Analytical Biochemistry xxx (2008) xxx–xxx www.elsevier.com/locate/yabio 0003-2697/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.11.019 ARTICLE IN PRESS Please cite this article in press as: H. Grievink, . K.M. Stowell, Identication of ryanodine receptor 1 single-nucleotide polymorphisms ..., Anal. Biochem. (2007), doi:10.1016/j.ab.2007.11.019 Available online at www.sciencedirect.com Identication of ryanodine receptor 1 single-nucleotide polymorphisms by high-resolution melting using the LightCycler 480 System Hilbert Grievink, Kathryn M Stowell * Insttute of Moleular Bosenes, Massey Unversty, Palmerston North 11-222, New Zealand Received 7 October 2007 Abstract High-resolution melting (HRM) allows single-nucleotide polymorphism (SNP) detection/typing using inexpensive generic heterodu- plex-detecting double-stranded DNA (dsDNA) binding dyes. Until recently HRM has been a post-PCR process. With the LightCycler 480 System, however, the entire mutation screening process, including post-PCR analysis, can be performed using a single instrument. HRM assays were developed to allow screening of the ryanodine receptor gene (RYR1) for potential mutations causing malignant hyperthermia (MH) and/or central core disease (CCD) using the LightCycler 480 System. The assays were validated using engineered plasmids and/or genomic DNA samples that are either homozygous wild type or heterozygous for one of three SNPs that lead to the RyR1 amino acid substitutions T4826I, H4833Y, and/or R4861H. The HRM analyses were conducted using two diVerent heteroduplex-detecting dsDNA binding dyes: LightCycler 480 HRM dye and LCGreen Plus. Heterozygous samples for each of the HRM assays were readily distinguished from homozygous samples with both dyes. By using engineered plasmids, it was shown that even homozygous sequence variations can be identied by using either small amplicons or the addition of exogenous DNA after PCR. Thus, the LightCycler 480 System provides a novel, integrated, real-time PCR/HRM platform that allows high throughput, inexpensive SNP detection, and genotyping based on high- resolution amplicon melting. © 2007 Elsevier Inc. All rights reserved. Keywords: High-resolution melting; SNP identication; LightCycler 480; Ryanodine receptor 1; Malignant hyperthermia; Central core disease Genetic testing has an important role in many diagnostic laboratories and can provide dramatic prognostic and clini- cal benets. Many genetic tests are available to detect and/ or type single-nucleotide polymorphisms (SNPs). 1 Most of these techniques, however, require an additional separa- tion step that makes them less favorable for high-through- put assays. Examples of such methods are single-strand conformation polymorphism [1], denaturing gradient gel electrophoresis [2], restriction endonuclease analysis, and DNA sequencing. Homogeneous, closed-tube methods for SNP detection/typing that do not require separation steps are available and are based on either allele-specic PCR using SYBR Green I [3,4] or expensive fluorescently labeled probes [5,6] or primers [7]. SNP genotyping based on allele- specic PCR requires three primers, two of which need to be allele specic. Thus, diVerent mutations require diVerent allele-specic primers. When using labeled probes for SNP detection/typing, only SNPs that lie under the probe can be detected. Consequently, multiple relatively costly probes are needed to cover all potential SNPs. In addition, the use of probes often requires extensive optimization. These con- ditions limit the usefulness of these methods for screening purposes. If PCR is performed with a 59-labeled primer as described by Gundry and coworkers [7], high-resolution amplicon melting allows genotyping and mutation scanning without probes. However, this method requires at least one expensive labeled oligonucleotide. High-resolution melting (HRM) was introduced as a homogeneousclosed-tubesystemthatallowsmutationscan- ning and genotyping without the need for costly labeled * Corresponding author. Fax: +64 06 3505688. E-mal address: [email protected] (K. M. Stowell). 1 Abbrevatons used: SNP, single-nucleotide polymorphism; HRM, high-resolution melting; dsDNA, double-stranded DNA; T m , melting temperature; MH, malignant hyperthermia; CCD, central core disease; RYR1, ryanodine receptor gene; cDNA, complementary DNA.
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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: [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.
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)
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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)
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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).
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
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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-
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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
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.
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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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ARTICLE IN PRESS
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