ResearcJi
`
`rt
`
`Quantitation of Targets for peR by Use of
`Limiting Dilution
`
`II
`
`.
`
`.
`
`limli~ll~~i:ift;~;n\~:~l~l\l;::;~~~~ :::~~~~a~l~~ ••....:•...:•....:I:!:I:!:I:!:I....:1....:•...:•....:•...:•...:.
`
`P.J. Sykes. S.H. Neon,
`1\1.J. BriSCfJ. E. Hughes.
`J. Condon and A.A. "Morley
`Flinders !\.fedical Center,
`South Australia
`
`ABSTRACT
`n"'f describe a general method t(J quan ..
`{(Ha! namber (~{ iniiisl{ targets
`liiOh{ flit
`present in a sampl« using linntiti8 iA!ution~
`PCRand Poisson s!wi.\'!in. The DNA tar(cid:173)
`get J;..·t the P(~R ;'FO:S tht' tt"ffrrangt-d unnu, ..
`nogfohuiin hein,): (~hain (Igfi) gt'ne derived
`fi(}!'!"? a. !euk<:.:tnir cione ihat H-'as quanti(cid:173)
`rated against a b{jckgrou.nd ;:{ !::....tcfSJ reay(cid:173)
`ranged i~ft ::;~etl.t\y frosn.
`~u.~Fnl(tl lyn~ph:~h
`cytes. The .P((R HfO~' op~/1nj2r.d .to ptovi:<:ie
`an all ·~.)r,,!tO';fr::.·· fJtd PO/fil at ~"('f~V l[i~\" f)AlA
`t{"lrgel rusmbers. P(7? an;pliji(:atlon ,,:r flu
`'N<ra:; gent -H,'(iS used as an internal('ontrol
`10 quantiiate the numhO' (i!"potemia!fv am(cid:173)
`pNli<.th!e genJ.)f:fif:)· pf esent. in a satnp/v and.
`henc» to measur« the extent. <:.~t" [)/\IA. f.ffg(cid:173)
`md"uiNI, A t>Fo-Noge peT<: W<iS fleet's!:<iI)'
`owing to' competition betwees;
`leuk:e!~tic
`and nun·!fuk:t.:tllic tfnv;lal('s, .~:;tud,): of ei~r;ht
`leukeJnic samples showed that approxi(cid:173)
`amplifiab!e
`two
`pOlentially
`fnllEdy
`leukemic IgJ-f tttr,gers cOlJJd fJ:f dfteC!ed In
`the presence {~i.f 160000 i..'tllnpe!ing non(cid:173)
`/<·ukemic g<·noni<?>.
`·Tllll ~Tiethod pre/)'erlu'd quantitab..:.,....· the
`lotal nutnher {~l i1.1.iti....d I\NA taJ~r;t"ts pr(','"
`(Ill in. q. M"unplt~. unlik.e tNOS.f other quanti·
`fati'vi m.l;:,thod,~ thai qwmuta{c PCR pru?
`{lets. It has }fide <:.lpplh:{}llon. b(~Ca.U,H:· it is
`u~.ch.1~ical!): ~hnpl{), does t[Oi requirf~ radio(cid:173)
`l.':lctiFi(v~ ttddrf ...l,'.':oCs
`the prohlt..,n (~.r fx('e,~'s
`{I..lrgets (~nd esrunate.~ {he C.aen{
`Cl)tt'~tJl;.'tin}~
`(:!i'LhVA degi'1.idc.uioti in a .sarnpl~'.
`
`IN'fROIlVCTION
`
`Quantitalion of DNA Of RNA, by the
`peR is a problem that is presently Ull··
`der active investigation by rnany viork~
`ers. Nearly a.ll nWihods reported to dille
`have lls(:d co··amplificati{!ll of reporter
`IJNA in the same lUbe and some fonn
`of quantihltion of the ampEfkd malt>
`rial 0,4,11). It is assumed that the effi·
`ciency of mnpliJicatlo!l of the reporter
`DNA is the same a.s that of the titrget
`DNA, .and caku.hJtionof the amount of
`target DNA inillaHy present isbased on
`the amount of repont~r DNA added or
`origimllly present and on themtio of
`the quanl.itit~s of targd and reptlrler
`DNA a~ determined in the a.mplifkd
`material by V;;\rit}U$ nK~thod~.
`We have been llsing the rearranged
`inlxnunoglobulln heavy (hain ([gH)
`gene as targ;d DNA in the peR to
`study patients Wjth,K:ut(~ lymphoblastic
`leukemi.a (ALL) in order to delec~ and
`qmmtltHte
`a nnnor
`population of
`leuk('mic cells within a larger popula"
`timl of nomwl1ymphoid and non-Iym·
`phold cells ( L2\ In a particular patient,
`all leukemic: cells win !);''l.ve the same
`rearranged 19B getK thar c:an act as a
`getlNk marker to dh>tingl..llsh leukemic
`cells frnm normal nOfl,lyrnphoid cells
`and T lympJHX'ytes, \-vhich !)<lVenot re(cid:173)
`arranged their 19B genes. and from
`nonn'll B lympho(:ytes, whid1have un(cid:173)
`dergone wui.oui:' and differentrearran2i>
`me~ts of their IgH genes. Qlla.ntitatl~m
`of the lmi4ll{~ r\;~arrangement of the
`leukemic done poses two pwblerns.
`first. only a few copies rna)' be present
`in a tl<;Slle sample taken from a patient
`in remis~il)n following treatment Sec~
`
`ond, germ· line IgH genes limn cells,
`other than B !yrt1phocYlt~S, and rear,
`ranged h~H ~(ene)', from nonnal B hm·
`pht;c::ite; wilT be present and, o\Ning f(;
`homology with the leukemic sequence,
`rna)' compete with it in the peR, Out"
`,lppwach to qU<llltitation has diffel'(~d
`hm) that of other workers in avoiding
`nse of reporter DNA. imd quantitation
`of amplified materiaL Rather. we have
`used the principle of limiting dilution
`\vh.ich is based on the use of il qualiw·
`Ii n,~ all·or·none end poiIlt and on the
`premise that one or more targers in the
`reaction mixture give rise to <l p()sitive
`etld poin" .Accurate qu.antimtion is
`achieved by perftmmng multiple repli·
`cates at s(~rial dilutions. of tlw materia;
`tQ be sssayed, At t~e limit of ,dilutioH:
`where Rome end pomts sre pOSlllve ano
`some are Mllath;e, Ine number of tar·
`gels present ;::m ~x; calculated from tlw
`proponion of negative end p(!inL~ by
`usirlp.: Poisso!) st<ltistks.
`
`llsing the l,\:~al'-
`a ttlrget for the peR,
`ranged 19B g~~IW from it leukemic clum>
`a~; an example. We also discu;;,s fU~B' re..
`hUed issues: optimization of the peR fey
`detect one or a few DNA targets; the
`effect of excess competing targets IiI
`the peR; interthen(:e in the peR by
`prjmel'S li)r an unrelated DNA segment;
`and the prohlem of DNA degradatkm
`in the :>arnpk. Although data fwm~(
`small 11umber of patients ~lre inclu(j,:d
`in this rmper, the clinical and biologi~,~ll
`inforrnmion ohtained bv Iirnilimr dibS
`f.i(m ,ma!v,i" from a 1<lnre l1uutber l*(
`patients ;,;'ith ALL will'\}C tbcrib>f~
`elM~'where (unpllblished dara).
`. .
`
`MYR 1013
`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
`
`Page 1 of 6
`
`

`

` MATERIALS AND METHODS
`
`
`
`DNA Samples
`
`PRL DNA was abtained fret nor-
`mal Dood celle separated by Lympho-
`prep’ iNycomed Pharm& AS, Oakey
`Norway} te contain predominantly nor+
`mal
`lymphocytes, and Ho DNA. was
`frorn a bone marrow sunyde of a pa-
`fen owith ALL, chained at diagnosis.
`
`Ho DPS provided asrarcead specific
`lenkernic [oHtargets and normal N-ras
`fargets, aad PRET DRA provided a
`sence al norpial oH and Neres tare
`gets. Ho DNA contained virtually
`180% feukemic oells,. whereas PREI
`Was.
`estimated,
`fo contaire
`appreyt-
`
`
`
`mutely 19%. normal B lyraphacyies,
`The optinigation experiments 1relied
`ay DNAconcentration of the Ho DNA
`sanaple (OO mg/l) to estimate the num-
`her of PCR targets presentin the dile-
`tings. Due tothe small amount of mate-
`rink
`avadlahle,
`an GDag_ was not
`possible. The concentration of
`the
`DNA wae obtained by ethidium beo-
`mida spotting against known BNA
`standards (Reference “&, Appondi« §,
`p6h}. Because.one human diplout cell
`contains & pe of DNA (S) 1 be af
`PRLT DNA world contain apprasi-
`
`mately 3.3 x OF Stray genes, appront-
`
`maicly 24104 rearranged IH genes
`and 3 x £05 unrewraneed lok. series:
`GNA from 7 other patents with
`
`ALL was extracted from fresh bone
`
`TEPECY aspirate samples for patients 1,
`2, Sand 7, from frozen. Fieal+Paque
`separated lyerphocytes. for patiear 4
`and frou stained, fixed. bare mirrow
`
`slides for patients S ard 6. The DNA
`conoentranion of
`samples 1, 2,3, 4 and
`
`7 owas determined by ODean ami for
`patients 5 and & by evddnan heamtide
`apating,
`
`PCR.
`
`16.6 mM
`contained
`PCRs C7)
`(NAG bSQ,, 67 mM Tris-HCh pH 8.8,
`lomM psriercaptioctianol, 200 pafml
`gelatin, 2mM MgCl, 0.) mMeach of
`deoxyadenasine. tybasphate, deoxy~
`guanosine inphosphaic, deoxyoyidine
`inphasohate,
`and
` deoxythyrmidine
`triphucphatc, TOG ne of each pomer,
`yarying amounts of ternplaw DNA
`(0.45 pe-l ue), and4 0 of Fag DNA
`
`Polymerase CAmpliTag®: Perkin EL
`amer, Norwalk, CT) in a volume of 25
`ul, overlaid with 23.AI hehe mineral oil.
`The ssingles were, subjected tan in-
`
`itis? 4 min denaturation at S4°C fol.
`nese
`cueneeeuneeeecy
`
`
`
`
`:33::%3$s:
`
`
`
`:&38e33e::
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`lowed by varying numbers of cycles of
`
`Ladin sorealing at 38°C.
`| au exien-
`sind at 72°C and | min denaturation at
`SAC. A trial 20 min extensian.ac 72°C
`was performed at the end af each round
`af PER,
`ip all experinents, gagative controls
`
`containing no templaie DAA were sub-
`jectedto the same procedures to detect
`any passible- contamination,
`
`POR Primers
`
`the Voregion:LH 98°BGTALGAG.ACG-
`
`
`Primers were synthesized on an
`Applied Biosystems Model 371) aato-
`rasied synthesis
`(Paster Cay, CA}
`
`Consensus primers (2) used to arnplity
`
`
`all loFbwenes in the rst mand of PCR.
`were FRSA-4 ACACGGCIO/THGACE
`TGTATTACTOP 3 forahe 3’ end of
`
`
`GPGACE %forthe ¥end of the F re.
`Sion,
`Paticat-specific primers (2) sited Be-
`
`
`preen fH and FRSAwere used wiarn-
`plify Ho feH genes onlyin the second
`round for the Ho DNA: Hol = 5° TOT.
`GCGASAGASTCTCTOCE 3? for the
`
`
`3 ead of Vy G2
`MAGTAGECA-
`
`AQOGTGGOTA 3” for the S’ end
`at J.
`Paticat-specitic IgA primers for the
`other sever. pavents will be: published
`
`ehsewhere.
`Specific primers were udedk Foy As
`ray om both Fire ane second Fourds: ©
`$y
`AAGETITAAAGTACTGTAGA3a
`First, round:
`(intrer
`2}
`- 3°
`NBi2 feren d)
`» S°CPCTATGOTGG-
`GATCAT.ATTICA ¥. Seeand round:
`NATF(eson T}~ S ATGACTGAGTA-
`CASACTGOOTGGTS 2 thar les. be-
`tween TS) amd NB-12 and im the’asc-
`gad round is used with NB-13.
`
`For singde-raund POR, Nuws was
`amplified by 234 and NBY2 and Ho
`Teh by Hel and Hod. For bvroared
`POR, Arae wits ampiificd by 231 and
`
`NAT? followed by NBTS and
`NAIZ
`and lenkersic IgH genes by FRAand
`LIH,
`iotiewed
`by patent specific
`Reimersae the seoonih round for Pe
`
`
`Gents 1!
`phor
`poh
`
`(VSe VBE balleratss
`
`
`
`
`
`Zos
`we
`we
`
`3)8) oa f
`
`—
`
`as
`ae
`
`a
`
`a
`
`ae
`_ a ES}
`AY
`
`ge
`
`a
`
`,
`
`an
`
`i
`{
`
`—-~cesRY, t
`\
`
`{
`:
`
`wet
`
`Po
`
`:
`:
`:
`
`sso
`
`NumberofmoleculesofPCRproduct
`
`oe82OomMmeowGS
`
`
`‘ea
`
`10
`
`19 *
`
`1907
`"0 0
`
`
`
`
`OEELEEPEREPOTSTEDnT
`eannenRA
`
`
`ntethpeeeee
`“30
`40
`80
`20
`10
`Number of first round PCR eyeles
`
`
`
`
`Fas aneHoonRE penehictsproduced by a Srstarsneed PERafter-varys
`Figure t Number af
`
`
`aPDSHHHCEofponpeting PELE RNA. The giimbers Hrparenthe>
`
`ins PC& eeyele Rosierin+theBee
`
`mphHoatlinted DNA.
`aapemied idagl
` Norers Pek prtPBLD aoe‘HoTenPC
`‘heodack (FPBED A <9
`
`
`» Ha teh POR producs
`
`
`“
`UPBEAT,
`
`
`Page 2 of 6
`Mal, TS) Na. 3 ¢$982)
`
`
`Page 2 of 6
`
`

`

`Quantitation
`
`
`Phreefold dilutions of samples were.
`prepared in water ar PBL DNA. and
`li replicates of eachdilntion. were ana-
`
`
`Ny
`ning the optimal PCR prnend
`presented inthis paper: The mena nurri-
` me
`het af targets required ie give w# PCR
`
`
`proract was. determined using the
`method of Taswell (16), which fads
`distribution thal best fits
`AGES al Sstioake Cee
`
`
`
`a2 da£4 contin vs
`
`RESULTS
`
`Preliminary Experiments
`
`Although ane or a few A-fas-ar Ie
`Taree cenuld be detected by ota apt
`miged.single-siags POR, a mining 2K
`portsbearshowed thea the.
`OepeteBO HON-ewken vfenplatetesoe.
`
`
`
`peased dete
`of Ho templates
`
`GF that ofhenvise obtainable. A
`
`gyo-raund PCE. straiegy using nested
`primers was theretore evelaped indi
`
`prove sensitivicy and spectichyof an
`paficatiea.
`
`Seeond-Rouad PCR — Number of
`Cycles Required
`
`for.
`
`Sy
`
`Serial dilutions were muds fo pro-
`dues aliqhots thal, comained varying
`numbers w He inh tarectsin | ug of
`FPBLI DNA. Each aliquot wag araph-
`fied for 45 oveles in the Jirer mand, and
`
`Uy te HE? dikwions af dhe praduct
`were made-in water to produce a din-
`
`
`tion conmialnag one ora few cap
`P
`aruphiedEDINAae st fing temp!
`
`tah Taewets, amplifiedpre:uct would be
`
`ewcted from the lddimes after 20
`cycles, froma the LA ddution afier 3)
`
`fescand Frathe FO° are fO-) Sie
`
`tong after 40
`eyeles: Araphified prod-
`
`
`Bet could not be deteeted far the 18
`and Ub? dilotinns. These results indi
`
`cated that approximately 107 Ish iar
`gets: for the second-round were being
`
`Page 3 of 6
`Ads RigTochrigres
`
`Was
`
`PRES
`
`w
`
`ep treTest anysectakddround
`
`
`tor herwe
`PUR ag
`and-rond PER cyahes
`
`
`in 8s experiment, 30 first-round gy-
`
`cles were sufficient in enable an. ever-
`
`age-af (3 ight
`targetsto be detected,
`The3santinatve stupects of ehe first
`poured were then studied in raare detail
`
`in arderte detersaine both the eprimal
`
`
`number of cycles andaisethe appropri.
`ate dilation ioause Berween. the first anid
`
`
`second rormads atthe FOR. See
`yt Ta.
`
`talutions of Ho DNA ware made
`ikl
`ml
`ugar PBLI DNA. Be-
`
`Bweee ct)
`ad AQ cycles of first-round
`sorformied, cach aligucd ofPOR were £
`
`
`
`
`|
`
`i
`
`$7.80
`
`44.28
`
`75.00
`
`22.8
`
`1.728
`
`0.375
`FHS
`
`
`
`10
`
`1G
`
`8
`
`4
`
`9
`
`10
`
`3
`
`c
`'
`
`Mean No. comes deleted
`| “Eastimetert fron ONA cancertrations
`
`
`28
`
`a2
`
`Pirst-Rowad POR ~~ Number af
`Cyeles Reduired and Eifeet of
`
`Competing Fenplates
`nenambers at fis
`
`
`
`
`cof electhns {Copies} af Nunes af Igh in the Presence ur Absence af Competiog
`
`
`
`Nuntber af Positives it10Tubas
`LN
`
`igh («PBL1)
`(igh
`“Neras
`Neras
`IGN (-PBL1)
`
`
`
`
`
`
`
`
`
`
`
`
`
`Seid was8 diluted 1S ms
`
`in ow:aterad
`
`
`
`3 ovcles
`
`hatkon.
`
`‘The resalts ace shows ta Rigure |,
`
`Bocuse.akeasepee secondraund
`
`Prsert Se Or &i fos
`
`
`mum dihaon of the
`that snl leads ts araphj
`rial
`
`
`the secornl round uydiss
`
`
`
`make: naraber«a
`
`
` ‘ get namber of VS
`
`
`SerOMES, Hy
`the
`
`uted bya mlaisay of 108
`
`
`
`
`yeles of ample
`ever, when otherif ighieee Were
`
`
`
`
`
`
`plificatws of Ho DNA vwas h
`
`
`cient and andy 106 Hodgkt WeRe
`
`
`duced afer 30-c
`SO
`!
`Based-on these
`LE
`
`
`
`as
`y
`fuse 45 oveles for G
`
`
`sufion and a LGdilution of armplle
`
`material between the Brst andes
`round, wopeachOTeneditionswoul|t
`
`
`Tar:~TR as
`
`ond round.
`
`Quantitative by Lani
`
`Analysis and Poisson
`
`
`Sexidblutionsat He DNA each
`
`
`the omunee
`
`
`cles, PH diivean:
`
`
`auibe was
`
`for amplificalion
`:
`
`
`{ rendts of ore such
`
`
`Figure Bs AR sehidhint bromide sine’
`produced by ite2 Rest-rocnd POE and
`
`
`
`Page 3 of 6
`
`

`

`
`
`
`Table 2. Seasitity of Deloction (Caples) in Dingasatic ALL Trahet Sasipies Usiay Two-Raand
`POR
`
`
`
`igh ¢-PBL1}
`Abras
`Patient
`
`
`Ratia Atrasiigh
`
`-PALY PBA
`
`eeveipeeneneccesacncen
`
`
`
`Se::::::
`
`‘:::::F:::::P%::::
`
`3::$ssS
`
`:S
`
`ES:::$3:33%3:3:
`
`
`
`AREERARERRRREEEEE
`
`
`
`tectatde (3.2 intrets). However in the
`ae
`
`presenoe of PBL DNA. fell pris
`
`AONE. WETe able tg detect mean Mrest
`namiers af 3.3 inane experiment ard
`Hhin a second, wheres with co-araphi-
`
`fication with N-ras: primers, the gk
`primers cand anly detect INCA Hinget
`
`numbers of 18. of 29, This
`feck of W-
`
`presumably reaulted fram
`the concerrent ammplifieanor ofthe 105.
`fold excess of N-res tarests provided
`
`by the BEN
`
`DSCCLISNEGEN
`
`We
`have presented a genera
`
`meathod. for cuamtttatsan of largets by
`
`PCR ig
`itingpdt
`
`
` 3 sidSeaF Poisson=shat
`
`ibhan
` Ss,
`the PER
`this approacl
`ae
`imized so that
`ny wall take
`
`place In an Cafl-orsrone™ fasion, ard
`
`one or a fewslarting targets will give a
`pasitive sesult. Whea theoptimal con-
`
`
`concentration
`ditions are known, largel
`tan be astimated by Pais860 statistics
`
`apphed to the sesulte fron: replicate
`tubes takerrat the hee? dilation.
`Limiting dilution analysis iswidely
`used. for quantiation in cell biology but
`iy met. commonly usedtor molecular
`
`
`
` thtate ‘tiple HEV molecules,
`unaasre ofthelgrepartwhen wedevel.
`oped the methodpresemed hover, and
`i
`ceviewy. of Has ice
`
`CALved
`
`abd hepee
`
`futon” Hows
`a heen per-
`
` HeralureSM,BSan
`
` Rag nat boon veri-
`he Teachion
`p atbirary has. been.
`
`muiber Gh -<
`
`
`8G-5nG ~
`
`oteeek
`
`:itivea an
`rend:point duesRot
`vonaiiigte 8 proper inmiung dilstion
`ye Sen-quantita-
`8
`ive results,
`Sens areaa of resdarch require a
`method ey.guariiiiate aml menber of
`
`
`
`m2
`
`3
`i
`i 4
`5
`
`5
`
`?
`
`
`
`‘a2
`
`>
`
`to
`
`18
`13
`BS
`
`544
`
`aa
`
`OF
`44
`214
`
`55)
`
`3
`
`a8
`
`824
`
`Le
`
`Og?
`
`Geometicmean of rates
`
`
`O§ sogtinnies af dial
`“eometric maan of all axperimenia.in Ho are shown.
`
`hake stuchad for patierds t-
`
`
`‘tproved psrosnible: fo
` Va nea af 5
`Le. fo detect
`3 poterendialy
`
`
`REEEN +
`
`soudation in sanyples Trara
`‘
`patients3 and f but polin patient], in
`whern the rcsulis, although somewhat
`
`variahia, suge nied a less ao degre at
`oe
`
`
`degradation, The
`Sten
`for patent J, although Faint appearsate
`ee lareelyinfant
`
`Facters Affecting Amplifiontive
`efficiency
`
`Campcting IgH targets, The effect
`of these oy reducing amplification of
`
`the speciiic target has already been dy
`
`lustrated (Figoee Cand Fable 1).
`Compeilion heteven primer pairs.
`Several methods fir guaraitaties: of
`PCR tarsets rely
`on the use of two
`primer pas18 fhe sar: tun
`BasSBRwhether ch
`
`
`
`
`
`tn. pwn exper:
`am shown in Lable 1,
`roeats, 17 aod 2.9 capes af A-rew
` a,ch
`
`TEE
`aeoe
`could, be detected,
`and in three saperi-
`45.6.
`WHo lel
`and Fh cnpies
`me ;
`
`detected 3te the bre
`PBL! DINA. The de
`
`nent were consistentwi
`
`aticpee ENS fer eachs.
`
`fivity of detection
`
`stic
`ALL pation!
`
`
`samples
`GF all
`samples,
`“E
`
`
`are Surtumar
`ved ti Pable 2. and these
`
`ine
`tegrity, af these DNA sanples WH
`
`
`veatigaied hy
`electroghorentgon.afs
`
`agarose gel (Figure 3). In this table, the
`resuks in fhe cahayms.
`referring to
`MUNIN: Meat nurmber of copies ale-
`iteled are calewlanx)
`from:
`the fetal
`nuroaher of copies present as haseed on
`the
`sstimated DNA
`
`
`ton of Morret
`High sensitivity fur de
`
`
`
`and [gH genes was.
`<
`dia S pa.
`Hents: and lesser semaitivity i
`3. pa-
`
`
`
`
`
`tients S, 4 and 7, Because the surriher
`af Aras comes detected depends on
`bath the total number of Copley we
`are present ard the Proporton adie
`
`are amipliftable, tte Ag of the sue
`ber 6
`é na detected ta the
`mupiberof ightarg tsdetesated give we
`
`
`proportion at mote addy amplifiadle
`
`fel targets that were achaally amphi
`fied. Inthe absence of competing nen-
`
`
`
`Jeuk mc Pehl targets
`ble to aniputy a@ mein ofBUS
`of the
`
`le keSHC dargets, and in the presenceof
`
`
`vorPORKOF Faves
`
`
`
`
`
`
`Page 4 of 6
`
`

`

`
`
`Research Report
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`be performed based on these data. Deg.
`ridation can be a significant problési
`especially where fresh samples are an-
`available and to our knowledge this js
`the. first reported PCR approach thai
`enables correction ia- be made fordt,
`js
`Limiting dilution quantitation.
`simple, réqpives few manipulations if
`samples and has widespread apphes-
`tion. Our usual approach is te perform
`& preliminary serial dilution experi-
`ment.
`ia determine the approximate
`
`polut at which some amplifications are
` ‘e
`likely to he negative and same posits
`avd then perform a detailed -exper-
`ment, perhaps 40 tubes in all invalving
`
`
`multiple replicates of difuthyas around
`this ‘poust. LP quantitation. is tg be per-
`
`formed in terms of the number of am-
`pliable wangetsof'anather gene such aa
`
`N-ras, it should be noted. ihat separate
`amplification redetions must be per-
`formed for eachgene and that the van-
`ance: ofthe final value will be contrib-
`wed wradditively by the variance of the
`estimation for each gene, More. repti-
`eales viii therefore be required
`for a
`giveri level of precising,
`
`The assumpuiorn is that the extraction
`Ss
`pray
`dies riot ynadd'y the DNA.
`
`However. chenucal modifications to
`
`ENA, suchas sinind breakage, depuri-
`fathon or jomnation of adducts, may
`render the DS “ incapable: of acting as
`
`
`afemplate far
`the POR, Quantitation by
`Jarnting ¢dation may Have-aunigne ad-
`
`varlage in Overcoming the potential
`problem of DNA modification beacause
`it is possible te also quambtate an endo-
`genous gene yielding a PCR product at
`sispilar size whichis present in known
`number in all celle and which alse. un-
`
`
`derpoes the extraction pracedure,
`In
`die present study Ue Voss gene was
`
`selected as the endogenous geneto cur-
`
`for degradation. The 8 pans
`rect
`studied fell into 2 groups (Table 2). 1
`3 patients, all ar nearly all of the 4:
`
`targets could be amplitied. whereas in
`3 pitiients a lesser: prapartiionof targets
`contd he ammpiified This suiesestedthe
`
`presence of « variable degree of DNA
`
`
`degradation in ihese 2 pationts, and utis
`was confirmed in the 2 mast obvicns
`cases by electrophoresis (Figure 33.
`
`Nevertheless..as seen In Table 2. in all
`8 patients there was an approximately
`constant ratia between the number of
`
`ampiiable IgH targets and the ounmtber
`ofampliiable N-rastargets. These data
`waagest tthat the number of amplifiable
`
`
`Neres genes, rather than the DNAcon-
`centration,
`is the hest indicator-of the
`rember of amplifiable genomes pre-
`sent, that Virtualbyall potentially amipli-
`
`fable leukemic JoH genes are ampli-
`fied an
`the dhsence of competing
`non-loukemic {eH genes and that ap-
`proximately half of the Jegkeniic Ig@H
`genes are soiplitied in the presence of
`competing genes. Quantitation.
`in the
`presence of DNA devradation can thus
`
`
`
`iargets agabist. a background of highly
`homsalogous targets, eg., detection of
`specific mutations in 8 population of
`
`normal cells, The biological problemin
`was the demcnuon of rare.
`our study
`leukemic cellsinva large population of
`normal cells, whichin molecular terms
`became the problem of detvetion-of 4
`rare upique Tell sequence against a
`background of numerots other bese-
`quences, The two-stage POR syst
`
`that was developed praved capable af
`deecting approximatsly twa Cy9.so)
`potentially amplifiable leukerme IgH
`
`SeqHeNwes against a background of ap-
`
`
`proximately [60000 total genatnes,
`These gennnies would provide: a vast
`exetss of sequences that woukl com
`pete with the leukermic gh sequences
`for
`the PCR primers because they
`would contain anproximately: 24x 108
`rearranged eH genes. frdoe normal 8
`lyvaphocvies and 3.x 105
`gemn-line.
`igh genes, each containing multiple V
`woJ segments.
`
`
`Quantitation of PCR targets by ioe
`
`iting dilitien can be compared with
`
`other methods far quantization which
`
`use an added imiermal or esternal stan-
`dard, whick is cartied through the an-
`
`ACKNOWLEDGMENT
`pliftcaticn, and which involve sore
`farm of quangtatinn of the aroplified
`
`Wethank Dr. R. Seshadri and Dr f
`producers)
`(38.11) Quantitation by
`
`Toogood for pravaling es with the pas
`limiting dileton does’ not require the
`
`tient Saniples used mt this study. Thi
`use of an added standard, sith the ine
`
`work was supported by the Nation
`herent QSUMPTGNS invadved, and the
`
`
`end potot
`is sivpie, nongquantitative
`Health amt Medical Research Counc
`
`and the AntiCancer Foundation of
`the
`and, nonradiogetive. Farthernere,the:
`Universities of South Australia. MLB.
`
`end point is Based an an all-arcnone
`is in tecerpt ofa Rotary Peter Nelse
`ggnal derived frora the terminal plas
`Leukemia Research FundFellowship.
`
`
`teau phased! the PCR, abd the tech-
`nique is therefore relatively rebust, ba-
`
`
`ing able thcape with widevariationsi
`
`amplification efficiency Wwithour affect-
`REFERENCES
`
`ing: the estimation of DNA target nun
` iBrisca, Mohed ©
`ber, One potential disadvantage is the
`
`possibility of coniainination, parhiea-
`
`larly Wa two-Siage PCR is peifarmed.
`
`We ase the precautions recommended
`
`by Kavok of aL(6) to minimiae the-risk
`
`af cartamination. Replicate negative
`
`controls areusedbut also, ia effect, di-
`
`
`iutkws below the critical Hoik of dilu-
`Hon act as additional negative controls,
`
`When molecides are quantitated by
`& from diagnostic
`Figure 3. Extracted
`
`ALDpatient samples, Approsimaicly 230ne of
`
` f
`PCR,is nevessary tovexpress
`the -re-
`
`
`DNAAwere loaded onto the
`accept for parent
`stitedn toons of-a denomimator such as
`
`
`
`Ewhere.only 4 noowere suiable..M- molecuiar
`re
`Soywith the stacsgivern ta kb. Lanes
`
`muss-of DNA suadiert or mumber of
`
`71
`eelis-trom which DNA was extracted.
`
`
`
`atsBinten208;pot 6
`
`Vol. 13, No.
`
`
`
`Page 5 of 6
`
`

`

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