.
`Ulllted States Patent
`
`[19]
`
`llllllllllllllIllIllllIllllllllllllllllllllllllllllllllllllllllllllllllllli
`USOOS399491A
`[11} Patent Number:
`
`5,399,491
`
`Kacian et a1.
`
`[45] Date of Patent: Mar. 21, 1995
`
`[75]
`
`[54] NUCLEIC ACID SEQUENCE
`AMPLIFICATION METHODS
`Inventors: Daniel L. Kacian, San Diego;
`-
`.
`-
`T
`. Ful V
`both f Callf.
`rmothy J
`tz,
`1sta,
`0
`[73] ASSigfleei Gen-Probe Incorporated, San Diego,
`Calif.
`[21] APPI- No.: 855,732
`_
`[22] Ffled:
`
`Mar. 19, 1992
`
`[63]
`
`Related US. Application Data
`Continuation of Ser. No. 550,837, Jul. 10, 1990, which
`is a continuation-in—part of Ser. No. 379,501, Jul. 11,
`1989, abandoned.
`e
`’
`.
`.
`........................ cup 19/34,&172}(12211G608i
`Int. Cl.
`[51]
`[52] US. Cl. ................................ 435/9121; 435/91.2-
`435/6; 536/2433
`[58] Field of Search ................. 435/6, 91, 91.2, 91.21;
`5
`.33
`36/27, 24_
`
`[56]
`
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`49 Claims, 26 Drawing Sheets
`
`t
`
`1
`
`t
`
`1
`
`l
`5
`
`
`
`H 1......-
`s &_m min—mm
`use
`
`In
`
`x.
`
`D”
`“Bani—l
`,- mini-mum”
`rm
`5' “fill-H
`
`’5-
`
`
`
`’W
`l—ummmh}
`
`Ariosa Exhibit 1020, pg. 1
`|PR2013-00277 -
`
`Ariosa Exhibit 1020, pg. 1
`IPR2013-00277
`
`

`

`5,399,491
`
`Page 2
`
`FOREIGN PATENT DOCUMENTS
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`.
`. ” 86 proc. Natl.
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`.
`. ” 87 Biochemistry 1874—4878 (1990).
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`.
`. ” 62 virology 46—59 (1974).
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`Mullis et al., “Specific Synthesis of DNA in Vitro via a
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`Watson et al., “Molecular Structure of Nucleic Acids”,
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`
`Okayama et al., “High—Efficiency Cloning of Ful-
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`2:161—170, 1982.
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`
`(List continued on next page.)
`
`Ariosa Exhibit 1020, pg. 2
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 2
`IPR2013-00277
`
`

`

`5,399,491
`
`Page 3
`
`FOREIGN PATENT DOCUMENTS
`
`8810315 12/1988 WIPO ..................................... 435/6,
`8901050 2/1989 WIPO .
`29014-439 11/1990 WIPO .
`9115601 10/1991 WIPO .
`
`OTHER PUBLICATIONS
`
`Both et al., “A general strategy for cloning double—s-
`tranded RNA: nucleotide sequence of the Simian—11
`rotavirus gene 8”, Nucleic Acids Research, 10:7075,
`1982.
`
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`tRNAITyr genes using DNA fragments produced by
`restriction enzymes”, Proc. Natl. Acad. Sci. USA,
`72:4754, 1975.
`Rossi et al., “An Alternate Method for Synthesis of
`Double-stranded DNA segments”, J. Biological Chem-
`istry 257:9226, 1982.
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`DNA Fragments Cloned into M13 Vectors”, Methods
`in Enzymology, 100:468, 1983.
`Guatelli et al., “Isothermal in vitro amplification of
`
`nucleic acids by a multienzyme reaction modeled after
`retroviral replication”, Proc. Natl. Acad. Sci. USA,
`87:1874, 1990.
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`Krug and Berger, Proc. Natl. Acad. Sci USA 86:3539,
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`.
`
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`
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`
`al.,
`Oyama et
`264:18808, 1989.
`Grachev et al., Nuc. Acids Res. 12 (22) 1984, p. 8501.
`Milligan et al., Nuc. Acids Res. 15(21) 1987, p. 8783.
`Gubler, Guide to Molecular Cloning Techniques; Aca-
`demic Press, NY, NY. 1987, p. 330.
`Krug et al., Guide to Molecular Cloning Techniques;
`Academic Press, N. Y., N. Y., (1987) pp. 316—325.
`Bethesda Research Laboratories Catalogue & Refer-
`ence Guide, (1988) Bethesda Research Laboratories,
`Bethesda, Md., p. 37.
`Cox et al., J. Med. Microbiol., vol. 35 (1991), pp.
`284—290.
`
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`294—296.
`
`Ariosa Exhibit 1020, pg. 3
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 3
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 1 of 26
`
`5,399,491
`
`RNA TIIRcET H
`
`5' W;
`I. Q FIRST PRIMER (+)
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`
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`
`Ariosa Exhibit 1020, pg. 4
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 4
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 2 of 26
`
`5,399,491
`
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`Ariosa Exhibit 1020, pg. 5
`|PR2013-00277
`'
`
`Ariosa Exhibit 1020, pg. 5
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 3 of 26
`
`5,399,491
`
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`Ariosa Exhibit 1020, pg. 6
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 6
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 4 of 26
`
`5,399,491
`
`'
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`Ariosa Exhibit 1020, pg. 7
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 7
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 5 of 26
`
`5,399,491
`
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`
`Ariosa Exhibit 1020, pg. 8
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 8
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 6 of 26
`
`5,399,491
`
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`
`RNA MT (4-)
`
`7
`
`'
`
`8
`
`'
`
`
`
`lRNose HDEGRADATION OF RNA
`9. —PRO(-) SECOND PRIMER EXTENSDN PRODUCT (-I
`m M
`IIMIEIIL PRIMER
`' L—mH SECOND PRIMER EXTENSION PRowcT I-I
`1mSYNTIESIS
`.. PW“ m “"5”" W ""
`-
`H SECOND PRIMER ExTEMSIoM PRODUCT (—I
`lRNAsmug
`I2. swmmm + W RNA PRODUCTI—I
`
`5-
`
`"3-
`
`FIRST PRIMER ISPLICE TEMPLATE (+)
`
`, mm cOPIEs
`
`Ioo-Iooo COPIES
`
`so TO FIGURE Ie,STEPT.
`
`Ariosa Exhibit 1020, pg. 9
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 9
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 7 of 26
`
`5,399,491
`
`.
`
`PRO
`
`I. 5 \Qm
`
`HRST PRIMER/SPLICE IEM’LATEI’rI
`
`RNA TARGET I-I
`
`DNA SYNTHESIS
`
`H6. /g.
`
`EIRST PRIMER EXTENSION PROOOOT I+)
`""'"""'\':'.'\'l"“"{'.'{'42{""":‘- RNA TARGET (—I
`
`
`2-
`
`lRNase H DEGRADATION 0FRNA
`PROI )
`3. SW FIRST PRIMER EXTENSION PROOIIcT (+I
`lANNEAL PRMER
`EIRSTPRIMEREXTEMSIONPROOOOTHI
`5' PM
`‘ M SEOONO PRIMER/SPLICE TEMPLATE I-I
`PM)
`
`4
`
`OMA SYNTHESIS
`
`PRO H
`5'wFIRST PRIMER EXTENSION PRODLCT (+)
`PR0 H SECOND PRIMER EXTENSION PRODUCT (-I
`lRNA SYNTHESIS
`IOO—I
`_
`S'mmgg'écxgpgs RNA PRODUCT (+) + 5-W RNII PRODUCT (-I
`l
`.
`PRO (+I
`5'WRNA PRODUCT(+)
`5m FIRST PRIKRM
`SECOND PRIMER (-I +
`RNA PRODUCT ‘4
`
`ANNEAL PRIMER
`
`PROI-I
`
`
`
`
`
`
`
`
`DNA SYNTHESIS
`
`
`
`5
`
`-
`
`e.
`
`7-
`
`8
`
`.
`
`5'
`
`SECOND PRIKR
`PROH EXTENSION PROOIOTI—I
`
`RNose H DEGRADATION 0F RNA
`
`PRO I+)
`SEOONOPRMER + '— H T PRIMER
`_9. PRO H EXTENSION WWW 5
`EfiENSION mm (+)
`
`
`
`10 9—um""
`'
`
`ANNE/IL PRIMER
`
`FIRST PRIM R/
`
`DNA SYNTHESIS
`
`Ariosa Exhibit 1020, pg. 10
`|PR2013-00277
`
`RNA PRODUCT(+) + 5. lmwllllllllllllllllll %W%T (+)
`
`
`
`
`‘\\\\\\\\\\\\\\\\V m m (—)
`
`PHI
`
`
`
`
`
`
`smogflfgflw +OH“) E'RRIE‘NEIIIEWIN
`EXTENSION PROOIIOTI—I
`Wfifim I—I
`
`
`Ariosa Exhibit 1020, pg. 10
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 8 of 26
`
`5,399,491
`
`5'
`
`5
`
`'-
`
`2
`
`3
`
`4
`
`5
`
`PRO H)
`FIRST PRIIER EXTENSIGI PRODUCT (+I
`"""""'12{"'{'\'{{'\'{'\'{'\'4{'\'{‘~ m mg I.)
`
`
`lRNose HDEGRADATION OF RNA
`,
`PRO (+)
`5W FIRST PRIMER EXTENSION PRODUCT I+I
`lANNEAL PRIMER
`5' m “9
`FIRST PRIIER EXTENSION PROOIIcT I+I
`’ m % SEcONO PRIMER I—I
`10M SYNTHESIS
`FIRST PRIMER EXTENSION PROIIucT (+)
`PR0 m
`5'
`- MSESOO PRIMER EXTENSION PROOOOT I-I
`PROF)
`
`HM)
`m FIRST PRIMER /SPLIcE TEMPLATE I+I
`lDNA SYNTHESIS
`
`RNII TARGET (—)
`
`F76.
`
`/h.
`
`(~
`
`RNASYNTHESIS
`
`6. 9% RNA PRODUCT (+)
`IXNNEAL PRINER .
`5'Wm PRODUCT I+)
`R —)
`m H
`CORD
`ME
`I
`DNA SYNTHESIS
`
`SE
`
`PRI
`
`7
`
`-
`
`8
`
`5' \\\\\\\\\\\\\\\\\\\\\\\\' RNA WOT (H
`
`
`1
`
`PRO(-4 SEWD PRIMER EXTENSDN PROHICT(-)
`
`RNIIse H [ISRADATION If RNA
`
`FIRST PRIMERISPLICE TEMPLATE I+)
`I‘m MR
`PM“)
`5-
`“’- L—mH SEcONO PRIMER EXTENSION PROOIIcT I—)
`lDNA SINTNESIS
`Pm H SECOND PRIMER EXTENSION PRODUCT (-)
`lRNA SYNTHESIS
`I2. swmmm + WRNARMTI—I
`
`I —
`
`_.
`
`”-
`
`60 TO FIGURE Ie, STEP 7.
`
`Ariosa Exhibit 1020, pg. 11
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 11
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 9 of 26
`
`5,399,491
`
`5'
`FIRST PRIMER (+)
`L Em RNA TARGET (-)
`lDNASYNTHESIS
`FIRST PRIMER EXTENSKN PRODUCT (+)
`5’
`- 8mm RNA TARGET I—I
`
`'
`
`2
`
`3.
`
`5‘— FIRST PRIMER EXTENSION PRODLCT (+)
`
`lRNose HDEGRADATION(PRNA
`lANNEAL PRIMER
`4. m SEOONI) PRIMER/SPLICETEMPLATE I—I
`
`5.
`
`FIRST PRIMER EXTENSION PRODUCT (+)
`
`(~)
`
`ONA SYNTHESIS
`
`I
`
`5-
`
`5. W SEOONO PRINER EXTENSION PRODUCTI—I
`
`FIRST PRIMER EXTENSION PRODUCT (H
`
`RNA SYNTHESIS
`
`6.
`
`IOO-IOOO OIPIES
`. Em RNA PRODUCT H
`
`lAIIIEAL PRINER
`5'
`FIRST PRIMER (+)
`7- gm RNA PRODUCT (—)
`
`ORA SYNTHESIS
`
`
`
`5-
`
`FIRST PRIMER EXTENSION PRODUCT (+)
`RNA PIOOOOT (—I
`
`RNose NDECRADATION or RNA
`
`5'_ FIRST PRIMER EXTENSION PRODUCT (+)
`
`
`
`FIRST PRINER EXTENSION PRODUCT (+)
`"'"1. SEOONO PRIIER/SPIIOETENPLATE (—)
`PRO H
`
`ANIEAL PRIMER
`
`3
`
`a
`
`-
`
`9.
`
`m
`
`-
`
`DNA SYNTHESIS
`
`HG. //'.'
`
`Ariosa Exhibit 1020, pg. 12
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 12
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 10 of 26
`
`5,399,491
`
`I
`
`FIRST PRIMER I+I
`5'
`' m RNA TARGET (_)
`
`
`
`DNA SYNTIESIS
`
`2
`
`FIRST PIIMER EXTENSION PRODUCT (+I
`5'
`' m RNA TARGET I—I
`
`RNuse II DEGRADATIINI 0F RNA
`
`3.
`
`5' -—— FIRST PRIMER EXTENSION PRODUCT (+)
`
`ANNEAI. PRIMER
`
`4
`
`-
`
`5
`
`‘
`
`5-
`
`FIRST PRIMER EXTENSION PRouICT (+I
`k SECOND PRIMER/SPLICE TEMPLATE H
`
`5’W FIRST PRIMER EXTENSION PRODUCT (+)
`
`SECOND PRIMER EXTENSKIN PRODUCT H
`
`PRO (-I
`
`RNA SYNTHESIS
`
`loo—I000 COPIES
`6. mm RNA PRIDUCT (—I
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ANNEAI. PRIMER
`
`
`
`
`F76.
`
`//.
`
`Ariosa Exhibit 1020, pg. 13
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 13
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 11 of 26
`
`5,399,491
`
`I.
`
`5' Q
`
`FIRST PRITER (+)
`RNA TARGET (-)
`
`F76 //(_
`
`lDNA SYNTHESIS
`
`FIRST PRIER EXTENSION PRODUCT (4-)
`
`
`
`n\\\\\\\‘\\\\\‘\\\\‘ RNA TARGET F)
`
`lRNase HDECRADATIINI OFRNA
`
`5'_ FIRST PRIMER EXTENSION PRODUCT (+I
`
`ANNEAL PRIIER
`
`5'
`
`5-
`
`ka'
`
`ROM
`
`FIRST PRIMER EXTENSION PRODUCT (+)
`
`SPLICE TEMPLATE (—I
`
`INA SYNTHESIS
`
`FIRST PRIMER EXTENSION PRODUCT (+)
`M SPLCE TEMPLATE I—I
`PRO (-I
`
`RNA SYNTHESIS
`
`IOO-IOOO COPIES
`W RNA PRODUCT (—I
`
`FIRST PRIIER M
`
`RNA PRODUCT (-I
`
`FIRST PRIIER EXTENSION PRIWCT H)
`PRODUCT (-I
`
`lANNEAL PRIMER
`5.W
`[INASYNTHESIS
`5'M3 RNA
`lRNose N DERADATION or RNA
`
`
`5'_ FIRST PRIMER EXTENSION PRODUCT (+I
`
`1mm PRINER
`
`PROI-I
`X
`DNA SYNTHESIS
`
`FIRST PRIMER EXTENSION PRODUCT (+T
`
`SPICE TEAPLATE (-I
`
`
`
`
`
`
`
`IO.
`
`Ariosa Exhibit 1020, pg. 14
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 14
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 12 of 26
`
`5,399,491
`
`I.
`
`5 m FIRST PRIMER/SPLICE TEMPLATE (+I
`
`RNA TARGET I—)
`
`DNA SYNTHESIS
`
`
`FERST PRIMER Exmm WT (+)
`IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
`
`§\\‘\\\\‘\\\‘\ RNA TARGET (.)
`
`1% HDEGRADATION IX: RNA
`5'W FIRST PRIMER EXTENSION PRODUCT (+I
`
` ANNEAL PRIMER
`5'
`FIRST PRINER EXTENSION PRODUCT (+I
`' M SPLIcE TEMPLATE I—I
`PM)
`DNA SYNTHESIS
`
`3
`
`4
`
`5.
`
`6.
`
`7
`
`8
`
`-
`
`9.
`
`IO
`
`‘
`
`5'w FIRST PRIMER EXTENSION PRODUCT (+I
`l
`SPLICE TEMPLATE I—I
`
`PRO I—I
`RNA SYNTHESIS
`
`IOO-IOOO COPIES
`5'W RNA PRODUCT (—)
`
`lANNEAL PRIMER
`5'm FIRST PRIMER I+)
`
`5-m FIRST PRIAIR EXTENSDN PRODUCT (+I
`
`DNA SYNTHESIS
`
`RNA PRODUCT I-I
`
`RNase H IECRADATION TX: RNA
`
`5'— FIRST PRIMER EXTENSION PRODUCT (+I
`
`
`
`
`
`
`
`
`
`
`F76.
`//.
`
`lMEAL PRIMER
`5'w FIRST PRIMER EXTENSION PRIDUCT (+I
`
`X
`
`PRO (-)
`
`SPLICE TEMPLATE I—I
`
`DNA SYNTIESIS
`
`Ariosa Exhibit 1020, pg. 15
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 15
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 13 of 26
`
`5,399,491
`
`HIST PRIMER (+I
`5.
`I. mm RNA TARGET (-I
`lDNA SYNTHESIS
`
`FITST PRIMER EXTENSION PRODUCT (+)
`5'
`2. mm m men (-I
`
`5'_ FIRST PRIMER EXTENSXIN PRIXXXIT (+)
`
`lRNoseHDEGRADATIONOFRNA
`1mm PRIMER
`Xkflm SPLICE TEMPLATE (-I
`
`FIRST PRIMER EXTENSION PRODUCT (+I
`
`5'
`
`‘
`
`5'WRIST PRIER EXTENSION PRODUCT (+I
`I
`X
`
`RM SYNTHESIS
`
`PRO (-I
`
`lOO-IOOO COPIES
`6. m3 RNA PRODUCT (-I
`
`3.
`
`4.
`
`5
`
`'
`
`SPLXIE TEMPLATE (—I
`
`
`
`
`DNA SYNTHESIS
`
`7
`
`8
`
`'
`
`lANEAL PRIMER
`FIRST PRIMER I+I
`5-
`' Ems RNA PRODUCT (-I
`IONA SYNTHESIS
`5’M FIRST PRIMER EXTENSION PRODUCT (+I
`lRNoseHDEGRADATION?RNA
`
`RNA PRODUCT (-I
`
`
`
`9.
`
`5'— FIRST PRIMER EXTENSXIN PRODUCT II»)
`
`MEAL PRIER
`
`x m (_
`
`FIRST PRIMER EXTENSION PRODUCT (+)
`SPLICE TEMPLATE (-I
`
`Ariosa Exhibit 1020, pg. 16
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 16
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 14 of 26
`
`5,399,491
`
`I
`
`FIRST PRIMER (+)
`5-
`' Em RNA mm H
`lDNASYNTHESIS
`
`2
`
`FIRST PRIMER EXTENSION PRODUCT (+)
`5'
`- m RNA may I.)
`JRNose N IIGRADATTON (P RNA
`
`3.
`
`4.
`
`5'_ FRST PRIMER EXTENSDN PRIDUCT (+I
`
`5'
`
`lAIRIEAL PRIMER
`
`X
`
`PR0(.
`
`INA SYNTIESIS
`
`HIST PRIER EXTENSION PRODUCT (+)
`SPLICE TEMPLATE (-I
`
`5.
`
`5'
`
`FIRST PRIMER EXTENSION PRODUCT (+I
`xm SPLICE TEMPLATE I-I
`Pm (-I
`RNA SYNTHESIS
`
`ICXI-IOOD COPIES
`6- mum RIIII PRODUCT (4
`
`1mm PRIMER
`FIRST PRIMER (+)
`'
`5'
`' % RNA PRIDUCT (-I
`lDNA SYNTHESIS
`
`7
`
`8
`
`FIRST PRIMER EXTENSION PRODUCT I+I
`5'
`' m RNA PRODUCT I-I
`lRNose HIICRADATION OF RNA
`1mmPRIMER
`' W SPLICE TEMPLATE (-I
`DNA SYNTHESIS
`
`9.
`
`IO
`
`5'_ FIRSTPRINER EXTENSION PROIXICT (+I
`
`5'
`
`FIRST PRMER EXTENSION PRODUCT (+I
`
`Ariosa Exhibit 1020, pg. 17
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 17
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 15 of 26
`
`5,399,491
`
`F/G.
`
`/0.
`
`5.
`5W)
`I Q SPLICE TEMPLME—HRST PRIMER H
`
`RNA TARGET (—)
`
`'
`
`5'
`
`””0 H
`
`ONA SXNTIIESIS
`
`2
`
`'
`
`FIRST PRIMER EXTENSION PRODUCT I+I
`
`“““maa'xaaaawaak RNA TARGET I—I
`
`3
`
`4
`
`5
`
`lRane II OEORAOATION 0F RNA
`5' PROH— FIRST PRIMER EXTENSION PRODUCT (+)
`lANNEAL PRIMER
`FIRST PRIMER EXTENSION PROOIIcT (+)
`PR0 H
`5-
`' fl SPUCE TEMPLATE-FIRST PRIMER (—)
`PRO (‘)
`
`ONASXNTRESIS
`
`EIRST PRIMER EXTENSION PRIIOIIOT (+)
`5- M H
`' m SPLICE TEMPLATE-FIRST PRIMER (-)
`
`PRO (-)
`
`RNA SYNTHESlS
`
`IOO-IOOO OOPIES
`6. W RNA PRODUCT (-)
`
`PRO H
`
`RNA PROOIIOT (—)
`
`lANNEAL PRIMER
`5'My SPLIOE TEMPLATE—FIRST PRIMER H
`lONASXNTIIESIS
`
`5.
`
`PRO H
`
`“RSI PRIMER EXTEm PRODUCT (+)
`llllllllllllllllllllllllllllllllllllll
`
`n\\\\‘\\\\\\\\\\\\‘ RNA PRODUCT (_)
`
`JRNOSO ROEORAOATION 0FRNA
`
`7
`
`-
`
`8
`
`9.
`
`m
`
`
`
`
`
`PRO H
`5'— FIRST PRMER EXTENSION PROOIIcT (+)
`iANNEAL PRIMER
`FIRSTPRIIER EXTENSION PROOIIcT (+)
`5' m H
`' m SPLICE TEMPLATE-FIRST PRIMER (-I
`
`
`DNA SYNTHESIS
`
`
`
`
`
`
`
`Ariosa Exhibit 1020, pg. 18
`|PR2013400277
`
`Ariosa Exhibit 1020, pg. 18
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 16 of 26
`
`5,399,491
`
`5‘
`
`PRO (1.)
`.
`M FIRST PRIMER/SPLICE TEMPLATE (+)
`lDNA SYNTHESIS
`
`ONA TAROET (—)
`
`FIRST RRINER EXTENSION PROOIIOT (+)
`"‘0 "9
`5-
`- M. ONA TAROET EXTENSION PRODUCT I—T
`iRNA SYNTHESIS
`
`IOO-IOOO COPES
`5' mm RNA PRODUCT (+)
`
`60 TO ANTOOATALTTIO ANRIEIOATION NETIIOO
`
`ANNEAL RRIIER
`
`5.
`
`RNA PRODUCT (+)
`W SEOONI PRIMER I—I
`
`ONA SYNTHESIS
`
`RNA RROOIIOT m
`5'
`- W sEOONO PRIMER EXTENSKJN RROOIIOT (-)
`
`DEGRADE RNA
`
`'-
`
`2
`
`3.
`
`4
`
`-
`
`5
`
`
`
`
`
`
`
`
`6. _ SECOND PRIMER EXTENSION PRODUCT (-)
`iANNEAL PRIER
`
`PRO (+)
`
`-
`
`8
`
`5- m ‘9
`
`SECOND PRIMER EXTENSION PRODUCT (-)
`
`lDNASYNNIIESIS
`
`FIRSf RRIIIER EXTENSION RROOIIcT IIT
`
`SECOND PRNER EXTENSION PRODUCT H
`
`F76. 20.
`
`Ariosa Exhibit 1020, pg. 19
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 19
`IPR2013-00277
`
`

`

`'
`
`I
`
`2
`
`3.
`
`4
`
`'
`
`US. Patent
`
`Mar. 21, 1995
`
`Sheet 17 of 26
`
`5,399,491
`
`F/G. 2b.
`
`.
`
`A)
`
`DNA mm H
`
`5 IN FIRST SPLICE TEMPLATE (+I
`lDNA SYNTHESIS
`FIRST SPLICE TEMPLATE (+I
`5'
`’ M DNA TARCET EXTENSION PRODUCT I-I
`lRNA SYNTHESIS
`
`
`
`GO TO AUTOCATALYTIC AMPLIFICATION METHOD
`
`IOO-IOOO COPIES
`‘
`5 9mm RNA PRODUCT (+I
`
`ANNEAL PRIMER
`
`5'W RNA PRODUCT (+I
`
`FIRST PRIMER I-I
`
`DNA SYNTHESIS
`
`
`
`
`
`5' W FIRST PRIAER EXTENSION PRODUCT I-I
`
`RNA PRODIET (+I
`
`5'
`
`REMOVE RNA FROM SECOND
`PRIAER EXTENSION PROOIXTT
`
`5. _ SECUID PRIIER EXTENSION PRODUCT H
`
`7
`
`'
`
`8
`
`
`
`
`
`PRO (+I
`5'
`FIRST SPLICE TEMPUATE (+I
`
`
`' m SECOID PRIMER EXTENSION PRODUCT I-I
`
`
`RNA SYNTHESIS
`
`
`ANNEAL PRIMER
`
`
`5'Mi FRST SPLICE TEIPLATE (+I
`
`SECIXO PRIMER EXTENSION PRODUCT (—)
`
`DNA SYNTHESIS
`
`Ariosa Exhibit 1020, pg. 20
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 20
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 18 of 26
`
`5,399,491
`
`5'
`
`mo ‘9
`
`I.
`
`F/(z‘. 2a
`
`FIRST SPLICE TEMPLATE-PRIMER (+)
`DNA TARGET I—I
`
`lDNA SYNTHESIS
`5.
`”I0 “I
`'
`FIRST SPLICE TEMPLATE-PRIMER (+)
`2- m.- DNA TNRCET EXTENSION PRODUCT (—)
`IRNA SINTIIESIS
`5 moonsgng RNA PRODUCT (+)
`
`3.
`
`
`
`GO TO AUTOCAMYIIC AMPLIFICATION METHOD
`
`NNEIIL PRIMER
`
`5'
`
`RNA PRODUCT (+I
`
`.
`
`4. w FIRST SPLICE TEMPLATE—PRIMER (+)
`
`P30 (+)
`
`DNA SINTFESIS
`
`5.
`
`PRO (9
`
`FIRST SPLICE TEMPLATE-PRIMER
`EXTENSION PRODUCT I-I
`
`REMOVE RNA FROM SECOND
`PRIMER EXTENSDN PRODUCT
`
`
`
`
`
`
`
`-
`
`FRO (4,,
`
`EXTENSION PRODUCT (—)
`
`ANNEAL FIRSI SPLICE
`TEMPLATE-PRIMER (+)
`
`
`FIRST SPLICE IEMPLATE-PRIMER (+)
`PR0 (+I
`5.
`
`7- L FIRST SPLICE TEMPLATE-PRIMER
`lDNII SYNTHESIS
`5.
`”I0 ‘9
`FIRST SPIICE TENPLIITE—PRIIER (+)
`3- m- FIRST SPIICE TEMPLATE-PRIMER
`
`
`pm (4,)
`
`EXTENSION PNIDUCT I-I
`
`EXTENSW PROM)“
`
`RNA SYNIIfSIS
`
`Ariosa Exhibit 1020, pg. 21
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 21
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 19 of 26
`
`5,399,491
`
`‘
`
`-
`
`5«
`
`ONII TIIROET I+I
`W EIRST PRIIER I—I
`
`10.1 ms
`
`/-76. 2d.
`
`‘
`
`2
`
`5'W D“ W ‘*’
`FIRST PRIIER EXTENSION PRODUCT I—I
`ISEPIIRATE STRAIIJS
`5'_ WI TARGET (H
`3- — FIRST PRIIIER-EXTENSION PROOOOT I-I
`NNNEIIL PRIIER
`
`4 \n
`
`-
`
`SEMI PRIIIER/SPLIOE TEMPLATE (+)
`
`FIRST PRIMER EXTENSION PROOIIOT (-)
`
`5
`
`'
`
`lOIIII SYNTI£SIS
`5‘Mm SEW PRIER EXTENSION PRODUCT (+)
`1m SYNTHESIS
`
`FIRST PRIMER EXTENSION PROOOOTI—I
`
`IOO-IOOO OOPIES
`6, SW RNA MT (4»)
`
`OO TO AUTOCATALYTIC MIFICATION NETIIOO
`
`
`
`
`
`NNNENL PRIME
`
`7
`
`~
`
`5-
`
`RNA PROOIIOT I+)
`FIRST PRINER (-)
`
`DNA SYNTHESIS
`
`5'
`RNA PROOIIOT (+)
`8- W. FIRST PRIMER BITENSION PROOOOT I-I
`
`OEORIIOE RNII
`
`9.
`
`FIRST PRIMER EXTENSION PRIDUCT I—I
`
`ANIERL PRIMER
`
`PRO (+)
`
`H
`
`5» m “"
`
`'0 Sm SEW WSUCETEWTEH‘)
`-
`FIRST PRIIER EXTENSION PROOIIOT (-)_
`lONN SINTNESIS
`
`
`SEOOIO PRINER EXTENSION PRIOIIOT (+)
`
`
`
`
`
`FIRST PRKR EXTENSION PRWCT H
`RNA SYNTHESIS
`
`
`Ariosa Exhibit 1020, pg. 22
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 22
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar.>21, 1995
`
`Sheet 20 of 26
`
`5,399,491
`
`I
`
`“
`
`2
`
`'
`
`5'W DNA TARGET (+I
`
`FIRST PRIMER (-)
`
`DNA SYNTHESIS
`
`H6 26
`
`5‘m- D“ “I“ ‘*’
`lSEPARATESTRANDS
`
`FIRST PRIMER EXTENSM PRODUCT (-I
`
`3
`
`5'_ DNA TARGET (+I
`' _ FIRST PRIMER EXTENSION PROWCT (—I
`
`ANNEAL PRHER
`
`,
`
`FIRST PRIMER EXTENSDN PRIDUCT (-)
`
`”906)
`5'
`4. m SPLBE TEWLATE (+)
`10M mums
`SPLICE TEMPLATE (+‘)
`PRO M
`5'
`5- M FIRST PRIMER EXTENSION PRODUCT (-I
`lRNA SYNTIESIS
`DO—
`CIPI
`5' $2283! RNA PRODUCT (+)
`
`
`
`
`
`GO TO NITOCATAUTN) AMPLFICATDN IETHOO
`
`6.
`
`ANNEAI. PRIER
`
`
`
`
`
`
`
`7-
`
`8
`
`5‘
`
`RNA WOT (+)
`S W m PRIMER (—I
`iDNA SYNTHESIS
`
`5'
`
`RNA PRODUCT I+)
`FIRST PRIMER EXTENSIGI PROIlICT H
`REKIVE RNA FIN FRST
`
`lPRIAER EXTENSICII PROMIT
`
`9. _ FRST PRIER EXTENSION PRODUCT I—)
`
`I0
`
`5‘
`
`PRO (+I
`
`1m PRIMER
`SPLICE TEMPLATE (+I
`
`FRST PRIMER EXIENSKIN PRODIET (-I
`
`'
`
`‘
`
`FR) M
`
`.
`
`1% SYNTHESIS
`
`
`
`RNA SYNTHESIS
`
`FIRST PRIER EXTENSNIN PM (-I
`
`
`
`
`
`
`
`Ariosa Exhibit 1020, pg. 23
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 23
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 21 of 26
`
`5,399,491
`
`I. m FIN PRIMER (+)
`DNA TARGET H
`
`DNA SYNTHESIS
`
`[C76 3
`
`'
`
`'
`
`5'
`P309)
`2. m FRST PRIMER EXTENSION WT (+)
`lSEPERATE DNA STRANDS
`
`DNA TARGET I—I
`
`PRO (+)
`S“— FIRST PRIER EXTENSIIYI PRODUCT (+)
`1mm PRIER
`5'W FIRST PRIMER EXTENSION PRODUCT (+)
`sewn PRIMER M
`low was
`5 5mm FIRST PRIMEREXTENSION PRIDOCT (+)
`lRNA SYNTHESIS
`
`PROI+I
`
`SEON) PRIIER EXTENSDN PRONJCT (-)
`
`TOO—I000 COPIES
`5' 83m RNA PRODUCT (+)
`
`
`
`GO TO AUTOCATAUTIC AMPLIFICATION NETHOD
`
`ANNEAL PRIMER
`
`5' SW RNA PROMT (+)
`lDNA SYNTHESIS
`
`SECOND PRMER (—)
`
`5'
`
`RNA PRODUCT (+)
`SECOND PRIMER EXTENSION PRODUCT H
`
`105mm RNA
`
`— SECOND PRIMER EXTENSION FRODIKST I-I
`
`SECONI PRIMER EXTENSION PROIUCT I-I
`
`
`
`
`
`
`
`3.
`
`T
`
`‘
`
`6.
`
`7
`
`'
`
`8
`
`'
`
`9
`
`'
`
`lANNEAL PRIMER
`‘
`PRO (+)
`smflTSTPRMER/SPUCETEMPLATEM
`lDNA SYNTHESIS
`
`,
`
`PRO (+)
`
`IO.
`
`5
`
`FNST PRIIER EXTENSION PROTlICT (+)
`SEOOID PRIER EXTENSION PRIIUCT (-)
`
`RNA SYNTHESIS
`
`Ariosa Exhibit 1020, pg. 24 '
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 24
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 22 of 26
`
`5,399,491
`
`AMPUHCATION SYSTEM - AUTOCATALYTlC CYCLE
`
`100—5000 COMES
`l. m RNA PRODUCT (—)
`
`ANNEAL PRIMER
`
`2
`
`'
`
`5'@
`
`FRST PRIMER (+)
`
`RNA PRONICT (—)
`
`
`
`ANNEAL PRIMER
`
`5
`
`'
`
`5'M FRST PRIMER EXTENSION PWDUCT (+)
`PRO (—)
`SECOND PRIMER/SPLICE TEMPLATE (-)
`
`
`
`
` PRO (-)
`
`DNA SYNTHESIS
`
`FRST PRIfR EXTENSKN WT (+)
`
`SECOPDPRIMER/SMCE TEH’LATEH
`
`HG. 40.
`
`Ariosa Exhibit 1020, pg. 25
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 25
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 23 of 26
`
`5,399,491
`
`!
`
`RNA TARGET WITH 5' END OF TARGET & TARGET REGION IDENTIGAL
`5-
`FIRST PRIMER (+)
`' 5% RNA TARGET (—)
`lDNA SYNTHESIS
`FIRST PRIMER EXTENSION PROOIIOT (+)
`5'
`2‘ 55mm RNA TARGET (—I
`IRNose HOEORAOATION OF RNA
`FIRST PRINER EXTENSION PROOOOT (+)
`5-
`1 EM RNA TARGET (-I
`
`lANIfAL PRIMER
`5-
`FIRST PRIMER EXTENSION PRODUCT (+)
`4- 5M SEOONO PRMER/SPLIOE TEMPLATE (-)
`P1909
`
`ONA SYNTHESIS
`
`5
`
`5'
`FIRST PRIMER EXTENSION PROOOOT (+)
`- M SEcONO PRIMER EXTENSION PROOIIcTI-I
`lRNA SYNTHESIS
`
`IOO-IOOO COPIES
`6. mm RNA PRODUCT (-I
`
`RNA PROOIIcT I-I
`
`ANNEAL PRIMER
`
`FRST PRIMER EXTENSION PROOOOT (+)
`5'
`- m SEcONO PRIMER/SPLIOETEIPIATE I—I
`lDNA SYNTHESIS
`
`5'
`
`FIRST PRIMER EXTENSION PROOOOT (+)
`SECOND PRIMER/SPLICE TEMATE I—I
`
`FROG)
`
`RNA SYNTHESIS
`
`Ariosa Exhibit 1020, pg. 26
`|PR2013-00277
`
`lANNEAL PRIMER
`FIRST PRIMER I+)
`5-
`' @Sm RNA PRODUCT (-)
`IONA SYNTHESIS
`5‘m FIRST PRIMER EXTENSION PRODUCT (+I
`lRNose HOEORAOATIONOFRNA
`4
`3
`FIRST PRIAER EXTENSION PROOOOT I+Y
`' M- RNA PmoucT (-)
`
`7
`
`8.
`
`.
`
`9
`
`,0
`
`H
`
`
`
`
`
`
`
`Ariosa Exhibit 1020, pg. 26
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 24 of 26
`
`5,399,491
`
`.
`
`MPLIFICATTON SYSTEM-GENERAL KTNOD
`RNA TARSET WITH TINT-TARGET SEQETEES 5'10 TARGETREGNN
`NTlATDN TECHNN|SN I
`
`I
`
`PM(
`
`
`
`PRST PRIMER (+)
`
`RNA TARGET (-)
`
`INA SYNTHESIS
`
`5 a”
`l
`5'
`”Pow
`
`FIRST PRTNER EXTENSDN PRGJUOT (+)
`.“ngeamawuumwaa RNA TARGET I.)
`
`lRNose HOESRAIIATION 0F RNA
`
`5'— FIRST PRIMER EXTENSION mmucT (+)
`
`5.
`
`”‘0 ""
`
`PRO (+)
`
`5'
`
`lANNEAL PRIMER
`FIRST PRIMER EXTENSION mm (+)
`m SEcONO PRIMER (-)
`lONASYNTHESIS
`
`FIRST PHMER EXTENSON PROOIIcT (+)
`SEOONO PRIMER EXTENSIOII PROOIIOT (-)
`
`SEcONO PRIMER (-)
`
`OIIA SYNTHESIS
`
`SECOYD PRITER EXTENSKN PRODUCT (—)
`
`
`
`
`
`
`
`,
`
`'
`
`2.
`
`3.
`
`4-
`
`5
`
`'
`
`6.
`
`7
`
`'
`
`’
`
`9.
`
`l0
`
`‘
`
`II
`
`lRNA SYNTHESIS
`Io -IOOO
`S' m§sxg§m RNA PROOOOT (+)
`lANNEAL PRIMER
`5' SW RNAPRWUCT (+)
`
`I 5'W “m ‘*’
`lRNuse H WADATION (1’ RNA
`5'W SECOND PRIMER EXTENSDN PRODUCT (-)
`lANNEAL PRIMER
`5‘WW HRST PRIMER/WETEMPLATE (+)
`10MSYNTHESIS
`
`SEcONO PRIMER EXTENSION PROOIIOT (-)
`
`5'
`
`PRO m
`
`HRSTPRIMER/SPLIcE TEMPLATE (+)
`
`'
`
`HG. 4c.
`
`SEIXXO PRIMER EXTENSION PROOIIcTI-I
`
`RNA SYNTHESIS
`
`Ariosa Exhibit 1020, pg. 27
`|PR2013-00277
`
`Ariosa Exhibit 1020, pg. 27
`IPR2013-00277
`
`

`

`US. Patent
`
`Mar. 21, 1995
`
`Sheet 25 of 26
`
`5,399,491
`
`RNA TARGET WITH TON—TARET SEOLENGES 5'T0 TARGET REGION INITIATION KCHANTSM ll
`
`5'
`PM)
`1 Q FIRST PRIMER (+)
`lDNA SYNTHESIS
`WORM
`
`FIRST Mlm EXTENSION WT (+)
`IIIIIIIIIIllllllllllllllllllllll
`
`
`‘ “‘\\‘\\\“‘\\\\\\\w RNA TARGET (.)
`lRNOSc HDEGRADATION0F RNA
`5'W!
`lANIEAL PRIMER
`
`‘
`
`RNA TARCET (—)
`
`5'
`
`PRO (+)
`
`PRO (+)
`
`.
`
`5
`
`SECOND PRIMER H
`
`
`
`‘ FIRST PRIIER EXTENSION PRODIICT (+)
`
`lRNA SYNTHESIS
`
`IOO-IDOO COPIES
`5'm RNA PRODUGT(+)
`lANNEAL PRIMER
`RNA PRODUCT (+)
`W. SECOND PRIMER I-I
`lDNASYNTHESIS
`RNA PRODUCT (+)
`6W SECOND PRIMER EXTENSION PRDDIICT I—I
`lRNYIse H DEGRADATION or RNA
`9- W SECOND PRIMER EXTENSION PRIDIICT (-)
`lANNEAL PRIMER
`5'WW FRST PRIM