EES OEEpolymorphic 1 > restriction : 1 sites Primer Position 1 -5’sequence 4(cid:4) /   . amplification | | primers {  s for 7 ’3*4 } bp 1 ) mtDNA 1 \ region D-loop | - <+4;(:0+4;4(> (cid:14). “AC . @@ (cid:14).. .(cid:14) .(cid:14)@ CA D-loop |  <+3%%+0+3%’,> AAG ( @@@ j AAC @.@ .@@ @ . (cid:14).. .(cid:14)@ @ Sequencing | ; primers ?7L+- <+4;(,0+4;4(> (cid:14). “AC . @@ (cid:14).. .(cid:14) .(cid:14)@ CA ?7L+(cid:29) <+*’%,0+*’4,> @@ @(cid:14)@ @(cid:14). . (cid:14).. @@. @ @ ?7L’- <+*’(,0+*’’,> .(cid:14) .(cid:14) .(cid:14) (cid:14). ’ AAC @ (cid:14). ?7L’(cid:29) <+*%3,0+*%*,> ..(cid:14) @ @ @.@ .@@ . @ @@. @.@ ?7L4- <+*%(,0+*%’,> TAA 1 . AC (cid:14). ATA « . AC . ?7L4(cid:29) <+*:%,0+*:4,> @(cid:14)@ @(cid:14). (cid:14)@ @ CA @@ @ AAG ?7L*- <+*:(,0+*:’,> @(cid:14) . CAC (cid:14). CAA ?7L*(cid:29) <+%+%,0+%+4,> @ . (cid:14)@ AAT ( @(cid:14). (cid:14).. .@@ ?7L%- <+%+(,0+%+’,> . . @ . .@ ’ AAC .(cid:14). (cid:14)@ ?7L%(cid:29) <+%*%,0+%*4,> @@. @(cid:14)@ } AAT (cid:14)@. @.. ‘AAT @. ?7L3- <+%*(,0+%*’,> CAC ..   ..(cid:14) .(cid:14) CAC ?7L3(cid:29) <+%,%,0+%,4,> @@. .(cid:14)@ } AAT @(cid:14)@ @(cid:14)@ @. .@ ?7L,- <+%,(,0+%,’,> (cid:14). . CA* .(cid:14). (cid:14). CAA

)*+* . Ross ¢ yet ¢ tal. / , / Experimental ( | Gerontology - /0 123345 4404644(cid:30)7

Table | + | (continued)

Primer Position 5'sequence - 4(cid:4) ?7L,(cid:29) <+3(%,0+3(4,> @@. @(cid:14)@ .   ATA ( .. @@@ .@@ ?7L:- <+3((,0+3(’,> CA" ..(cid:14) @   ’ AAA ( @ . AAG ?7L:(cid:29) <+34%,0+344,> (cid:14). CA" .@@ @@(cid:14) @(cid:14) . GAA @@@ ?7L;- <+34(,0+34’,> : AC .. AAC (cid:14)@. “AC CA .(cid:14)@ ?7L;(cid:29) <+3%%+0+3%’,> AAG ¢ @@@ ; AAC @.@ .@@ @ . (cid:14).. .(cid:14)@ @ /   . amplification | | primers { s for 1 (cid:17)(cid:3)%+,:B(cid:17)(cid:3);(%% ) polymorphisms  + <%((%0%(’,> .(cid:14)@ (cid:14). AC . . CAA * ..(cid:14) : AC  ’ <%’:40%’3’> ..@ .@(cid:14) (cid:14).. .. @(cid:14) TAA .@@  4 <:;(’0:;’*> @ CAC .. ..(cid:14) CA CAA @@ AC  * <;+;30;+,%> @. @ CA @@. AGA ¢ @@ ..(cid:14)

/   . amplification | | primers { s for 1 (cid:17)(cid:3)%+,:B(cid:17)(cid:3);(%% ) polymorphisms  + <%((%0%(’,> .(cid:14)@ (cid:14). AC

defining 1 restriction s 1 site, +3(3%(cid:21)(cid:30) , for t ‘the | - longevity-associated | & | haplogroup ( » (De | - Benedictis et j cal., +;;;>(cid:28)

/ oR aedamplification was performed in +((  reaction volumes containing 3,  tris-HC  (cid:15)9 :(cid:28):) +3 y mM. <(cid:13)9 > ?(cid:26) ) +(cid:28)% ) mM. nnMgc   ) ((cid:28)(+= <(cid:27)B(cid:22)> Tween; ’(( ) ~M of * ’ * ’ each ¢ (cid:8)(cid:13)./) ((cid:28)+ LM « [ of ¢ nee eSF each | AAAS LN| primer; 1 “47 fetwo | ) units | nee ATEs Tag | Sen ee Meee/ polymerase ( “Xe pO”: (Bioline | DE(cid:30) REND AEM pe, London, | DE>) ((cid:28)+A((cid:28)% saao) pg | , DNA." . The | pe RR- following « owe Sn.cycling « ELEN DS AIST: conditions | MN PO> Were ¢ NIN NN> employed; : —— ee; samples | ; were | “epeeheld « ne| at ;3(cid:2) for %   followed by 4% oo i —)cycles | =~of | ;3(cid:2) for + “ewemin, %%(cid:2) mee esfor 4( reys, and ,’(cid:2) for 4 } min. J . After t r the { 3 AMOS> final ¢ l cycle, t wey, the “SI ee> samples \ “ee; were | » held < lat 7 ,’(cid:2) ed’ for‘ %  ,and t int ee| then < Lat +%(cid:2) ‘until removed from the / yeoR “9 NEmachine (Perkin-Elmer ;3(( ae eee gyThermal “ycler). Successful amplification | | Was | ; confirmed | using | - VYgel electrophoresis | / On | | a += EEE Neagarose | gel | pe ——estained | | with Meee peeethidium | | bromide | <((cid:28)% ) g/ml).

Following : ‘successful ; | amplification « | of 1 the 1 »mtDNA | region | <(cid:17)(cid:3)+4;(:0+3%%+> ) from alll « | of the DNA | “ee Neesamples, | each | of pee"the ’3*4 bp / oR ERA NEE ESproducts ome KM, were DID ODE EE EEindependently | Df RENEE BEDsubjected to

++33

Hinfl, OEE! POSHpal, nn =!Hpall, D>Mbol, Rsal, BamHI, EE AEEHaell, in inHincll at 4,(cid:2) > >and .(cid:10)8(cid:24) at 3%(cid:2) > >as LEED TAYoutlined pee Speepreviously (Torroni weeet al., +;;*>(cid:28) mdThe digested pe ND EROSsamples oe ETwere er“run on +(cid:28)%= EI IIEMetaphor NL”agarose J NOgel <-(cid:1) RD DE A ANE OBioProducts, Maine, D?(cid:14)> for ‘peesgreater resolution. After ae epee NEverification | “oeof | the. - owt eenposition -and : | sequence | enoof | all | renee 4| the | ot in nn —)- polymorphic | oe Rerestriction | sites | .encoun- tered. | in | this | NE; study reby. nucleotide |   they epeewere EO EONSincorporated - | into | haplotypes | . by virtue | of "the ee PT DOpresence <+> or | —eeabsence <(> of |the yeevarious ee EErestriction | sites a aapand  J Peetable aeof genetic | we RN poehaplotypes « Che NENA SOME; constructed.



Purified ] / oR | . products | ; (Qiagen 1 QIAquick | / OR | . purification | !(cid:2)(cid:3)> ) of s f samples « ; displaying  band | | pattern | as| that enews| deviated | ~"o| from “ii ~<. that | ae eee| predicted | ~~ || by t the | » published ; ee EE ETE| sequence ' ree- were $ ee PE TENE: sequenced on la. 4,4(cid:14) . automated | | DNA : ee EEE EDEN YP. Sequencer, | , using 1 —r the « 2 wi peewee> appropriate s ee> sequencing | ; primers : eee; shown i in Table +(cid:30) to. identify | the | “aosexact pe eeelocation | DIE-and | Oo MeN SPP| sequence | “rRof * the | Cp nepolymorphic | restriction | sites —~— Ap oaencountered | < eee JCurran | et i -al., +;;3>(cid:28)

2*<* Statistical | analysis