3533

曰ecfmphor色sjs2001,22,3533-3538
KazuoUmetsu1
Mas2shiTanaka2
IsaoYUasa3

NaruyaSaitou4T3keshiT目keyasu2
NonyukiFuku25
EmikoNaito6

KazutoshiAgo7、NonNakayashikiB

AyaMiyoshi9'DepartmentofForBnsic

MedicinaYamagataUniversity

SchooIofMedicina

YamagataJapan

2DepaItmentofGeneTherapy，
GifuInternationaIInstituteofBiotechnoIogy0
GifUJapan

3DepartmentofLegaIMedicina
TbttoriUniversitySchooI

DNAvariations

Ａｎｕｍｂｅｒｏｆｍｕｔａｔｉｏｎｓｉｎｃｏｄｉｎｇａｎｄｎoncoding尼gionsofmitochondriaIDNA(mtDNA)haveprcviousIybeenstudiedIntheprBsentstudybwesimultaneouslytypedsixmutationsitesinthecodingregionbyuseofampIifiedproduct-Iengthpolymorphism(APLP)anaIysisThemtDNAvariationsof2471individuaIsfrom20popuIationsof

Japanese,Korean,Chinese,andGermanwereexaminedandcIassifiedinto18haplo‐
typesTWoofthesehapIotypes,Ｂ１(estimatedancestralhaplotype)ａｎｄＣ１,weredis‐
tributedamongaIIpopuIationstested､HoweventhehapIotypesA1,Ａ２,Ｂ２,Ｂ3,ａｎｄＣ２
ｗｅｒｅｍｏｓｔｌｙ尼strictedtotheMongoIoidpopuIations,whereashapIotypesB5andC5
appea｢cdalmostexcIusivelyintheGermanpopulation・PhyIogeneticanaIysisbytheneighbor-joiningmethodreveaIedthattheJapanesepopuIationswe｢emorScloseIylelatedtoeachotherthantotheotherEastAsianpopuIationssurveyed.ThemuItipIex
APLPmethodissuitabIeforlarge-scaIescreeningstudiesofmtDNAvariabiIity
becauseitisbothrapidandeconomical
Keywoms：MitochondnalDNA／SinglenucleotidepoIymorphisms／AmpIIfiedp｢cduct-Iength
poIymo｢phism／PoIymerasechainreaction／PopulationstudyEL4536

ofMedicina

Ybnago,Japan

4LaboratoryofEvolutionary

GenetiＣｓ，NationallnstituteofGenetics，MishimaJapanｌIntroduction

TbchnoIogyCorporation，TbkyqJapan

TheoccurTenceofalargenumberofmitochondriaIDNA(mtDNA)polymorphismslikely｢eflectthehighmutationrateofmtDNAwhichisthoughttobecausedmainIybyhigh5JapanScienceand

6Depa『tmentofLegaIMedicine
NiigataU､ive｢sitySchooIofMedicine，Niigata,Japan

7DepartmentofLegaIMedicme，
FacuItyofMedicine，
KagoshimaUnive盾ity，
KagoshimaJapan

8DepartmentofLegaIMedicineD
IwateMedicalUniversity
SchooIofMedicina
MoriokaoJapan

9DepartmentofFo帽nsic

MedicinaFukuokaUniversity
SchoclofMedicine

FukuokaJapan

IeveIsof幅activeoxygenspeciespresentinthemitochondrion[1lThemtDNAmuta‐
tionsobservedinbothnoncodingcontrolandcodingregionsarebeingwideIyusedto
characterizehumanevoIutionandforidentification[2-41Fortheformerpurpose,thenucIeotide(nt)substitutioninthecodingregionseemtobemorcsuitableforstudy，
becausethehighmutationrateofmtDNAinthenoncodingcontroIregionresuItsin
compIicatedsubstitutionsthatmaymakephyIogeneticanaIysisdifficuIt・SeveraImethodoIogicalapproachesareavaiIabIefordetectionofsinglenucleotidepolymorphisms(SNPs),whichincludedirectsequencing,RFLRSSCRandtheuseofallele-specificprimersandsequence-specificoligonuc1eotideprobesTheRFLPanalysisisoneof

themostwidelyusedtechniqueSfordetectingknownpointmutationsinthecoding｢egionofmtDNA[２，５１Inthismethod,someambiguitiesremainwithrespectto
theactuaImutationsiteMuItiplexsoIid-phasefIuo｢escentminisequencing{61andtheimmobiIizedsequence-specifico1igonucIeotidepmbe-basedassay[７１ａ尼ａｌｓｏａｐｐｌｉｅｄｔｏｔｈｅａｎａｌｙｓｉｓｏｆｍｔＤＮApoIymorphisms，HoweveKthesetechniquesare

ＣＯn℃spondence：、『zKazuoUmetsu，DepaパｍｅｎｔｏｆＦｏ臆nsIc
Medicine,YamagataUniversitySchooIofMedicina2-2-21ida-

Nishi,Yamagata990-95850Japan
E･ｍａⅡ:kumetsu＠ｍｅ｡.idyamagata-uac-lp
Fax:＋81-23-628-5273

Abb”viations:APLRampIifiedpmduct-lengthpoIymorphism，
９ｂｐ９－ｂｐ厄pｅａｔｓｉｎｔｈｅｎｏnccdingcytochnomeoxidaseII／tRNAlysintepenicr℃gion;ｎt,nucIeotide;SNRsingIenucIeotidepolymomhis、oWILEYLVCHVer1agGmbH69451Weinheim,2001

expensiveandtime-consumingRecentIy,wedeveIoped
thesimpIeampIifiedproduct-lengthpolymorphism(APLP)methodforthetypingofSNPs[a91Theprinciple
ofthismethodisbasedonanattachmentofanoncom-

plementa｢ysequencetothe50-endofoneoftwoallelespecificpnmers・Ｔｈｅｕｓｅｏｆｓｕｃｈｐｒimerspermitsthe

amplicationoftwosize-differentproductsdistinguishingtwoalIeIes．

0173-0835/01/1609-3533＄17.50+５０/０⑩宮。□。－の－．コ三

SeiichiKashimurag
GotaroWatanabel
MotokiOsawa1

MuItipIexampIifiedproduct-IengthpoIymorphismanaIysisforrapiddetectionofhumanmitochondrial