Free Motion to Vacate - District Court of Delaware - Delaware

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 1 of 45

'

United States Patent
Chee et a. l

t9 11

[ I ~ IPatent
[43

Number: Date of Patent:
a992

57576 ,9,1
Aug. 18, 1998

1541 C O ~ ~ ~ E F I - A I DYISUAIlWLTION A M ) ED ANALYSIS SYSrW FOR SEQUENCE

rY0 9Y105M

WIPO .

EVALUATION

9 I l 9 Y1995 WIPD. 5J95 W O ~ ~ ~ S S WIPO.

p&l

lnvcntcux

Mut S Cb- 3199 W a v e Sr. Pah
M o , C 6 943CKk Robert 1Lipsboh aC ¶O W o Aim Ave- Palo Alto. C l aX

UlW3 PUBLICMXONS

94381 I 1 AppL No.: 327525 21

Drmanac d a 1 DNA m Kkfumbtio. by hybrid& a t i o M shacgy for Effck5m Lage Scalc Squcadng Scicacr 28): 1649-1652 1 9 . 93 Sbooska a rL DNA scqusaing by Ilytdklion :100
basudbya~bilSCdlDCtbDdPNASI:

~oo~p-~m.i~~. Drnmnac d aL h algaittun fur the DNA Scqucocz Gcncralion faun k-Tuple W d ContcoU d a Minimal Nu& b a ofRandom Fragmcnu. L of BiShucfmc & DyDmnicr % 1065-llm. 1 9 . 91 .SollIhum a d Aaalyrimg and Chmpiog Nuddc Add Sequences by Hybrimon UI Amys of Oligonudcotidcr. EYahlafion using E3puimmtlls Mod& -omin 1: 3 1WS-1017.1992

-

US. P

m DOCLTMENIS

4.141.043 # 9 1 Bra. I8 4.965.725 1M993 aL 5.KQ36) 3tI991 M6a 5 W 2 3 1 4n993 Drmmr ad.. 5273.632 12/1993 Swd3-n d A. 5 m z 6 1 1099s WWP u d . 5.445934 M99s ? a & J . 5 4 0 7 0 1 1 9 5 Wek a d. .7.1 119 . 5.492.806 ~1996 - a . h L 5Z,6 S S 4 4 Ql996 a st . 5 J n . a l 6n996 Holma

.

- - -

A wmpum ryscm far analyzhg n d d c acid suplcom is Tbz wmpula syslcm is uccd 10 pfmn multipk mclhodrfa dumnining uchcwn basu by analyzing L c 43516 fluonscracc inlcnsirier oT bybddizd Ducldc arid prober The rcmk of individual qaiinmn arc iuqmvcd by pr0GcSsiD.g nuddc acid equc~rrrmgdher. Compatjvc 43~18 ~ysisdmuhiplo~ObnLaprovidul& by 435% plrying r d a c n a scqucnces in one z a u and urnplc scqucnm in &aca w a dirplay dmM4356

pro-

-

43%

1

Affymetrix v. Illumina C.A. NO 04-901 JFF Trial Exhibit

DTX3

1

Case 1:04-cv-00901-JJF

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Case 1:04-cv-00901-JJF

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U.S- Patent

Aug. 18,1998

Sheet 2 of 26

SELECTION

204

-

0
SCANNING
210

I-D. OF MUTANTS. GENE SEQUENCE. ETC.

FIG. 2A

FIG. 28

Case 1:04-cv-00901-JJF

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U S dPatent

A U ~ 18,1998 .

Sheet 3 of 36

-

FLUORESCENT

Case 1:04-cv-00901-JJF

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Page 5 of 45

.

.

US. Patent

A U ~ .18,1998

t3eet 4 o 26 r

SUBTRACT

341 w+

306

(INSUFFICIENT INTENSIN)

I

312

CALL BASE 1 COMPLEMENT

/

314

-

316

4
4

CACULATE RAJl?2:3

1
C A U BASE 1 OR 2

318

COMPLEMENT

322

CACULATE RATIP3:

1
COMPLEMENT
328

324

> VALUE?

NO
ASSIGN 'X' TO BASE (INSUFFICIENT DISCRIMINATION)

FIG- 3

Case 1:04-cv-00901-JJF

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U.S. Patent

Aug. 18,1998

sheet 5 of26

SUBTRACT BACKGROUND

406

REFERENCE PASS
T

I

I

CALCULATE REFERENCE BASE RATIOS

SUFflCIENT

412

" SAMPLE PASS
I

I
I

I

CALCULATE SAMPLE BASE RATIOS

416

Q +
AND SAMPLE FAIL?

YES BASE = N CODE = 9 .

FIG. 4A - I

Case 1:04-cv-00901-JJF

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F G 5B-I I.

Case 1:04-cv-00901-JJF

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.

. I

pJ
'd

C

.
.

.

5

%
630

'10
532

g

5

P
INTENSITIES
NIL + NIR ..-N-L + N-R N-L N-R

2.16 1 3 8 1.15 1,39 1,41 1.39 1.31 1.95 1.52 1.20 1.30 1.60 1.71 1.78 1.50 1.45 1.63 2.50 1.45 2.18 2.04 2.05 1.61 2.77 2.04 1.71 1.80 2.18 2.03 2.22 1,88 1.85 0.22 -0.48 0,lO 0.02 0.03 m0.36 0.64}0.06 0.21 .0.10 0.10 0.02 0.17)~0.11~0.12
,-0.28,-0.73 0.21, 0.03r0,02;0.0Q 0.54 10.43 L0.32,0.10 0.30,O.lO 0.07 -0.2710,06 0.43 10.32 .0.10 10.30 0.06Io.ie

8
G
%

S:

I

FIG.58-2

Case 1:04-cv-00901-JJF

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U.S. Patent

A U ~ 18,1998 .

Sheet 13 or 26

Case 1:04-cv-00901-JJF

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US. Patent

A U ~ 18,1998 .

sheet 14 of 26

Case 1:04-cv-00901-JJF

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.

U.S. Patent

, ~ u g1%1998 .

Sheet 15 of 26

Case 1:04-cv-00901-JJF

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U.S- Patent

A U ~18,1998 .

Seet

1 of 26 6

5,795,716

Case 1:04-cv-00901-JJF

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..

US. Patent

A U ~18,1998 .

sheet 17OT 26

5,795,716

Case 1:04-cv-00901-JJF

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US. Patent

A U ~ 18,1998 .

sect 18 of 2 6

SUBTRACT BACKGROUND
1

604

REFERENCE SUFFICIENT INTUJSrn?

REFERENCE FAIL

612

CALCULATE REFERENCE STATSTICS
1

64 1

618

+4
SAMPLE

SUFFICIENT
INTENSIN?
YES

SAMPLE FAIL

SAMPLE PASS

BASEi BASE,

+ BASE2+ BASE3 + BASE4

I

-

EITHER

REFERENCE OR

FIG. 6-1

Case 1:04-cv-00901-JJF
. . .

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.

-

.

. .

U.S. Patent

A U ~ .18,1998

Sheet 19 OT 2 6

626

CALCULATE RATlO COMPARISONS
I

I

ONLY YVT IS GREATER?

mno

RAJlOS ARE

636

-4

BASE = X

I

Case 1:04-cv-00901-JJF

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US. Patent

,

A U ~ .18,1998

Sheet 20

o 26 f

, , , , /

LABEL REFERENCE

I*
706

1

COMBINE REFERENCE AND SAMPLE SEQUENCES

F

FRAGMENT SEQUENCES

k

708

HYBRIDIZE SEQUENCES

+G

SEQUENCES

i'12

REFERENCE SEWENCE

SAMPLE SEQUENCE

EXXIACT DATA FOR

FIG. 7

Case 1:04-cv-00901-JJF

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US. Patent

Aug. 18,1998

Sheet 21 of 26

IB
D&le sereclionb)

Edit

I

L & C Seplarres (o ReleR e m Links Fmm Sequcnun R e m L M s Rom Ralsrsncer show AII Links

806

81D

FIG. 8

Case 1:04-cv-00901-JJF

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90

91

FIG. 9

Case 1:04-cv-00901-JJF

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U.S. Patent

AUK.18,1998

Sbeet 23 of 26

no.

FIG. 10

.

-

IAFPOOOOOI55

Case 1:04-cv-00901-JJF

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FIG. I I

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U.S. Patent

Aug. 18,1998

Sheet 25 of 26

"I ' ,
9D

, ,

I
1208

FIG. 12

Case 1:04-cv-00901-JJF

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tcga~ataatctatgtcctcgtcta~tatgtcataatcttctttacttaaacggtccttttacctttggtttttactatcCONSENSUS 90 100 110 120 130 140 150 160 rm mgagata~tatgtcctcgtcYactatgtna~~~ltl~aaacggtcct

tcgagataatctatgtcctcgtctactatgtcataatochnnatactoaaacggtccttctacctttggtttttactatc tcgagataatctatgtcctcgtctactatgtcataatcttctttacyaaaacggtcctxctacctttggtttttactatc

t c n r g a t a a t c t a t g t c c t c g t c t a c t a t ~ t c a t a a t c c ~ c ~ n c t o a a a c & t c c t y c c ~ y k g g t t n y t a c ~~ O S ~ R E A - M E N T atc

"--

5
Q,

ccccttaacctccaaaatagtttcattctgtca tgctagtctatggacatctttagacacctgtatttcgatatccatgt CONSENSUS 170 180 190 200 210 220 230 240
---

rn

ccmct t a a c c t c c a a a a t a g t t t c a t t c t p t c a t a c t a ~ t ~ t

aawcycaacctCCaaaatarrPnrrPrmtct~a~~lanot~amm~na~acacctatat~tatnca~at
ccccttaacctccaaaatagtttcattctgncat~~nnetcta~a~~tta~acacct~tatttcg~t~tc~&~t cmccttaacc t c c a a a a t a g t t t c a t t c t g t ~ ~ t a ~ t a g t c t a t g a u t a ~ c t t t a g a c a c c t g t a t t t c g a t a t c c a t g k

5 2

bi

ccmcttaacctccaaaatagtttcattctgtcatacragtctatgagtauctttagacacctgtatttcgatatccatg~

FIG. 13

Case 1:04-cv-00901-JJF

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5,795,716

1
COMPUTER-AIDED V I S U ~ O AND N ANALYSIS SYSXM FOR SEQUENCE ]EVALUATION

2
AecordiDg to onc rrpm d tbe mvmtiw. a
s y = i a o b n s c d t o i ~ a n ~ ~ b a ~ e n u d d c w d aqby the steps d :
5

-

RIGHIS Non(IE

bpuaiug mnl6plc probe intmsitiu. cad^ of lhc.p& intcnsilia being ssxchlcd witb a pmbc;
m e = = 4wkrc o ~ g ~ofm u lpmbc e ~ * i n l - ~ is k s - & e each c the t i p l i o w ~ ~ h subsa~-

Porriwrofmc~crialin~ir~on.r~e**c Of a mh mL 92ER8m

N a i o d hutinncr o Hcatlh f

m

COmGm N

10 o l l i n g ~ I ~ b c l m l r w r u P b u c a ~ g w m C c u ~ n d m C mulriDlc&h&u r-- r - ~ Aaadiog to ont qea5-1~s p x d h c invention. a hi* a

prokin&yt0~~a1~upobch~en~tor9

A p 6 0 ~ tbe d i r c l o ~ ~ e t k @cIIt d o a m m l d of h the unhcwn huc Acmdimg to mo!bu spaiSc of mntains m A a A which is mbjcd to m tp o t c U i 0 ~1s the w o n pobc ilnji of r sa~~@c c c q u c n a ~lrr g h ncrfo q I h e Eopytigbl oarnu hs no o b j d o n lo lk x a v a ~ c " m p p c d 80 pk iomsirics of a refscqncnce rcprcdnrtion by anponeof U ~ ~ p * e n l drmc p a b l ol A-ding u y d anocba sped6c rtped d (bc invmlon , disclosure in m y thc Sam it t p p in the Patcnt d d ~ pmk h c d i c r of a sa@c aqucna a~ cornparad to TWcmuk 0 5 c e parenl 61e or I but o t k ~ i r c slatistia a t a t pmbc inmuiris of a r c f c ~ o e ~ p u c ~ c c s rcscnu all cp orights wh a0 from d c i p l e cquimca0. hccordiagto.rmbarrpcaofthciov~n.o mabod is MICROFKE MEND= d i d o s 4 ofpmccssing rC cand smnplc nuckic add

+

MimachcAppcndiocsA lo d g 6vc (5) rotaling m Iramrs M i ~ ~ l berewith d ~ d ~

BACKGROUND OFTHE m

O

N

'Ibc pcscnt invention rrktcr m thc fidd d 0 0 u , ry Mcrc spccificaUycaUypEwnr (bc w=P*= s s yfor vk-g bidosicrl=q-well as for cvrlvatiag d a u q d u g bidogid s q n m c u

-

IS

supenas t o k d ~ cLh; variarionr t u w u i m c t bythcsnpsck providing a plunlity of nuddc add probes khI(d a c n a ouddc xid q u m a wi(h r firs! the
labcling UK sample n u d d e add sqwnoc with a = a n d nu&cG and hyK&ing Wd nod sarrrp]. ou* Stque(hC Aaaadbn to vd aralhu a m d d the invention 1 campis u r d rm d v e analydr sod mnltiplc rcpKDm b & skF of:
~

& .

Dnica f b g ad m n y s of matuials on a suhrmrc arc LaDPm Fo cranrp)~. R 3 awlintion W09U1058R b c u p a a ~ c d bu& by rcfa== for al pnpoxs.dcrPibu &qua l for sequencing ff chccfing nuclcic &ds and otba ma(crLLr ArraysforpCaouninglbcKquationsmy befamutin -diw ro mdbodr *t fcr m e PP

*

=

GSDmSDm

-

~ y i o g r u ~ o ~ I c T u c ~ 0 ~ ~ ~ t ~ ~ ~ m a 6 r a ~ o n a aisplay dcvb.-hnd

n h g : & n i q u u W a r c d inUS PAL 1143,851 a d No. US. phi a&icalion S u No. OW249.188 betb incapnlcd badD by rcfwcna f all purpma u * c m g one aspca of the lcchniqm b Ih5dn. nu may of nudcic dd p b s is f a known l d o n r @a chip u ~ b r m f t A f l u ~ n r c n l l y a . labdcd nudcic acid i tbEo bwgbt i r ~ m . owith tbc 4s ~ a ~ d a s c ~ n u g ~ ~ ~ ~ ~ . ~ ~ i I e i n d lorarimr whuc Ibc labdcd nudcic atids bound to rbc chip Based u p h c hctitics of h c probes at hcrc l d o n s . bccrrmrr possible w enraa infrrmation such the monoma scqucna of DNA a FSA Such systcms have bun

dbplaylllg =' 1 on said display dcvia;

-

-urnDc in -ad

=

-

ofrirmlly EOegthc SUIDA h d x r mdunanding of b c na~m adv+omgo of aod the invcntiom bacin may be n&xd by refmace t me o of & &don & a& .
drirviDgr

RG-lh is an illwnfioo d (he sohwarc f a Ihc w d ~tofoosfaanmplrauaysofDN~!btmytc~lwd spacm;HG2BShrsmlcrthcglobalayollldrchip
~os~awJdaca~~rckvaut~,cysicfiho~.mC fDnncdioU~wdsyslcm;adFIG,2CPush.Lcrcoogcnc (rdcvanl to ccrclin - . ) W. snd d b u me &ding of@ w &ips: gcnaic E h a r a u a i s t i a ss FIG. 3iomoea high lcvcl Boa of thc Ltcn Jy ratio t
Lnp~~cdmmpltusyQsyaa~candodmcmodrncaiaito and p m UIC v& amount of i d FIG. 4A illume higb lcvcl E m of o m impkmm. tion now u x d and madc available by Lhac pioncalng talion d the r d mcmod a d F G 4B shows an I. ~ o l o g i u a a d y s i s tablc fw uu wilb the r d a a ~ mabod: c
cvahmr an*

, -

i c a BRIEF DES(RIPIVIN OF THE DRAWINGS r i n g m S FIG. 1 illurOa~u an o v d syncm for fmming and &yzing mays o bidogiol mavripL such as DNA a f

.P53

F%.i~~ill~(hcbighkvd~wd.oothaimplcm m b t i o ~ tbc r r f a c ~ s c of mdod: W. sbows a dam 58 bblc f uw witb k r d - a a mdhod: FIG SC sbowr r and AD impend rmnpurcr-s&,d fa graph tbc kmmnhd saroplc haw inlmdh~ minnr Lbc dcromining Ibc scquma of mud& ad& is dkcioKdTbc solmalucd d c a b e iu&cs; a a and RG. 5D shows w m p u system provib. ammg ornu rbilrgs. +vcd mxtbcdS of a d y z b g fluorcsom imagc m a of a cbip 6S cthr lgaphr of d m in the dXa ublc: FIG. 6 illusLntu thc high lcvd l o w of the statistid mntliniog h y b d d i d nudcic acid pmba in adcr to d l hws in sample nudcic acid scqucoccr mc(hod.

SUMMARY OF-IHE INVmlYoN

-

ao

of

Case 1:04-cv-00901-JJF

Document 444-3

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Page 24 of 45

5,795.716

3
RG 7 il l lhc pDofiDg pocusbg of a r f eand sampk d

4
I06 may hdudc the bardorarc
to m d ~ n r s u

maslcrnOandmd~caavs~y~~mpmr~=d m G 8 ~ t c s ~ ~ x r c m d m c ~ " P sp 1 ~ ~ ~ c e a r r y c o l a y ~ - - ~ o n ~ c - l n Q w o m u m r * o r C D ~ ~ a d ~ n , , f mhSLiar.cf6dcnl~u-ArPritbmc-f-i.

c &d wpm=;

mulrip)c uL: - (

sFIG.L~~!mayor~ymbcloca~amC

~p~silrlm~hsboum~ogcmaf~~ed~urrnti~.n F l G 1 ' I b s ~ c m% gcncrra i 1 UOM o~~~sucbucbromc-on-&5SmarkrrfM ,j.bffrbrisllioOf*rny& dcaed 10 'Ibc mah 1le.a~ wcll rs wlcaedMdon&g to ~ 1 1 i u u a ; r l u m c ~ s i y r a I i o f h ~ d C~ i g ~ d t b C c h i p s f i ~ m s y ~ l K l . a . . r e i n a ~ Cd S crlliog a m m i m in solmions with ruping coocaoltiolrr; s y w 112Spbn-3 system 1l2 iedvdcs tbc
moMdowfaassclmcd bau; RG1villuslra~d*~y~-o*fff
f 1 0 . 1 2 ~ s m L % t b c R Z ~ c n ~ h# n d a m e m d ~ u w d l o ~ ~ . g s o f ~ ~ a ~ w n b L I + I V b Q ~ ( h S i D I C n s i c y N i o ~ o dp ~ l y m m o n a ~ a c b i p W F u ~ c ~ h o

M.t mmhey

cll)cdUcnnawbmcmd
~ ~ ~ ~ ~

~

u rizcrU2lododuabgbt-ll6a~dacb~al~cdl I l t o a w h p b ~ ~ la c h i tp ~ 4 ~ ~g l w c fh ~ 4 @ n k ~

~

~

~

p

gr~narimfoorpotmcntwmpla~dfavpo~tmcnS ~plctd~-hbeliebt-dmcrobrtrau~ad thchuometnorllalrdrlivcm~DcboaI.pp~ nmph rima f dcpmtccrion o &mod ~ g i o m Ibc chip a f of

D

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O

N O F EMBODIhmn

T

R

E

~l o S c ) c c l o d ~ m g ~ ~ ~ m m D g b ~ d 118 fa c q k g lo dqrotmad ~cgioprs w& ar f a a
~dmha~onrAIlcpnfionsucpdaably W c d by m appqxiady pgmmncd -a 19 1. which may a nuy nc4 be Ibc pmc m q n u z as the

CoNrmms
LGmd
IL bunsily Ralh Mcrhod

u~s)dinmrct~ladmnlrmaLiqk
nKsubslrat6fahddby~ssystwXl2arc
opb'ocrlly diudhlo smaUu ciaips and spored to mpktd mupar'Ihc nuy a may not be w m p l m to onc a mm d h e moLosLs o tbc mbshk liic m rcaporrmmnlrcdwimaWnrhasrtluorrsccinkbcl (imdicrlcdbyams9criskhRG 1 ) a n d p h c e d h ~ g ryxcml2O.San~g~~again~lmdcrlbc direction d an a ~ t c t y digital mqmlcr

m ~ r f a e n cMclhod c

N Surisrical MtlhDd . V Poolin8 Rcc&g . VL Compu;rtivc Ao.lysi
a====P's MIL e d i c c c

a

1G s n d

-

T b c p u c n l i n ~ o n p r o v i d a m c & o & d ~1 2 2 ~ ~ m y ~ m a y ~ b C l b t ~ c ~ t c r a s ~ e bybridihon intady fib faa &ip mnbirhg bybidizcd 3s -s u used i n sycdbck rmrt makbg. a d mksk nudch: dd p m . In a ~ h t i v ~ the &sip. 7bc UI in&& r daraion devia 124 Sa qmacnl flamxrna d u firm a biologid auay. bll l a such a a dd miaorcogc m CCD ( M e a a p l c d s B e liks may also lqxucm ozha &fa mcb u n & a u i v e Ma) IhaI is used to ddm the imriotrr whcrs kbW r u q d o r (=)bas band to mc snhsunk Tbe ompl( d in-icy dam FW puqwscs o d t i w mc p a f s l invcna'wbdetoi.sbdDg~daomnpl~sysrcm -oUIkuimage6lMs)s)WH&iokcucd a n-ob, b W a l 1 c a p r o r . the Ilwmanec -5 'bad r d g n s s c h i p m i r l c r y n m tkpobu outhernip. ~ such u lab& tbc nuddc Kids. and scats Ihc hybridized mekk w o n cmob a ochs rcbrcd ralllgc) as r foodicm o parition on t h mbstntc S i f ~ sdd p r k Such a syami s N l y dcsmkd in US.p l a ae 0081119.188 wbim has l m i n c u p a hi@ photon munLc will bc o k m d what b c labclcd W D Su. RlCdbyrclum4Icxallp~gp0~~H0w~~cx.(bCp-tzznIw = p m b U b O U D d m o n s u t r o a g t y t o t b c a n a p d ~ 1s r invention may be used s c p l ~ c l y from Ihc o v d s y N m and &a du mono!& ssqncna o Ihc polymcn on the f fa-g daU gcnaalcd by such ryzrcmr. Nbsa;acirbKlrVD as a fimdoll dposilion. itbccomu FIG. 1iUmUafa a mmpumkd system lor forming and m k in d d a m k fhc sequcmu(s) d -51 or rhc s u h a l e b t arc c o m p b t m y lo the ~ t a -2ins arrays o bidogiul mswials such as RNA a f
DNAA~mpulplMis~todam-dbio-rn

Ibcbnanc6lcWispr~as~lor.r~sir

a + c o + ~ a w ~ s u r b u a n I B ~ ~ ~
cqwJmkmcbdingappropdatemcmor),aodr~.'Ibe

m a J b s w & d r ~ ~ o f A m p u ~ & i ~ ~
ioa~dunbodimcDltkb+ysissyscmhtnwdtm

~ m p u v r s y ~ l 0 ) ~ ~ k ~ ~ 1 1 s m u s SundW an ag i m a q S v a b L ' I h c ~ ~ t l iWcrditm ~ r i b t l1 &andaistics or8 gcmc d i a e m dkr inpas rcgasdl p v & a various mcrhodr of .nalyring-lhcchip &sign fitr ing me dairad I any. ~pliwdl~. mmirmgc ~ I U poviding 0.1prr m.n c putsryslcmrmyobldDinfcuuutiw~gaspai6c ~ t ~ ~ ~ m y h m b a

b

e

~

u

,

~

~

~

pnaicwqanaofimpcahamanntcrrulcuinlcrnd r n ~ i n a ~ 1 a s ~ a s D N A f f R N . 4 h b s s c 102 such as GcnBank The output of Q c w e 60 FlG W pwidcr a simpIi6cd iUnstration of che w d s y a a n ~ ~ k r e r d ~ h i p d c d g D ~ m ~ 1 0 4s om c r a r c ~ ~ l v ~ w ~ q J a a t i o n o b o n e r m ~ ~ t o f & tta form o. fa f c rkM ths imtwtia hr sbawe in FIG 2A the Nnem 6m idcnu d t ~in PCT i appliarioD W O d U ~ 0 0 9 ~ & d compna ur the gm* squtncc(s) w s I w& k of c a L ma. intacninapaiPlluadysismntp~2'lbexgucoocrofof nc design Hcs arc provided lo a s y s m 106 lbsl 65 inlnu( may. f pramplr be nmrml u mutanr palimr of bEhip f a &rig= rhe litbogrnphk masks use4 in the fabidion d a genr germ that idmrify h d y . a provide f e m Y S of J D O such a DNA Tbc system or pOCCSS ~ s ~ h f d o p ScqucDcs reltctiOn may bcptLWidedVia~rd

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 25 of 45

inpllofrcxl61crormaybcha~s0~tlas~u GcnBant Al acp 284 &e sysvm d m l c s Ibc gmc lo dcunnbc a assist 1hc I in ddnmining WIG& pmbcr M rvouldbcdainblconIbcchipaodprcnrjduan~1C %vou-~nth~chiofcomc~Addfl~cmbcrta 5

. TABLE I s o ~ u c d
4x30p--ma~

amogcmcnt on rhc mq,
~U~

pamiu %suing- oT gcnaic d l h b t h cf cdge dfcm care o f

In the ~ISI b m r the lop row of Ibe probCS ( b g -chip" a
mth ~ n c ~ b C I ~ l l C . t h d ~ h ~ f c u ~ ~ d - ~ + )

spmairarldthe~

s b d d b i ~ d th l q c l DNA x q u c e e Howrrrr. il the to ~ G ~ i l l i B I r ~ I b c g b b l l y ~ t d a c h i p ~ ~ 4 u kmmp~xdddplcmi~slvbcrccadtuitmay~ g d ~ b a s a T t U P ~ ~ u m ~ p m c d I b c kbJtd mu- a wiU ~ a b i h d ~ m w g tome ly d i f l c ~ a t 6 h g s f r r & c ~ ~ q o a e U n i proba in Ittt c ~ l ~ m a m n d admu 3. M aamplc llIc ! l ~ ns
B o w n i n ~ M a o d s t m w s h t U m u n i ! i s w ~ ~tloeplor~Eouldbindwi&thcpobuiDcdlumn2 d ndfipk ocllr whim ave uras on Ihc chip Ibal may

d c n t o a ~ i o n ~ ~ a s u ~ t c l t u I ~ ~ U i D I l ~ r a uy l l i . a r d a m d y d n r k o l ~ c d a r c a u d r d o n &ad ~~ l a molaule o mlcprlu of imtuea Ead~ i compost9 r unil s Om~ircoenm thesrmDd~chi~"rboSe1ohdlecbc' d mulIiiglc cdh Bslmay bc pbcsd in#orasand columns L as a wild-type b k 'Ihut the mumt ss&cd one cmbodImenl a scl of h c d d olb indudes the a SXGlT rbould bisd SUOLWIV the rvildrvpc ID

irs~whidlnuynotbind~~~m"yOfmc r n n I a i n ~ o b u b n ~ . u c h d i s ~ o c&sin~2becPlscth~~~eTbuslnlbccDmd pf multi#scrcddatcdccllr&uscd6urie~tamd I h ~ ~ r a d p D b o C ~ ~ m l ~ 1 OIteD . T h t ~ )

synr&sir arc daigncd AI stcp 208 & SO^ ~ i the Wm ~ ~ l ( i kGc s;'Jfor a mask U p aud byan infamalion to mak rhe DNA or E;or uamplr if b e wiI&typc probe i T-ACGT. tbc p s otbs po cbipr'hr &c I i PO8 conlld L"&&c T - A m 3'-ACa T - A m and T-ACTT m y be Ihc ' ~ t i o n ' p o b t r C c l l ~ 2 4 i r ~ b c ' b ~ "~ d n ~ f a s u b r ~ a ~ d ~ m a r t ~ c f l ~ ~ ~ d ~ ~ o rogmts-gb aflow CCILtbe synthcrklempas~otll~c the mnuh m probes (also d c d m " b m - ~ o b c ) . c thnkdconbiusaoprc&s.kbclcd~sbaJdwr ~ ~ ~ ~ ~ ~ ~ I U ~ ~ ~ O . I b i n d t o & c h i g i ~ l h i ~ - n ~ . m ~ b ~ ~ ~ ~ p ~ v i dofrohararrislls~~insnnningachip&urynm&aod ts~ ~ l b t a . b e u a d I o m u n u t & r ~ ~ ~ n d ~ ~ ~p t o w d l o a h t d c d r c ~ c p o ~ ~ ~ c ~ & c c x y . Inoncembobmcolnu-t&g.proeauraavailas s a m i n g o f ~ m i p . a n d r u ~ c s k d a I a t h m ~ i n a fiJc I . may l t r be m i l h i to &d bl ae wqrwa i d able includingscqucnccG g , b l a k t i b g . qd-fi~ing. as ~ k l w . O f c o c l r s awidcr;logc dLymtmaegiu r Lion. A slcp W a c o m p l ~ t synan -ding to UICpmmt m y bc Dscd a a d i u g O the invation bcrdq withour I invention lnilizcs tbc bycul Mumation lad Ibc B u a r ~ r rbc. deplting fromIbc smpe of the iavcntioe Fa pobcs may bc Bcd on a subsuatc in am & d o m 40 ccna i n f ~ ~ ID w h a t c thc lqtaidizcd nDddc add m o o pmbw oo b c dip b o n g fhc impa~am picas of %abmion wbuc a cornput- sysic & U to ~ a dd r d o n obtained from DNA chips m the idm6ftation O f &c pQ l d o m and comlatc &- o-cd from thc & . mmnanr~capas deIanhtiw of gdc w g u c ~ o f ~ and OpI-&g is the p ~ c u s s tiliw a d d i h n d ~ o b fa pupiprla rmpcm. of o FIG. 2C illnsrrrlc~ biding o a pdiculaUrgcl DNA tbc f sutpc~cd mmatiruu AS a simplt uampk of opl-xihg. 4s lo an m y of DNA pmbcr If4. AS sbwa ia t i sin@= hs suppose tbc wild-type mgct scqucncc is S-ACGT example. ihc Idlowingp h arc formcd h tht l ~ (ody q A m - 3 ' sod It is svrpedcd Uat a muyrm w q u a r r brr a Doc pr& i S ~ O W D IbC wad-tnw pr0bC): s fCl p ~ d Tck t m I a r i o ~ &c mddhcd base position at

zzz mn*&

a

D

O

~

~

O

D

C

&

~pmc~crthaIkchipwillkrynthcdzrdarima
fjlir~g stmtcgy. menring rbarpoh d f a u mommm x c usd and U I the monoU i pmicion 3. muting I& n tOrigbLdrhcpobcarcvuicd In opl-g. cnn pobcs x c l d for pcb suspcacd a

sr

Y-AGMm

z z
AM'IQX

~ ' l h c ~ r m ~ ~ t k d a r X t h c m o w j c a b a ~ t ir a wil&ypc k c Tlu followingshows Ihpx&sIha may ss be generated IQ this c x ~ p l c : & a h o w n 6 t s a o f p o b s M c r b y onlywebaremmC

-

T & L ~I
*--P-=.n
4x3

p ~ ~ d a ~ O d ~ ) ~ ~ i d F . b I y o f ~ ~ . f IbaIposaion in mc Dudtic acid squmcc.
60

o#-miq

~ a n u m e y d P ) a b c l o d ( ~ ~ ~ t p B d

with the scauuux S-TCrrGCAb cxposcd ID the m Y it is a .

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 26 of 45

5,795,7 16
7

8

urcb probe rbal i d i f f c ~ t s from & o ~ b u probu. %trb'ons" f u the unknoan AIUloogh of ~ddiriody.mcjmagc&d&a'bWccllwhCEb %Iah"pobcsa-ykwWkaJb ~ ~ k ~ S C d a S I b C B ~ b ~ t y ~ f I k b a & & ~ p u~ d . s u c h ~ l a d . m p o k r a r c ~ 0 ~ y r y l u h C r W bc By analyzing mC five d m a a o c iwasilia usodatcd k d f qnaliy conkol md drsign w w . a w i U ~ a q ~ ~ & ~ b a u - ~ . l t b c f ~ ~ ~ ~ sp a ~ai b c n ~ o f~ k b e b a s c i i r p r J e n M J d d a dl c ~ t xqucnuiafmnadonfromglCb~ubgthCmdhodrd by f l i &= n -4 thc b v a l c m disdmcd krein u p m f o ~ ~ d L c ~ p o P I a a d m + ~ c c l l =pr===-w-'==bYrrdgnin~~'== ~ ~ & ~ t i o n ~ ~ , ~ ~ c b y m c m w i o l l the SOaWing cDdar b r s r a m u m i o n ~ s i n ~ p r i l l b c r d ~ m u ~ I0 a u c i u ~ y r d m c m u ~ b r e Asasimplc~cofQintcmtcm~Rliomabodsup Orap u pon.~al~cst(IPgd)kammv~pcoe~ A A W IIJC scqucocc A S -T (SEP W

C G

:
w
Y S

C G

N & l ) . S ~ ~ c h r t a a m p I c s ~ i r ~
a & ~ ~ ( d b ~ ~ C m b = ~ ~ ~ ~ t h ~ ~ c d b z K

R

2 c
AoG AUno) C u T o CmG

g&& ",
OH-)
pY m 5 wm i .

podtMa~-ga-ofpoasmay~=@=+" on the tbc mmplc~m=mt~y o n pro& synd
l o d d a ! t r ~ m i n m C ~ x p u ~ ~ ~ t r t B e ~ lo ~ 0 n p O S i l i D ~ 1 o . y b e ~ f ~

*

X

hC.G.uTIII)

.. .
d
~

it m w l d bindmosl sbongly wirh the targd soqucnc~

ARcr(bc~e~e~airtbsldhpkidi2cd~Ihc chip. d scacmcb suppose Ih~fdloaringdnacrancci~tcp
~ P r r r r ~

~ o s d hc l codarn lo t b ~ ~ A C R -&L 3'-Tm45 '3-TCK48 B w v c r . ccodc N has bccD Icdcfiad a d eodc X hu kco ddcd I blcnrity R n h Mdhod L 7hc i n u s i r y ratio mabod is a memod ofaUiugb z a in a -s 1 nudcic &d rcqucmx='Ibc intensity nlio mdhod y kmaaaaanatsarbco ~ i s g o a d ~ b c r r bv ~ ~ ~ n c r I d c ~ ~ h r d ~ d2.'Ibepbolonm~ntsinlhecxmpLrbcr&ycrcpc&c fIucxrsmra inonsilics d b m m&hu and bytrid saAative (ad d d t ) and povidcd far illumrtioD aa mismslmcrlf mtxisiuufkieotdkuimbab.hchucP dry& mdhod assign%corrupoedingambiguity Ecdr w , p ~ I n p n a i c C t b e a d ~ a u ~ a v i U ~ a dqcndkg oa k apcrimcnl pvanrclen and 0 c Ihc UnkQVwo hpa. scanam ulilizcd [email protected] invllrityratia mcmod will bc d w 5 i Although cacb duoruoc~rr hlcnsily k from a pIok I I ~ C as being u E U, idcntify 0n.z &own sd k c in a sampk nucldc &d sququurcc In partia. t mdmd is used ID p r o k may k cbarMaizcd by heir unique mutation base k k idcorify many or all me b c s in a nuckic s d d SC~IICDD. r3 so 'be baws may k aid W b v c l faUDaiDg ietcndtiu

~

i

D

~

i

~

~IU*
Ad3
C+8

to

rrgioa o at scqucnce m d Ibc p d k mulation The f u "hi~tation" probes wilI be I same as l wild-rype pok h h

b r c will be i&oliScd by evaluahn a rp f ~ U U ~ ~ W ' P I ~ d * r n ir a loearion wboc a kklcdrcrrpa should n u b lo thc mip i d sinw no pro& 3 ~cser$. F r - c s a 1 nrppasc a DNA s q u c n u of inlaen or mgd wqnsna conuins Ihe wqworr S-AGMCCrGC-3' with a ponibk mubtiw ZI me hw suppmc 5-mpmba re lo b synlhaizul fm tbr mgct e A npcscmalivc wild-typc probe o S-1TGGA b axqdcmrnllry to tc f b

Tbc &own
~OUI

au-

Cb32

m

" ? b u r L u s c A ~ b crotkiatr.ldlydmcrmaPiompmbco f ~ . dcsuibdasbavingam~rasity
whi'l!
5J

T+K

m-tb (hc muralrw ~.uchmulolion~i.rmriryirrcdodby~ ba&grwnd a "blank" a l l h d t y . TMs is dooc a fds lowc
A-343-103

aaz4frradiBcMIbavrrbclbhddtim~foUoM: T - T ~3A ~ ~ C G A '- ~ T-ITGGAmd Y - ~ G A H mc flwrcrccnllymarLed sample stqis uporcdlo chc b c fOW ~ u t i o a probs Ihc in* ZbOUld bC 8)

C~S-M
G+3Z-2=30

h i g h u t f o r m e p o b c ~ t ~ ~ s l m q $ y l o m c m~mshwiolcnsitks~cs~cdindruro&.gc+daof ~ c wgucoa.Thcrd48IbcpbcS'TITGA*W~ i~trosiry.'Zbc~vebrrawddbCk~Ioll~ h i g h s intclrriry. rbc unknown baa i ihc sample will . A-t43 g C ~ ( n lk~d c d an A wtafh W S c l pobet M l k mmp!ulwJrayt thc sample s c q o c n a o 6, -0 3 7410 7bc mutation a n idcnrieal~o wild* the proba

T412-z10

accplUhy&aw~o~ofbtfoarACG.orT

+

C-36

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 27 of 45

duwcrccmc immsitia d hybsid malchcr and bybid mi* 7lu scwnd higbta inlcndty bascis thcn a m p a d lo Uc malcbcr. Hoarnu. if tbc b s e carrrpon@ to a pmd chird hiehut bar'lbcntio o f ~ G4 mratio of AG is is . b y ~ gives a bwcr intensity bus smismaicb ( r g - as a d rabies men w h t the ratio m 0 6 2 1 1 ~hc o rcsnlt d adybidb6on). iDocsrrdidcotifietion d g r a m than tbc ratio -06 (4>12). tbc unlrmm tusc k called by tbc mw-on probu co&g tbc m n c a t i ~ ~ ~ 30 bru will & For Ca w n bowcva. tbc h o d is Ahpmbu an compkma~tvy Ibc-c to 1 uqucnrr k owhrl fa c o v r i v c a s u s m a t ofbyhidimion w r y ~ c s q ~ ~ i s B U c d a s b . v i n g d t 6 c r a ~ n sodas.nindicrtardxqua~spedficpdkmspoafi* Gu T-rcsuhing i n a swple S C ~ U C D C C of Y-ATGTSA a m n p l c mc iulcnbty ruiDmemod lur b a a usd w daa-

~

~LA~GTA-~(SEQ~~NO~)~~P~K~~~~NPAC+ miac~rmbiguiticrandmiwallrMlokrcyditlacnt fcu G or To. uh ~ t o s q u c o a . d ~ d l c d ~ ~ ~ c o u p d i o n .ndxpdXvcoerr of UIC%~CUKC I bns llro ! Thcntiowtoffiu mcpcviwraunpler wascqull101.2
H ~ ~ ~ ~ ~ r . t k r ~ ~ ~ ~ f f ~ i U g C D O a U Y D S C d l o t e a d jh n u ne d ~ ~ ~ t a s r i ~ ~ ~ ~ ~ ~ - D t u i a e d b y ~ l r y i D g bj s al t poducc optimsl re& f rbc specifUc &@ dcsip aud o u byhridintion conditions. sample ppad~aand post(cgRNlw v t ) . tag& Use. dU100gb tbc ratio cutoff uscd has hybridizab R~UCDOCd Mah bcco ksamcfa cadma60 mqdxm.tbcratio d m r p JD Tbc r d mdbod k a x o c d d of calhg bascs in a IRlY dcpe~dingon Wbdba Lbc ralo mmyisom in& s-lc n d c a d d xgucna. n c refmethod UIC i g h c a m n d bigbotmhd hi&&. dc lamity barc h dcp;n& may liuk on dircrtnioatim bctarc~ah c BUOXSFIG. 3 illusimt~s high lcvd dm of k inlrm&ymtio Ibc ccorr.M r & I of bykid d m wd hybdd mimvl&tS. mdbod A! slcp 302 Uc four& inrcpdticr m a d i d by all inlcn;inl 41 and m a d m is moch I S xa5i1+c a ak++xi&ation CS subolaiq tb; b d g x u n d u The mibod cornpans Ihc probe intcnntiu of a r d ea&haccinkruiry.RcrcnMy.ilabauin~~~~ry ismmku wqucncs lo Ihc prah Intc~~ritics a @c s c q u c n r r h y of Uun ccuprrl m =o. tbc bpw in&tyi ~d q d IOa 4 sigdicdnl chngu ur.h&'al ar porsMc mulatiom. 37x1~ psilive nnmbcr to prcycnt &virion by zao cr nc&c arc two implcmenlations d tk r a a mcU10d diwbscd d a c m d u s in lmusc d-ns. A l s I c p 3 0 4 m c ~ i o l c n ~ ~ ~ ~ Y r n ~ b p b c a bcrda . YJ s i r p E ~ r h b a r c b c h c n a r s o d a l c d ~ a n ~ f r o m l w ~~ ~ . t h e r c f c m a m i b c d ~ b c d c M k d a s ~c braarirhthbi@[email protected] bdnSmsdlo~oncunholunhreiad~c~cic bigbca 3. and famh bigbcrt Q n s hh i r y d bare u d d srpcncc h pxacricz rbc IC&KUI b npa ro identify 1bb.a 2Bbrrc 3 2 b c 4% ~oraU&cbarainrnucldcnddscqnme A L r u p % t b c h i ~ s s l ~ b r a ( h a l ) i55 ~ ' I h ~ b . s c ~ b r . o U c d ~ ~ g t h c ~ c b e s 1 scc 8 it bls d r i c n t imasiity IO rall me u h 0 bru i n t c n d k d a b ~ c a mthc p t c i n b d t i a of u T h e inlcnsiIy k dab3 by -niug if &e i o h s i y of a saqle sqnaoc. RcfaaMy. Khc probe iDlcpsitics d lhe rf eand &c nmpic s c q x c u arc fimm chips bau 1is grntutbana prukmmbcd ba-cad diaacclrtofT. 'Ihs b.-lmd differanoff is r number ltvt baving Ibc sam chip wild-rypc. Howorrr Lbc r d m ~ ~ ~ I h c inknsily a base he* mun k ma fbc m squcncc may a may rn k clsdly lbc same at I b C chip bac%xanndimcmii in adcr lo d y d mc u b w n wild-typr as ii may have &) I+ Shc bares a k amcpadtion iu tbcIFfoolrrand sample b r k hc backgrwda d w basc ~ o I C D mun bc . S~~ scqucoca wiU cacb bc assncdd vah up 1 four muurion 0 grcalu Lha thc background di6ucacc a n d or tbc b w n is nol cdUabIe ~ . o d d ~ e c l l T h c ~ n b u c i n t h c Y r n p k If I inkmiry of base 1 is no( p a l a ma. me bck- 6s lcqucna is d c d by m u p r h g pobc inhsitim d h k ground diffawcc rums. h e ~ X C is mc vmple aqucwc lo prok ioknsiticr of thc d u c n a code N ( i d c n t intensity) a shown at ncp 308: s . wqucncr Fa a a pc sUppsc lbc cbip~ikl-cypco~n&s aml

-

frDm

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 28 of 45

- Icfcrewc q&a-k bdorr. b u s s Carn~numtiOn k probe 3 i d d b b k bJr the muation b m c fbc rmn*iDn prokr'i~ririawinbercTcnultorrmC"tci.xinknsit& i Ct mcir r+ve -on barer k r timplc cumpb of ODC hqdanmllliw of m e r d c ~m c m d suppmc g m c of etrwt (a@ u tr) bat lbc

-& u S-AAAACKXMA-3' (SEQ. I N e 4 ) . Sup D pasc 1 ~ C M O C x.qucocc has h e sulucna S-AAAAC CGhAAA-3' (SEQID NDS). s / i bdiflas from lbc txgct GE h
wpucrrcbytbc~CdbueTbe~~~egu~ic ~kula~dcrpoaodtopobaonacbipvith~cllaga a vpuc~cc being Lhe &b o r i l d ~ aSc ~IUUIU ~ w g u m ~ i r n n ~ t ~ ~ ~ ~ ~ ~ s a m c s ~ c a o( cf r or ~ ~ d I I I C ~ ~ u h l c ) k ~ ~ ~ c m c ~ I h c r p e d s q u m a a ~ l a a m u a t i w a t l b c ~s e c P i t h a r s n 5 c i ~ i ~ i l y t o c r l l m C u n k n o w n b u c . B bau @ l i o n h S - A A A A a W - 3 ' (SEQ ID NO:42 15 thir Uampk d C Icfacw~ & Q p b C HDWCYD. ~ I C s d l l c u m p I c s q ~ a i s d u , ~ ~ ~ ~ . p m e d ~ on~ c h sv m i y S ~ ~ i t b k ~ u h G b = b b e ~ ~ r d j p r v i t b l b c ~ ~ ~ iolcmity,whLd1is79iolhiscumpk?hisubaauw~c ~ ~ ~ ~ d r i p ~ ~ f t c r h y b r i d i u t i o . ~ ~ ~ h f ~ ~ ~ i , , ~ p basebhtmsitiu ~ r o e i , , ~ rauaUy q x c x n l Ibc m r a p l ~ siricr (not m ddat+wcrcfoumd for k~opc&vc u cpmplo "IIUIU~~~D~ pobcs. The ban iDkmirJ W
-u p ly*

.

T.nxc ->

T-IGA!=-> I 2

~ ~
T-w

9 S-~oot 80 ->

3 m IS ' - a

Allbwgb ucb nuor-oc pObcs m y bc '
m C y

daaminiagifbintcDsiyisgralu~a~ backgmund d i i f a a a culoft Tbc background yoffbanlrmborhatrpcdIia~~sitybcbrrc Sqk ~nrsol*cr mua k tbc b a c k g ~ b t m s i l y in & d 10amDdlyolltbc.nLmoam~'Ibra.dlckio~Iy SMCr* I 1 , rssod.lcdehme rclmawild* m a be gcao+ lban s t s 0 T->33 ' C 4 I bacigmund d i l f m a c d hunknown bau is not & ma SCOCT -> 8 ) rICT-> 6 rallablr If& background dif?amacufoEis 5. d ~ brulavpdg c a5iDd*cd prim l b d mMd-cypc brr s u 5 c i c ~ ~ hknhmliry from. pibe. & by mcir hqm -don but w, so W 0 * 9 ~ . P W ) k ~ d . o c d ~ ~ ~ 3 d ~ N ~ k ~ h s ~ ~ ~ f " b l c ~ s b ~o l g l e p 4 0 6 . ~ ~ s l ~ p ~ m ~ ( f d ) o ~ a i a s ~ ~

ispbadinalrrm~3d~c.nJyr~I.Mc

Acslep~meroriodrhbrrc~cndryrrrod.tcdvilb
kkm?-

m

k

A-D I2 c->9

G->m

c->lo
0->en
T->b

A-> 11

tber~~typctocarbd&cposuicbprau+ cahldrnnIio~fIhcbare~S*dtb& rcfnw~wil&trpcroasclfwiUbc1andhabontior

u

n u s base A O a c rcf-oc ~ wguarin be d c s a as to havingao i o U w j -nbc f & , lo &htcasiry of the mlstion pmbc 6 t h mc I C . U ~ ~ O I I base A. 'Ibc
brc~itiu FlG. 4A illwracr tk hi@ l e d flow o one irnplancnf lation of ~ c m f u m c m c l b o d~orilluskation puposcs. b e

wiU~~rd~wilds~pekGsoIbcfdbrviagnlioruc cahld: GA+=nl=72

w i n ~ ~ a c ~ ~ I . l l c b r r c i D I c m i r y ~ I c d

GC+7%-99 GG+IW9=1.0
G:T-&7911bSd 'Ihcsc mi01 a c placed in column. 4 thrmgb 7 of IIIC r anaIysh u k b. N slcp 418 cbc higbot hw i,,.tcDsity r r r U prim sample q n a is cbakd to scc if it has su5cim1

mabod wi0 DDW be d c s a h d pr ulliDg Ibc m b m n bast in UC sample scqucna by using tbcsc I

65

~ - * .

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 29 of 45

5,795,716
13

-

.

14

plmd in r n b 14 or &c analysis M e . The ratio C G ? intensity to can Ihe unknown basr'Ibe higbot brrc i n h (od- 6 Lo dm ll) b p b a d in colmnn 15 d tbc s i i c k d d by d c ~ g i f i h e i o ( m s i r y ipzatatlum iys r %bSfiy. thcld0'flT (durn0 7 lo d u m n) ~ ibC ba&gmtd ~XCICDC+ k IbC hicutoff , hw i m c n t i r J m a b c p a t c r & a . h ~ d ~~p~rcd1Dwhmn16dtbc~tabkTb'csc~Ii~6uc ~ cutoKuIbcunLMlwDbosbrmcdLMr 3 c;jadmed ufdlows AgainifmCbdrgrotud d i a ~ a a c c ~ i rb e. 5 M+7316.4=1.1 b+a imcndy. arhicb i s G in (his asmgl+ hn suEcbt CC-b9.9ll3=43 k~ns'ry (58>5) a r P (pas) is placed in abmo 8 d lbe , C G. %lt( M O -l b . 4 C k h w k a slep 4* n T:T+W14W.4
a 1 ~ ( f a i l ) k ~ h m l n m n 8 d 1 b clomontmowkis-by~@glbEararioaof ~ ~ ~ ~ Ac step 414 E m a t i a d l h c higbu~ s bkusiy of16c bc dor pcdd-d lo mo *cr ~ c t o e a c b d * ~ c ~ ~ A( st~439ifalI ~ ~ d o Ibcntiosofnrim (colmom Urn 16of d ~ h i g b & k h ~ t g s h r c U a t J l k l m d b c& t w & m e ) u c ~ ~ @ a ' - -dlowa& ~~

fa-

n t i o r d d k ~ - L m h h j s b c I f hdv b G SO chc f 0 n d g are dad.ledr GA+5m=6.4

15 , p

G.C+58RB=U
~c-cwa=i.o

c u r o f f . m e ~ b a r e b ~ ~ d t h c ~ o f ~ c ~ a -w D 432fddC JOC & rd~~~~~(arshow~incdumnZ)~beplsccd i ~ o ~ h 1 7 a r 1 d a c o n 6 d + o a ~ o f O ~ k ~ i n
C U I ~ I I C # I ~ R ~ ~ I ~ ~ ~ ~ ~

AI -434
1 -

U b ~ b assigadan~uityitJsodcindicalcsUu~hse

if 111-h ntios of 4~ da. fhc

lcs than a

* ; rmuning k c i s i al

F%%&s 3 llld ad QC ansly* hblc h e unbown hap U sra'gncd b e Eodc N Cm&m i~Imsity)assbown sep41BAn'N'isplaad a~ in cdurm, 17 of &e analysis tablc M t i d y . a contih a code of 9 is pkced in la of& analysis cable wbcrc r h ~ V n l i d c ~ ~ have L f&&g C cadcs b C &gS:
D I

may be any ooc d the bascs Uut has a eompluutnbry of g r Uun Ihc 1 ~ -~ ratio cut& and Icp bw tbe uppcrrrrio~du~rtstcp&.~if&ntioof ntios iff&A. C md @ arc all p I u rh.s the lowe C G latio -toif aodlccs lbpnIhe vppcrntio mtdi. the unteown buc M a l d bc mg c thc code B ( A g -nciA"). 'hr i nd I i is becanw Ibc ntios of ratios uc complcmntuy to tb&
-~VC

h K as f o U 0 ~ 5

JkA+T

.2

hbrblrkmrr-+ ~0b.b~ & ~ s i h m i -dbcd-brd

a

. . ,..

-)~.u==aQka
3
4

Wbr&U-dmmb.r

6 sq,k-&,&,.m

W i n h s J b .
M

9

Z?-yh
qLmo--U

w

-

~lhtco&~oc-areuscful -8 d y d s of chc r f e-

loriodiecDg
mcmod.

''

so Ibc unknown base would bc c l c ar being dhu C G. ald a T whid~ s i d d d by IbC m A C wtk B. 7% . i a m b i i code d d be plwxd in mlumu 17 and A c o n f i d c ~ ~ ~d 3 wouId bc placed in mlnmn 18 of& code analysis able AI sicp 438 a1 lcad w e of lbc d m o ntiac is grcsla f than tbe uppcrdo d o f f rod fbcuntnown bau is d c d as Ibe bast compbnaeq t d ~ highat n60d x % k . TbC o c mdc f u UIC base c o q J ~ n t u yo mC highest ratio of l ribs Would k phccd iu whum 17 and a cwfidcna codc of 1 w d d bc p k d in COWof the a d y s k hblc 18 AuUmc l lbc pllqmys of this uamplc lh6t the lava a s n k cutoffir 15 and Lhc - d o ~ 1 0 6 3. Agnio. Ute % mtios of ntim arc as followr

k g & c r r i s a . ~ h c d ~ 3 a o d a ' ~ i n f o l u m n ~ o f t b eG 0 I . 0 sW q r i r l a ~ c me unlaoum b m u k ~ c me code N . d ~ ~ 4 . 4 fim6dcolimdry)at~md ncpQPAn wkpbccd AS all the ntim ofrsrios a UOI less t m UIC b 40 Q m ~ l ~ . D d a m ~ ~ ~ c o d ~ 0 f 4 i s p l ~ i nccua h K . o b c n ~ k k c a l l e d t h e h r c r . o l l l p ~ ~ r y ~rm t 18 d Ibc analysis table. to rbc h i g h of &The high- ratio o n i b f tm f i ~ ~ 4 7 4 ~ C + a I y I h s l m p 1 e w q v c l l a c p o b u f au d ~ which b r conplnnenuly base G Thus. the i l CC u . bavc sumcicnr intcosiry to me nhm wrning u&own bve ir d d G whim irphcd ind m 17 md & m c ~ i r a ~ i n ~ 3 ~ . ~ ~ i e c d ~ m o - s~ od ( ~~i r p l a e t d ~ ~ u n m l s ~ ~ c ~ a c n -6s I.blc dw m h o w a basc is arrigncd fbc code N rablc. ! ~ ~ ~ b I y ) a s s b m a ! n C P 4 Z d A u ~ i s~clamplcrbowsboa,IbcunknownLauinIbcampk ~ d l D m l l m m 1 7 ~ a m n f i d c w c o d c d 2 i s ~ i n w bmud& add sequence w s mmply cdld as bx G. m a 18 of th and* lable ~ l t b o u g h m m p w h t a l i a n " pmbe m me -

m 42S t rsrioaof Ibc rdhcnenriosto tbc m c m i o r k
for
cnEb

-

. . - -

bususe tbc r s s k "itatiaa' probe had the highcrl buc I~FC ZX . do Thus brs Rtio A.h u ratio bwnx o w h cUm 7nutzaion- p b u (cdumn4 to d u m n 9) is pLccd iu calumn 13 ol Ibc FIG 5AWD;Yn I hi@ lcvel flow of lmma impb k .nd~ds table. The ntio C C ( m h 5 s d m 10) u mcohriw of fkrcfcrcnrr method. As in h e

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Case 1:04-cv-00901-JJF

Document 444-3

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5,795,716

IS
in~plunc~mio~ ' this nnlsocamparacbc pms letis of 1 s - c e t MIC pDbs o bwhia d.a - L H m m . thh impkmcDr

1 6
dcolrmldtinghf~raim.oucf~ucbbrwas Awn h t k d a n ( a b k T W S . t k f 9 6 ) ~ n t i o s w o u l d k calculaIcd i tacfr poddioll in Lhe 15 .xqocncc:

+
n

&

t

i

o

~

m

~

y

~

m

~

p

z

v

tiosiwW~~probcin~cf.rcrlto~d. s CmIb=CY(A+C+GcT). G ntbG.'(A+C&+T) ~gLnwr.vr~r~batcadlin~ As J shpk aaupk of mir implanmIaIim d tbc Tratm=~+C+Tl nl mdbod a XCSCMC~ sqnsna bu 1 Ac@Go~24LAl;lru,rPaSdbetkvildsrpcrati?.~etc uqwccofS-AAM:CCAKWXWCA-T[SEQJD nCioancgmPSyelruLMm~dcr10~ mc NO:6) and. r s~mpJcruprma bas a xpuo lo inltrmi d u BS f . pbctm fMlnrs mry v q mddy fmm (0 nhprrsidc J ~ W of 5-~~m--3 (SEQ ID N: O% W ~ C ~ S B c m v u a t b a r c b ~ d c d i n o i ~ . t h u r i r ~ d w om f a g b m

~i e~ +~ c ~ i + n

**

~

t

~

~

s

~

p

~

o

x

m

~

m Rd-.ifa=pbclm-u&~olvu~arju~fa ~ ~ ~ l r r d ~

.

~uetrld~&ipw3hQrd(fcoascquemcc ~ I D c x ~ I h r p o b c i ~ u d o w ~ kiog l chip oriWppem ~ ~ I . I i i 0 0 x c - ~s k dchc d r 'obe-~rmJizcb" ae mdhod will br dcsaikdas idmlifying Uk mutation ~ ~ ~ ~ . . msc unbmvn k l b e inkdy is dle&xl For ihsmIiw purposes. rtds bplcmambn of 4 c r - iolr;unr to-dl &= d

~~-

zyStqN& yz

i

l

~

*

c r r o a ~ b ~ u i i l b n L ~ d a u u b l e 3 n n n

i~1~.58<~~~~~28..nd~~lD~aZJ&~llbough
tbrdautabk amllimmcircdsatbna irrequbcdformir implcmcDWionhmonsdthc&mUb)c~uc podvccd by dcpsinFIGMarcsbawawiIb tbcamc rdacnu aumenL 7 % gcndon ~ d a &U table i pol s mcourg. b o a m a ..od is horn to rid W e be in u n d u s a x b ~ mdboh l l ~ c cbc 1 ba posilici~ a~ b posirioo 241 in mC r r Cand umplc scqecnocr.whk3
krbownLboldiacbcblnLabL At step 502 tbz bar in!cnsitia of the rdacncc and slnqrk wqvcllm arc adjusted by subm&g I r bckb g m n d a 'blank- cdl intmsiry Iicm each brrs inrcndty-

rrlpll;llcb-in fpora;ioloncffor;cb b a s i s IMcrably-if atme i n ~ k h ~ ~ r r e r p n l rhoanfsuedaunbk t o fit stcp 520 if cab0 tbc refor sample sequmca wa~basciDlrPsityissctcquaI~oadposjtiYc failed to ham sut6drPI immui~~. &oull ikc bra i s rmmbcr to prevcal division by zrm a ncguirr numbs. Jo asiipcd tbc fodc N ( i e h s @ ) as shown at slcp Ihc~tablc~SOIAisthcbackgmuodsll~bw
L -

lowa cast " - in tbe I& hand 061 n s tbk bISC IS ~ ~ ~ r t h C r d t ~ c ~ a n d ~ ~ c r a r i o s l l ~ d d & d ~ s u. htcnsilicr in cbs dab lablc am not &&kd by their 510 and Slit asqcdhelq. 'Ihc ht+ diabc~oa d g mmplwoll sod (bc tdd w s I ~ m u r i ~ n fmm m s r ~ o p r rb- M me datl ublr C m positioohaloiaunriryd385.?hcrcl-ail~ A~stcpSUcybpoDitimbcfi&to~c~iltbcretr i n l c p d ~ 0 1 3 E S k c h s + t d b y d U u m i ~ i l ~ ~ l e n r i r ybr x d i B c n n c c s J u t c r t b a n l a u r a o d i 6 a e n a ~ ~ a n d s ia

-

s a n d ialasity is a & 'IhnS. U h e i a c a r i r y u

r m n d y sll I h s u b m bask with me refwil& r~pcmus(bcg~la&anrbcb+ditf~~~tatrcdi
(0

u

~rbcuniwwnbaceism~Z+ll~c. If dle hre bttsuiry 8wab I d k -

wii&

l ~ p c k ~ p ~ ~ ~ ~ ~ d ~ a e i~E ~ ~ f~ ~ i s - 0 3 l2 ~ c b ~ l c s ~ b u n - Q . U r b e l ~ ~ ~ d d d L b s ~ ~ ~ ~ M d f E i l t o b r v c s u f S d c o t W ~ ~ M~d t A s t h e r c i s a h s c d a f a ~ a b o v e ~ r ) , shown m ncp 54-5. DthcrwirS It scp 308 d~ Ibcari)d-rppc m u p p o d i B u ~ d 4 a b r t c d i B ~ ~ ~ M w t h t J 0 1 ~ e r requma w d pass by having d d u d inrcoricy. d i f b u a atoU. m y be mutlIi011a fbir poshion. f U s ( c g 5 1 # d p l ~ u ~ p r r t a m a d o n i b c d p ~ d I mcrs is ncithtt a base f l b w c me uppu mba&dbucInMdtia o f t h r d ~ = ~ + c l l c c i . o r d o diSm nar a k c diKf f bdw tbe I o w a

Suppow thc SIC oppr diBanoff n 0.15 and c u M is 6 s a\ s- a m bv lhc h o l h - , . ~tiaain~5CA,cbcmmslionpodlion(lsbcled w i b a rCIcMcc 0). UY G dilTamccis 028 wbi& is a U U 0.15. UK nppa da U i-8. SimJnfy. Ibs A
cities

a c lowa di8-

w'namal&~kinincsnririnE.chposiliwio~bcrd~s~~ dilf-nncAf,thcchcatrl@onisassigacd&r. has four brlgr-d sutmac%d baw wm~irirss~ codc Q? fbe rcfumo wild* baw JS sbowa ar ~p 52% ZFX&W YriB 1'l'bc mtio of the iDItndty d mch baa lo AIdcp mIhedo of lbc higbca bsdrgrourdslbmcled tbc nun d thc iotasirria ofrhc p d M c b= [dlfour) is b u Llcnsity in the sample w Ibc bsckg~~und subtrscted

wqu-

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 31 of 45

5,795:716

17
rcfamcc wild-type base intensity is calculated. For
and a '%I&

18
d. Mac bccavw CAI l As muruion p ~ k

&e.slthcmnudonposition201ink&~raMc.h kidcnli6abkby~d~b;+n.~mutalicnpobca' h i ~ t a c l g r r m n d r o b h a d e Q ~ ~ i n ~ a m p ll e x i t i ~ l v i n b c r c f m s d W 2 ~ t h c ~ ~ t i u " d l h c i r h c ir nl (bare Q brdrgmu~a urbmaca~clc~c~oc wadrerpmivcD boa. typcbucdmdl~.is385(brscA).%tbeRtiod j As~~~dlksclcisriolnrthdsuppana 57lr385isdpr~and1~1uhrinL48usbwoin~&h g c n c o f i n l a c s r Itargct) has rhc scqucnce S-AAAACZAAAA-3' (SEQ I Nm.4). Stlpp~sc doD a tahlr m a s q n c n a ba I r raqDc~cs h 5'-AAAAclX;AAM-r A1 Np 535 lbcx ntios are m e to a ntio at a 16ghlxaingpmitio~'IhcRli0f~~a*pmitlwisstlb( S E Q W N O s ) . ~ ~ a s h d = r P g n w q ~ m a b y ~ a c d f r o m m C ~ f a t h c P ~ w ~ a c m t L F! o ~ ~ u r d u E n c d b ; l r S u p p m c h m b c r d u r a ~ ~ l r
c r a r n p l r a t I h c ~ n p o d r i o n x l i n ~ d . r a l i b l c ~~ s s u s p c d c d t o h a v c t b c s a ~ ~ a I h r ~ d c scqmcc-uaglf a aTbrscmmtica .c UIC uPdrrL'd bsrc tbc ratio at *tion 201 (which quak 1.48). I hts b c a t position lo 9-AMACGUAA-3' (SEQ ID NO:4). ST pow tbar hmuhipk czpa'hcnt~ d c m e c r q aIS f d tha~ arcatantcan k e o n h d a t l ~ dcaacd by dvr;Og

,

n i st p i s i o n 242 (amjdn cqullr 1.02) k rubmacd from ro

-

bsrc la that is not dcledrd A step SYI. n c b t a t i o pobu h a m fm tbc r c ~ m sccplcmx ~~ a may be born anly we aperimcot the dhr apin=nu position is Kdgncd h c codc of thc refaace wild-rype base a shown at q 5YL s w, may b v c ditfauu "nuldon- pokr chip wild-lyp. t i k g methods. and md like Ahbough rach-I l This wmnd iqlcmcolalion of thc rdamac M o d i s p-Cruableinsonv~ccr~i~caLriomxra~n~pinnrirybha+krhcpobcrqbeidcnfificd rok rbc ~ D b intensities m y k e by meir unique mntalkm iobsitiu d ncighbring prober 'lbc 6m + o n idmri6ed bj lb&r r c q R x l k brrcr s f0lloWS: m y nc4 havc dclaicd LbcA toG m&n in t i hs

II mc pa-

.-

Tbcadvanugcalmcrcf~~~.~~~~bthIImcOmed 3s
basc anbcQUcdcvm i o L b c ~ a o f s i g ~ l c v d s d a o a - b y b i ~ o a ar lcmg as ratios of h i t i c s arc

*
Y-~Jc-> A
'

= +
JGACT->A 3CT->C Y M C T -> G SUlCI -> T

fairlyconsirrc~lhcxpcrimcn(toupcdmcnLIn~caicc the ~nmbs misdls and unbiguilicl L dgni6cady of zc&uceh while Ibc numbs o f c m v x calk is actually 40 in& making the duma mdhod v a y usdi~Ifar idemwing u~didatc mulatiox Tbc ref-mtmod has

%-lux!c ->
I-lmC+T

3'-XZit-> G

d s a b c c n u w d t o c o m p a n f m c ~ b i l i t y d c r p c d - Thns.bzccAd~rcJiYe~y9uwccaillbcIksaibdaS having am i o t ~ w h i c carrcspwds to* hlasiy ofme h a~unUintqmsofh&~. N Slatitlical Mdhod . rs rnubtio. pobc with the muwion base A. Ibc sIalisticaI mclbod will now k d a m i as calling lbe OW^ base c Tbc Satkikd m c h d is a mclhod of ralliDa b e ~JI a ~CSYSIIO by uJmg this + . L SASQIIC nuc~cie aid q u ~ h ~c ~ i mahod ~ l~ l i 2 ~ tbc c a l in F G 6 illuI. rbc hiPb kvd Bow of tbe nstis(id UK aatidcal v & n anorr u h u to ull Ihc hucr m a o A ~ S ~ the fm-bau inlen&cs asso&cd wih mbb 6 ~ B 2 'IbCldIhc rla6stid m & e is prdmbIc what data bw multipl. upuimcorr h avaihble md i &uisfairly X the sample s q u a a aud c d of the mul~ipk k I a rdmm cxpzhAs m z sdjmtod by NbInxlin~ b & p u n d ar the mnsinmt =ass the upcrimcntslhet r d ~ c d o m p ~ s e tbc wobc ~Icositicr a sarwle seDucDa to mtistia d v & of c . " W iu~mdtv b cdl born& base ~ . R d a s b h . if ~ ~ o f r d - ~ u c o b c i n m u E k p k ~i~ a s e i ~ k a s i t y b ~ ~ 0 1 c s U u m 0 ~ ~ l u l l o m 1 h ~ ~ b km&ty b td qud lo a small pozitivc numkr to pm,d For s b p E t y . tbc statisticalm&d d bt & s d d as b d o g n w d m ~ o n c ~ b a u i o a s a u p k w c l e ss d i ~ o o b y z c m o r ~ ~ n m n t a % ic Atncp6W Uuimclldtia oftkrdnu!ad&ypcbasr acid wqucDcr b pndirr. L c h o d ir used to i b I i f y in thc &lipk c a p i m c n ~ &c&d to sac ij-they all rrr m y & . Ibc b & a in a nudcic acid reqncnn U have ~ d c ~ a intasitv t dl thc om b l r e TIIC d r o Ibs ~ n ~ w i l e l . ~ a b - v u m b o k ~ inccnriricsme tj-ifmc intensilict of a srmplc -& . to smisiss oa hobc inlcnsiliuofarefamccseq-inmuU+Icupuimmu m rdercacemSldqpzbascof.n~klpatcrlhana Garrally. thc pke btemitiu of IKompk q u m c c md I prodclamid backpuod d h c c a& The Wild-* t. thc rducocc squuwr cxpaimc~u from c+s hnving art probe. shown ~ ~ S S C I far therdcraa scqucw is r-TGGC the same chip wild-typz Howma Ihc rdwgum and Lbus the G kinIcndIyis thc arJd+pc brrc inknsitymay m m y n b e q u a t the chjp wild-* a o as il may T h e s t q s uc m d o g w s to slcps in Ihc otbu w o mcmodr b v c rmnationr 6s d a c n i A t % a tbc w p o s k jn Ihc n i a c x c a n d ample u t Ifthciulcndtydanyo~~~dm~rdamad&ypcb~f scquurtr wiU bclcsaiarcdwiIh up~ofwmulariw probes is nor g r u l a duo l t w o u n d diaacna curoff. mt b

*

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Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 32 of 45

5,795,716

.
20

1 9

d-ypc~tsfa~bhavcsut6cim!~nsir).as u n p l c s u p l e ~ t f a i l r C o h r v c ~ ~ ~ r r ~ ~ . ~ z ~ e p W O t h ~ a m p 6 0 B m C wt 3 p 6 1 6 . ~ u n r p 6 1 8 I b c ~ s s q u c n a . dr e ~ pas by hriag d d m t i& n. r ~ b y b a v i a p ~ $ c r d ~ ~ . N n e p 6 l ~ c r l ~ o n s ~ ~ ~ o n d 1 c ~ o rXm n c p ~ ~ . r c p c r f o s a d o n ~ c f ~ ~ A d
m a a e d b m Q ~ ~ s o f ~ ~ ~ ~ cs cg ci l p~ a id ~ t ~ k d ~ d t i a ~ O f l l l C ~ ~ m p l ~ ~ ~ m n p B i m M d U w ~ ~ ~ c i o k n s i t i c r . E M h d c ~ c pnrr i o s d ~ b ~ ~ d ~ ~ d u c b b ; r r c ( o r pnirrvrmbPfombxkgmtird~bssciDhDsiriu U b z s a u n d t h t b a ~ s ~ B l c m a & c d * ~w~itoncerJdrypcwaltbuiatbe& pc4bkbDcr(dlluu)mBLuYcd~gf~ntior.

~Wcbrrcr.In(hiraampk.chcGb+rcin~msiLykIhc o l t c f m i a & b a c r l i x c ~ & q ~ I h + n U b ~ ~ L h Jd-~cbchCdTbrseibldllcrbdngtheamd20 l k a c r r s c M s c P r i l d - t y p c h a i r c l l k d I b c ~ + p e n t i ~ mtb rbnr being lbrcc ' t i raliorThus. tbc f o l l d g o fd ~ T h e r u i m o ~hciu~tusiy f duchtau.btbcsam n h iosuc MhdaIsd: f Ibc inltnsiricr d lbc pmsiblc baScr ( fan) uc d dabkd. giving one wild-type ntio .ad Wkf ~rati-+C+GttT) m rnc nk o l l l d ~ k 15 C ~ + c X i c ~ A &MA+C&T) G mlio;GI(A+C+G+r)

BC UIC "&a" mtios. ~ G n r i o i s b c a o l ~ r r u i o d A C 1 ~ d T 8 t IDo s S u p p o s c t b c ~ ~ W l c r e d h ~ ~ i h r C m c - ~ c r l ~ r a c w f o u r n t i o ~ U i c J ~~it a c s a m p k s u q p * u f o n o a r b~ ucb d m c c u p u b c n r 'Zbor ii the nllmkr dxfmna: A 4 3 10 aprimcaUBntba would bc 4nr&s c=ak&lcbThcsc C+m r U i m ~ ~ y r a l ~ i n a & t o " n ~ ~ I h t Et26 Lypr;ryd . pr Ibc Moton couno m y vary wividdy from u. T-+100 cqaimcn: to U L H-cr. if& pcbc inmrllicr 10 & T C tbc mrrapondbg m l i s would be as follW2: b n & no1 vary w y bw u to bc ^. - ~ A 1ati0=31~10r5~26+100W64 r~& iotmsitia do nm ... .At step612 &ticrarcprcplredforUmxahs dwlaIcd c nriu=S01(31Ot50+2,1~~.10 f a - 6 d l b t r r f ~ ~ a p U b c n b Al SUIC~~CCPC M a , ~ 1 0 + 5 ~ 2 6 + 1 - . ~ ~ an cach
~~

C ntidCA+C+Gcm Gnw(A&+Gir) T nti=TI(A+C+Wr)

T nlipn(15X:+G+T) ~bmr;aioGistbc~~lioandratim~CdT

r c r - ~ l w ~ b r ~ o c t r a d r v i m o n c ~ Trak=l~310t50ti5+IWI kc dm Tbc .nd mDdud devian'w arc dollald frr & tbc ad-typc ntiar Ibc srcp "h I u rdumoz a Ibc m c a n ~ ~ d ~ ~ ~ n ~ . I ~ o c sf co p e o & m d ' o h c ~ ~ ' * ~ ~ - d mcom9. Rti05. r g e iO U-E-~S &gad k d N -6sntc=@) d o i i o u r for c x h o tbc hsa Uvrl h m tbc wild-typc f a ~ ~ p * 2 ~ ~ d - t y p c and uoma" -nada chc foDorviq walld bc c a c u l ~ wimtbctampkwqocacc~ca=paaiw~dap=s M a n and stmdd dcvisioa of A ntici r i o m . 7 b c M c r l -ions bdudc feurpduturhcd Mcan and srandd dcriatiom of C ratios standard deviation culo& one arsrs&w 6 t h tach baw. M ~ ~ d ~ d a n l d c ~ n d G r a ~ i o s a l l n u tberr i s 8 6bnddclcrution culoff f o r d o f thc b a s s A C G and 7 'Ibc loaliscd W d d dcvhtbn . . M W and -dud ddatjon of T ratios cuss allow Ibe U b w n bau lo be d c d wh higher i wbrx Ute man and amdard dcvidon o the G nrios arc l dsa lsown as l h c wild-ryp m the wad pcdriw taalnc e x h m& dcvLmtiw cutoff ran bc s d dad doristioa [email protected] de d dcvi, lo a d i K d valur Suppore rhc standard dcvhtioo doffs tion of the A C and T TS and standard dcviraiors uc " arc hcl a follow s also bwn mUdvdy rs the -&d and s k n d a d A sm&,d& , +h . ~.

-~

-

7

--

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 33 of 45

5,795,716
21
vbac lhc Olba bw nb dcv. rut& is Ibc standard M
a -

22
chip. h g c u a d cbc s a a gcmatcs an h a g c 61c by em f d g u&ali~ on U hjhidizcd chip and &kUlighl x
iogk8uorcasntligMthrlhauiUaL'Ibc&~g the B u r light ma be identifiedby Ihc wmclc~lp4 d
5

iondfa&cfl&-orhbb~&cabovc moparism SOIVU l k fd&g to Wu

~~

thcligbtPnrcample.mcmwrcwmccmofBp~n
is about530 n t n w N c t h a t o f r ~ ~ d dmdmmcdycis a 580 urn

~ f , e a t 6 + 4 0 ~ 3 - 4a )

w ~ i

C-rO1~myOLlW9QXbam

T-rOlWUybOOI-4)CIWU3

-Jlb=~crcatcsa!4imagcmformcdau%Icdatpa with ocb flu.cmsasf ma*a.indic~tinglhc hatiom w h a
lo the ccnrespocdinglg Lbclcd nndcir add b u d (o fbe . & c

whirb arc a l true a p w s s h % l A(ntp62Sifoaly&cwiJdrypcdodtbesampk xlpwa w x g m s u &an x k mrirrid crprrrion h unborn base it aPigDDd the code of h e d a n c e wildrypbawassbDam;Itacp6?41s

Bnscd upon a analysis d d fluvaancc in~cnsNa m and Ioati~.itbccomupossiMe~oawbf~~~mcbrr B e mwomcr scqucna o DNA ar RNA f
Pcoling p m u r h g rcduocr vminrioms u~oss individual

upcrimeas barnrc much ofthe r c ~ l wironmcnt u mmman. Al-gh pooling puusing has bon beEn~nbcd as At 632 if a ~ a more d the "oUcf nk of me c ts ~ c r c q u p l e c w u e g r c ~ ~ ~ h r i bdDgoscd~oimpmrctbcmmbiipcushgdrdcruw+ r ~ y c ~ upmsionr lhc antnown ba% is usyDed an and smqdc nodcic add s q n a c u the p m m y .Lrobe axle QL indidla thc nuknown tasc my be my me oftbe used fartwo d squurer.two ~ m p k a q u m -u~ c d tbae barn. iDdudiDg L rdr.rcnm wild- m m l i l q t ~ ~ by ~lili&g multiple &tmpdubk c utpe r s t y p r 1 n h a ~ r l b e " ~ ~ f u A ~ m d T r h~ m w

VL Compurtivc ADllydr 1 g r d a Unn their ecnqcmdimg sddid c q c u i o n Thns.thu~ownbaxprmldbe&mCc~~cmmlc 5hepcscnIinvclltioopmvidcra~ofoomparali.s o f t h c a b s e t ~ t c d b y m e r u ~ T . G ~ d A ' l b u rr . n n l ~ a a d Y i P l ~ o n d ~ ~ ~ a ( l . ' l b e thc unknown tax would be assignedthe code D (&g E-mdhod akwx k inknsiIy tatio. rclcrc~sc. d slatircial a "mot ( 3 mctbodraobew o n m u l t i p k d a r a 6 l r r i m u l ~ U d y . ~ pcrmiu dllfcrcnr cxp&cntal conditions. sample Y none of Ihc ntios a c m u d m mch m v e prrparslionr.and .oalyrir pnamclm lo be compared in &ia tcl apcrsions. thc u h w bsx is arrigncd B c t n of tb& effects a0 qthxfa a s alling. n c mdhod a h eodc X (hulX6cnl discrimination) as shown i s i q 6 6 3. m e srati& method pmvi& acnuate baa d b g % provider vdcation and cg & f u n a i o arhich ac~ rsqucnccs,as wdl as navigation and analysis bccauP it ntilizcs statistid & m fmn muk+lc r c f c ~ c s rialw r&g a lwlr cxpxkmautoall tbc unhownbar'Ibcstalk6cal mclhod FIG. 8 ihWrala U% main s a c m and d~ ass&jatcd pull has a h bcca Dstd lo iqlcmmt con6datcc utimatu and down menor frr oxpmtivc analysis and viaPlintion d &g d mixed siqucnccr. V-Pooling Racrimg u mdliplc uphuds (SEQIDN@).lk windows &own arc born an qprqwhtdy l x ~ Sun 3 Worhatioe Ibc preuntiuwtim povida p ~ l i prsssing which ~g Howevcr. ~ h rnmpxdvs mnlydr s c o * may also k ir a memod oi pcussin~ rdcrcmac lad suumlc nnddc add impl-cd oo o r+ to a p&md c=mpna.iDEhlding salvcaecr * I lo r;d- variations a individual IBM PCs and commhio. or o t l u wadnrarion c m h n c c In Ibc m s c n ~ v cmbodimm d r r s d e iarc bC;CiP t b & a ~ amp)c mudcjc rcid s q u u v a arc a mml6.A window 882is s h m having* &wn m u s for labdcd with d i f I m t flu-t matus m rpti glight r al rtn the fdkdng Iondons File 8W. Edit 806. V i 808. d i B c ~ WnVtlCDpmZ h n r a C the nuddc acids may b t T e EG&iK@I SIO. md Hdp 8lL Tbc mais d o n of !hc wiodow is dividcd into a r d u kMcd wilA &a iypa of marlcar hdndhg distinguish&lc radioaclive mshcrr cnccscqoursuu 814 i u d a s ~ I c u q n c n c s 816.'Ihc w Aflatbednnd zarnplcnuddc d d s q u c s ate .u ref- xqucorx zva i wbac bown aqucacs arc s displayed amd ir divided d o asdueru name subarcl818 lakdcd wilb diUamt color fluar~ccrd markus. the labclcd dcrma nnd smnple nvdcic dd s q u c x c ~ U c cam. am ~ m nndrcfcrrnaban submu BtO.'Ihrrrfcrc~ocmame Nbarrr b i n c d a u d p r m n c d t o g c i h c r A m ~ ~ ~ r f o r d d C d i D gL C ~ m v n w i t h t h c f i l c n a m a t b t c o m a i . t h c d a m a ~Uskbdcdprilh~&bpvidcdhUS. squcnccrTbechipwil~isidcn~cdby~6lcoamc appliotiw Sa. Na 08/195.889 md is hucby inaxpaled m with t cxlcnrion ".wW w h m t h c U i ~ t c a unit o tbc h 3 n by rcif a all rwpma chip. l k r d m a bae snbartx mntains the hw d tbc FIG f illWacs me pooling proassing of a ~ e f a c n a rcfncarrsqucnmA~pitalC823ifdisplaycdZOtkright of &c rf eq m a Ihx is tk cbp wild-r).pc fw rbe and sample nu& add squAt s(cp 782 a rcfcrcna nudcicddsquluconhm;lrhdprilh;dua&tctycs~ current analysis. W g b thc chip wild-rlpe sq cIns a P9LKmsxieAt rlcp700 r s q k d c add wqucna u associrtcd fin-cc inlcnsitiu the Mbcr duua s is mnLcd pritb a dye W upo.udhtim. cuds li@ of a s q u c u u s h u m W w h e chip wild-type may bc known o ~ m t w . v c k n g l h ~ ~ o f t h c n a r r s a m l d y c o f m ~eocsstba!bsvcnotbccn1cdontbcchip'lbcsc~uyar . nu& acid may nor b v c a d Purracmoc humilies 'lbc r d a rf rs c q u c n ~ o aampIc. mc Fr cna wqucnm ozkr #t tbc .m rvild-ry~c lasd far arc scqncolrmay kmarkcdvcithmoduniae. Als1rp706d3ckbcld r d m a xquwaamd ~ b Lbclcd a scgacnu comparisons aod m y be iu me form of rimplc c ~sq-arraDmbincdAhochkstcppoasi~g ASQI~filcr Sample scqnacc arca 816 u due sllnpc u unknown mnhwcs m the samc manna ns fm only one kbdcd wgueacc A( dcp 708 Ibe s q u m c s rue fnpcstcb 'lbc u p k c m a ! scqucna a m dirplaycd f muparismwith u the r d rcqucpm Tlac q l c xqucna m u is b p c o ( s d nndcic add sapatus ac t a byhi&izcd a z b m 6s d i v i d c d i u w a u o l p l c ~ a m c ~ b a ~ 8d s a m p k b s c 24 Fhip coMuniag probes ar & o m i scp 710. t s u b 826.73% ampk name snlnru is shown with At s p 7 U a scannu g w a b imagc 6 h tbat indicate &af mntaiD tbc the lootions h u e fhe laklcd mclcic ad& boMd t LC o wqucnfcr Thc Pmamc

add

.

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 34 of 45

5,795,716

2 3
cn6wr i n d i e I . mcmod used b
u call tbc ,

24
arqplc

FIG I@ illurpaps m@Iipki b w I @ wid^^ far F sckdul bases(SEQIDNW. SEQm NO34 tad SEQ ID d c o ~ the hlmsity ratio m & d r mcna vbcrc windows 1 . m N 3 ) Thee a . k c c htcndrp OS. m & n c * c r l h e d ~ ~ ~ ~ -q I denotes UIC ~ e D d . P Lirdcrlmcrhod(findiacrthcnni~onBcchip~Thc I O D Q r m d l H C r n ~ w ~ E P m ~ m ~ y b ~ orbnr UJC scqana h aple brw SULUXS ~ B I & S mc b a d me sample I wima ma-

iff

~ c ~ ' l b c h r u d U x e ~ c f u l ~ ~ p ~ bs e~ i ~ rdi m r m ~ ~ k d m C i ~ d - C I I i i h . s ~ a a~c

.

U ~ ~ h e ~ y s d S m U ~ w h i d ~ f u c h c mppr u tb e i a ~ - t y * u i b d J ~ r h i p ~ ) ~ a ~ I c o .afaimmOeIWaC~ scguc~~~dorvsla=JieDcd~a-gnIubm W m ~ W J s o c o ~ n s r ~ p ~ ~ I 6 2 & W la*I m C b ~ sboa wkdcd W*podcion ia crb d UIC avlcarabucaritban~dcvioci.~~-a ~ c q u e ~ ~ ~ ~ ~ 1 6 2 i 0 ~ . I b > ~ e r t ~ g y L T b o l , a n p ~ c b a s c s u b u u ~ b . s c b c c o m e s ~ g b ~ c d r a d objed r t k b t u r p i ~ i ~ g r b c s W t d b a r t r ll ~tbnavs kbce wnmining tbc base is displayed in tbc of RdmiDg a @ I FJO K is pplll down mcrm H @ O i @ nscr is abk I c o v UIC e q a r n drcfaC110S o s 118. esagc pad. 'Ibe b d s position m me nudeic acid ~ q u c e a k . L t o ~ y c d Q c b c ~ c ~ and~.mnl~~.~l~f~trccmp&oll~~~dbklom~ uar. iudoding fhc f0lIwi.g sac+. s q u c ~ m rhc chig to In plrll b s m u m c M tbc ma is able to load 6Ja t ~ t d s a p u ~ c r t b ~ b r v c ~ t i I t d ; U d sd r m i l ~ ~ ~ w q u - ~ W r ~ raclip-~acisachiparilb(ypc~~widJuch sapmar.samplcIupwmlo.oYLnladtcfr ~ r r l f ~ q u c ~ p . ' r h c & i p a r J d r J p ~ r ~ w id l d a r r a o c ~ ~ ~ ~ t o ~ c e h i p a t J d - t y p c ~ m hc6m aq-mdcdirrcld~s~pwax ~~cumplc..f(rr~bcwhtlinLedrd=et h s c h i p ~ r r f c ~ a ~ c 1 l o c ~ c r 1 1 4 ' I b t u s umplcseqwaathcnf~~.wmpa=mCb~~incbe uda isJsodaklobd6)crof~awwcldcacidwqumraas l i D t c d s e q - B r r a i n c b c s ~ c e p u ~ t h d a r t w d m xqucnm f d u a a n pnpoAS bcfditfncDt Fmm(bcdacacragucaawillrkcDkindlcalcd

d -

~ ~ ~ C - ~ ~ J & Z C D R ~ U C D C S ~ ~ W I I Y Jn ML C b~ t ~ y ~ ~ o ~ ~ diBUCD(: C W ID laMbP O L F *=iJy & Additiwdly, in this 0

Ihc nur k ablc t save scguc~~er uc sclcacd on b e o tbal

lncr k IbL t6 h e idcDtify s e e - h ~ p r o j ~ B e ~ c a a t t c ~ ~ i n r )( a Is ~~ ~ u c L Y ~ s . A ~ C T ~ U L ~ P ~ Q ~ ~ ~ ~ ~ ~ ; ~ ~ ~ ~ ~ z ~

* = ( r g , ~ ~ * b a

e~ the

' I b c p o j r n f J c ~ o o ~ s m y ~ c o f L e s c ~~ 9 u m c u . * ~ ~ * ~ c ~ a * t d o c r D o ( a s whae s c g ~ & m x t -1 forCO- -ro.pllrpow -~~arJ~rypcsu]-~-~~~~~ScqucDces tn k wc4 bofb rdaena and sample, JIC ~~~ref~C+d~cewgud-k~~~a-k by sc1-g m c salua in* dcpicc to mc nsa -my tbu mar

moe~ampIcrcpueaar.tbcsam@e scqumeu
P

08 &rJfJh. ~ I C labckd JS YE-. " - and "?on B. badrgrouod sub~apcdin.rmsitia d pobcs bving the i o d i d c d substimtion brsc. In we -eL~-bavr~sboarsinrod7hcpr~&

l n 906. it

( d o ) . ~ I usa is I& t o x inlmriry g+ C o r l visually o mmpart the i n ~ m h i a cacb ofthc p s b c of ~ s l calls.

~~a~MC~~z~~;iao~;J~
XqlKDOC by

-group of

a c o e w n

m -

dcviC+ialkrd-orsar&pk~amc~ca~. 1 pl . ll fhsuur is

a l h g r omariom h solmiom witb +g comxmab (SEQ I NOrlO. SEQ I W 1 SEQ iD N : SEQ I D D . . 1 O& D Y1 NOX3. SEQ I N&l4. SQ X N0:lS. SFQ ID NO:16. D I SEQ W NW7. dSEQ ID NCklSI. A window 1102 i s dy cPmpucwgucnm of 1104 u d A mulm~I ~ g u c o a s shown m&a J cbip abk to xcnmvc aud diqday linb in this mmu. 11M'lbc~xquaadifPusfrcm&cdripwilvilblypc Bplll~nmcnu~rv8O&&cwir&lctodisphy p ~ mlmcd ~Ibc mn g u l a r ~ n 1108 bmriry graphr almcd bercr Once is sclmcd r s 01 h i p r v i l d ~ l o d m u n n t ~ e ~ s a c a r ~ o n d ~ ~ ~ in d uOT"lop,D b15e lY l I &BC w w &BS d g h g lbpa~.

~~~~~$~~~~~~~ "=

-

=,

tow

iwhamplcpq-ubdngoneof@erdm

sck&g a cDlry

klinkd

plu

a

se~7dowD ir PIO- me .
inf-um
a d

-

Repl 1 inskudim 8 Z me
LC n D Qiz

-d

f i lo gd Q-= 16 WAC

ir rcrocisacd wirh a

mn e m

~ s ~ t e d W i1 b r r c E x 3 1 p c q i n g o f f ~ v C m d b ~ t b

m9mom..irr,c*y e&daolfoa sd~ ~ ~ ~ ~ W I ~ . ( S E Q I D N O i W ~ ~ u. d~ m~ c -m ~U ~ ~ u c - b o m O w , J ~ ) M lOOB by wogJ~TbcfoUcdmg mOar ~ p ~ m o f m C ~ E ~ ~ ~ d u r i r d i q h y d ~ 9 9 4 T b c g r f $ 1 shows ~c imc~sulcrfar of tbc h~bridizedproba u

solnri-~ Jr.2

-G g r

*ChiP fiYJblYILC *

*

s-qksdurb
IUO
60

--lODO 90JO
1 YUD

~ ~ ~ ~ ~ ~ b c 9 O S . l h c d k d k w i a ~ n 1 ) 1116 liDc 910. and hcbrcc mdtioa i show~l h9I7,. LIG s u In 1111

1111 1114

.

Blfn

-

-

-. . - .

-

.
,

F a ua+e. vmpk solution lll1 combs 75% Ebip wild-typc squcna. and 2 W wqmcr %

Case 1:04-cv-00901-JJF

Document 444-3

Filed 09/13/2007

Page 35 of 45

5,795,716
25
Now rdcrdng mUK bases d c d i n mdangular box llDll f n h e s q l c sdutions. tbobtcnsirynriDmabodcnrretly
'

26
ABI
rm
Chip

&~csol&n1l1bab~gabrscAar~&c ca;pyild rgpe s o p m a This is W h a w e q l c sobonon 1110 i 100% chipwild* s sup~uur'Ibc inten- 5 sity m i o m ~ m ~ L M sample &tion 1 1 as b v i ~ g ~ d ails 12 abaseAbc~mw~rruqPcro~i%908&pwil~
=PC-

~ ~ m 6 s = m c ~ c m c ~ n i r n v

m c iarasi~ ratio d s th idcnrihcd base in +soh~hnsll14~lll6ar~anIC.lshicbis.n " IWACcod= bating A a G @uriue%Tbis nlro
a ~ b a u a l l ~ I b c ~ d ~

8 1

-0

4

1 :
W

9

4

8

21

I 3

sUYDUPY

h

~ ABt~ism=> 993%
*(mu)

75%rp50schiparildlypcaqoencc.ndfmm25%0~
?hlls. s e istensity nth muhod corh ~BUItbehuiaUlit~ti0nSlatt Srmplc scdurionr Ill& and llP a ,d c d by the m
murrhon a

An1 ( ) m.6s Qlip(m6.rPr)-F~~Q

--

5a.i

-

iDlcmirgnti~lDCmOd~baviaga~nbxeGatthe spsi6ed lcmtin~. This k a mmd base c10 beauso &c
-4*n=pimar?y ratio

0%-

-.
~

- af me-& a

5 m-ry

e v c l y l . Ag eiucaly dkd mt buts. rLD me brv

uadfrrrdutionr ofIbc~YeDtion o f i h m one ri- - . atid s a - cn c mr -. -- - c -- -.> FIG. I ill-us 2 thc Icfucnre muhod ccarcdly c a h g a mmt basewime thc inlrnriry d o mabod incomdy d c d Ihc m U W base (SEQ W NO-36. SEQ ID NO-JI. SEQ ID NO-38, and .SIX) ID NO-39). 'Ibcre n due h w ~ q winwindows 12OL U 4 a 1206 as s h 0 U ~ k h ) d
w i n d m arc .nd 3 box a u l i n a m c basa ofinknxt,Wmdout U O Z r b m a tamplc uqncna d e d using du i o l a d y mb-o mdhod Howwcr. Ibehrein~cmzaninImbm1208anrinansdlyellcd

- -m

EJ.amplc 2
war
bdy
@ -

ecaa &@

ashgthe

olcu),

,

W m

lions Saqlcs w a e cvalnsd h m AIDS m u bdm and afta ddI ucalmmL Rcmlts wcn mn6rmd w& ABI Wencink FIG-U i l h l c r the outpn d W S E Q N Prog m m ~ i t h f o o r ~ ~ n t s a mmdfow-1 pkr samplu (SEQ ID NO:19. SEQ ID NOrtO. SEQ W N021. SEQ I NO-= SEQ I NO 23. SEQ ID NO-10. SEQ W D D NO=. WD N O - .urd SEQ I D N M 7 r Now me hrc I h n g c a t p d h ion whae a mutltion bas ariwo Even ad IWO addilioml mnhtionr () h c " S mumion has g. l

boamitknan~bh~wtbCmCLbOdnICdto~ pognm tba hpkmcac Lhc i ~ k a s i t mtia i a o lnnd y 4cL statislid mcmodr d rhc prcsrnt i n v m h . w d h B contabs Lbc hca& 6 u (-h I for CMlStiQN. Appcmcnctmabob-IhcrJcrwccy9~ir&barcCald1c d i m C a d D rn~Ilin solzrcc codc and bed& fils. Ibc psitic0 i d c l d by the rmangukr ka aiac rcrpecrivcty. for a p r o p that pcrfamr a m s h g W i m ltDdsholvsrhcsampIcsquaa call& us* fie stage for &c slatirlid m c h e o f24LUEQ'''f d a nvmodTbc daaa mabod cmroclly ullr ~ b c spacificdb8.x.a~b t i n g b a r c A n ~ ~ . f o r s o m c ~ t h c AppendixErn~thcwvlrczcodcandbudpfilafw VIEWSEQ'". which u a cDmpuarivc -sir a dvidd u m a mclhod is prcfaabk to tbc i n h d y Rtio mdbod inb'on p m p m accading to Ibc prrwnt iovcnrjorr.&pmhXaUX h COlUpdtCS@ inlc~rilicrd w l c SCgucDE dioa A-E zre witten faa Sun WaksJation. I~pmbcintcDsitirsdadm~~~~qu~~~~

~ ~ . Z h i s s ~ a l l y n h r c A a t b t p o d t i ~ 35 ' I b C ~ VIIL APPmDICEs h uq n i mcIhod i n m m d y callcd Ibc bssc -as C w i Io kcsusc UK P C I & immsity 1 s c c&d arim bmc C u much The Micmlicbc a g p c ~ d i c a(oqrylight A E p d x . + ) hi@= dun Ibc c(ha pmk intcmilin provide C+t smmu mdc and budcr fils for iuqlaW~llog W a l2M l o w s a dl'crcrrt squd c d wing I pfcscul h i ~ n n t i o ~ k Appendix ACOnbhS UIC YIITCC codc Ihe i n ~ ~ n s h a i d d AS the rdcrcna xpury o t k 6 a (scdl=) for CALISEQN. which ir a b e alling J

-

pr@y

me r c l a t n a s c q n a f c Howc1.u. it k impwtaa to have pobc iatcndties for a rducna sqID usc Uu nfer-

VJLEXM.IPLES
-1

5,

The intcnary d o mabod was used in scquma analysis d Various polymwptic HIV-l h a usiag r proleax c i hp Sb@e mamM DNA d a 382 nt region waa pxd whh 4 ditf=-l doncs (HXBZSF2, NY5- ~Pcd4mut18~ Rcsulo u d u h o m a n n B 1 scgu-T~C~LI~B , arc iLIusralal Mow:

a

~~~

ra a h e p l r w i s Pumarive and oo( d d v c ~ a u v a z i a OT the invention will b ~ m a p p a ID y ~ c ~ lbllsc of sLill in UJC u upon miol o & s a;wlosurr t f i M u d y by way o f ~ l e . w h i l e ~ i u i n v ~ o n l ~ t c d bil ari0,puliada r d u c n a l tbc ordudon of DNA (natural o ar u m a !he ~ bemabobr can bcumi in rhc analysis from cbip mcb d m ma&s synrboizcd h C r C 0 & such Z RNA Tbc rmpc of U u invention should. ~ ~ ~ S kR C I d-ncd D wt r c f m r r D tho above dsn@6on but M ih I instcad should bc 8 ' - -dwithrefto lbc appc~dcdndcd claims