischer,L.S.Lerman,DNAfragmentsdiVeringby singlebase-pair substitutions are separated in denaturing gradient gels: Corre-spondencewithmeltingtheory,Proc.Natl.Acad.Sci.USA80(1983)1579–1583.

[3] A.C. Papp, J.K. Pinsonneault, G. Cooke, W. Sadee, Singlenucleotide polymorphism genotyping using allele-specific PCRand fluorescence melting curves, BioTechniques 34 (2003) 1068–1072.

[4] R.Okimoto,J.B.Dodgson,ImprovedPCRamplifi­cationofmultiplespecifi­c alleles (PAMSA) using internally mismatched primers, Bio-Techniques21(1996)20–26.

[5] P.S. Bernard, R.S. Ajioka, J.P. Kushner, C.T. Wittwer, Homoge-neous multiplex genotyping of hemochromatosis mutations withfluorescenthybridizationprobes,Am. J.Pathol. 153 (1998)1055–1061.

[6] A.O. Crockett, C.T. Wittwer, Fluorescein-labeled oligonucleotidesforreal-timePCR:Usingtheinherentquenchingofdeoxyguanosinenucleotides,Anal.Biochem.290(2001)89–97.

cationofryanodinereceptor1single-nucleotidepolymorphisms

ARTICLE IN PRESS

Identi­fic­ati­on of ryanodi­ne rec­eptor 1 SNPs / H. Gri­evi­nk, K.M. Stowell / Anal. Bi­oc­hem. xxx (2008) xxx–xxx 9

[7] C.N.Gundry,J.G.Vandersteen,G.H.Reed,R.J.Pryor,J.Chen,C.T.Wittwer,Ampliconmeltinganalysiswith labeledprimers:Aclosed-tubemethodfordiVerentiatinghomozygotesandheterozygotes,Clin.Chem.49(2003)396–406.

[8] C.T.Wittwer,G.H.Reed,C.N.Gundry,J.G.Vandersteen,R.J.Pryor,High-resolution genotyping by amplicon melting analysis usingLCGreen,Clin.Chem.49(2003)853–860.

[9] R.Robinson,D.Carpenter,M.A.Shaw,J.Halsall,P.Hopkins,Muta-tions in RYR1 in malignant hyperthermia and central core disease,Hum.Mutat.27(2006)977–989.

[10] T.V.McCarthy,K.A.Quane,P.J.Lynch,Ryanodine receptormuta-tionsinmalignanthyperthermiaandcentralcoredisease,Hum.Mutat.15(2000)410–417.

[11] P.J.Lynch,R.Krivosic-Horber,H.Reyford,N.Monnier,K.Quane,P.Adnet,G.Haudecoeur,I.Krivosic,T.McCarthy,J.Lunardi,Identi-fi­cationofheterozygousandhomozygousindividualswiththenovelRYR1 mutation Cys35Arg in a large kindred, Anesthesiology 86(1997)620–626.

Pleasecitethisarticleinpressas:H.Grievink,.K.M.Stowell,Identi...,Anal.Biochem.(2007),doi:10.1016/j.ab.2007.11.019

[12] N.Monnier,R.Krivosic-Horber, J.F.Payen,G.Kozak-Ribbens,Y.Nivoche,P.Adnet,H.Reyford,J.Lunardi,PresenceoftwodiVerentgenetic traits in malignant hyperthermia families: Implication forgeneticanalysis,diagnosis,andincidenceofmalignanthyperthermiasusceptibility,Anesthesiology97(2002)1067–1074.

[13] M.Liew,R.Pryor,R.Palais,C.Meadows,M.Erali,E.Lyon,C.Witt-wer,Genotypingofsingle-nucleotidepolymorphismsbyhigh-resolu-tionmeltingofsmallamplicons,Clin.Chem.50(2004)1156–1164.

[14] L.Zhou,L.Wang,R.Palais,R.Pryor,C.T.Wittwer,High-resolutionDNAmeltinganalysisforsimultaneousmutationscanningandgeno-typinginsolution,Clin.Chem.51(2005)1770–1777.

[15] G.H.Reed,C.T.Wittwer,Sensitivityandspecifi­cityofsingle-nucleo-tidepolymorphismscanningbyhigh-resolutionmeltinganalysis,Clin.Chem.50(2004)1748–1754.

[16] M.Krypuy,G.M.Newnham,D.M.Thomas,M.Conron,A.Dobrovic,Highresolutionmeltinganalysisfortherapidandsensitivedetectionofmutationsinclinicalsamples:KRAScodon12and13mutationsinnon-smallcelllungcancer,BMCCancer6(2006)295.

fi­cationofryanodinereceptor1single-nucleotidepolymorphisms