_
`Umted States Patent
`
`[19]
`
`IlllllllllllllIllI|||||||||llllllllllllllllllllllllllllllllllllllllllllllll
`USOO539949lA
`{ll} Patent Number:
`
`5,399,491
`
`Kacian et al.
`
`[45] Date of Patent: Mar. 21, 1995
`
`[75]
`
`[73] A-33381188!
`
`[54] NUCLEIC ACID SEQUENCE
`AMPLIFICATION METHODS
`Inventors: Daniel L. Kacian, San Diego;
`T-
`.
`bo h
`"why J
`tz’
`‘Sm’
`t °
`G¢11'P1'0b9 Il1¢0I'P0I‘8ted, 3311 Dieg0»
`Calif-
`[21] APPL No; 855,732
`_
`F11ed=
`
`[221
`
`Man 199 1992
`
`[63]
`
`Related US. Application Data
`Continuation of Ser. No. 550,337, Jul. 10, 1990, which
`is a continuation-in-part of Ser. No. 379,501, Jul. 11,
`1989, abandoned.
`34-
`In .
`.6 ............;........... C12P 1
`5
`12
`68-
`9/
`t C1
`[ 1]
`’(%7I$211//Oi
`[52] U.S. Cl. ................................ 435/9121; 435/91.2;
`435/6; 536/24.33
`[58] Field of Search ............... .. 435/6, 91, 91.2, 91.21;
`536/27’ 24'-33
`
`[56]
`
`References Cited
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`Primary Examiner-Mindy B. Fleisher
`Attorney, Agent, or Fz'rm—Lyon & Lyon
`
`U.S. PATENT DOCUMENTS
`
`[57]
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`ABSTRACI
`
`Mullis ....................................
`5,130,233 7/1992 Malek ................................. 435/91
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`tions of substantially constant
`temperature,
`ionic
`strength, and pH are provided in which multiple RNA
`copies of the target sequence autocatalytically generate
`additional copies. These methods are useful for generat-
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`49 Claims, 26 Drawing Sheets
`
`
`
`H 1......
`s *E1fl'T1M
`nmuna
`
`=‘ -7'-'°’-T-.n.:.".E.i."l's"""".‘
`
`M
`
`’~‘
`
`DH
`TEXT‘-l
`= ' ’:."“‘“"""“’
`1"“
`5' lZH
`
`5.
`
`°~
`
`*
`
`5.
`
`1
`
`‘r
`
`
`
`‘ ’
`9 # }l$E!TZI-)
`
`Ariosa Exhibit 1020, pg. 1
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 1
`IPR2013-00276
`
`

`
`5,399,491
`
`Page 2
`
`FOREIGN PATENT DOCUMENTS
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`. ” 86 proc. Natl.
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`. ” 49(3) J. Virol. 686-691 (1984).
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`Guatelli et al, “Isothermal in vitro amplification of nu-
`cleic acids .
`.
`. ” 87 Biochemistry 1874-1878 (1990).
`Moelling “Characterization of Reverse Transcriptase
`and RNase H .
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`. ” 62 virology 46-59 (1974).
`Brener “Mechanistic Independence of AMV DNA
`Polymerase and Ribonuclease H” 14(6)
`J. Virol,
`1494-1502 (1974).
`Myers et al., “Sodium pyrophosphate inhibition of
`RNA:DNA hybrid degradation by reverse transcrip-
`tase,” 75(l1) Proc. Natl. Acad. Sci. 5329 (1978).
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`lytic RNA”, Gene, 82:83-87, 1989.
`Lowary et al., “A Better Way to Make RNA for Physi-
`cal Studies”, Knippenberg, eds, Structure & Dynamics
`of RNA, Nato ASI Series, vol. 110, New York, Plenum
`Press (1986).
`Murakawa et al., “Direct Detection of HIV-1 RNA-
`from AIDS, and ARC Patient Samples”, DNA,
`7:287—295, 1988.
`Mullis et a1., “Specific Synthesis of DNA in Vitro via a
`Polymerase-Catalyzed Chain Reaction”, Methods in
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`Watson et al., “Molecular Structure of Nucleic Acids”,
`Nature, Apr. 25, 1953, p. 737.
`Khorana, “Total Synthesis of a Gene”, Science 203:614,
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`
`Okayama et al., “High-Efficiency Cloning of Ful-
`1-Length cDNA”, Molecular and Cellular Biology,
`2:161-170, 1982.
`Watson et al., “Genetical Implications of the Structure
`of Deoxyribonucleic Acid”, Nature, 171:964, 1953.
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`Freeman and Company, 1963.
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`John Wiley & Sons, Inc., New York, Blackwell Scien-
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`genes of reovirus: Sequence of the cloned S2 gene”,
`Proc. Natl. Acad. Sci. USA, vol. 79, pp. 7644-7648,
`1982.
`
`(List continued on next page.)
`
`Ariosa Exhibit 1020, pg. 2
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 2
`IPR2013-00276
`
`

`
`5,399,491
`
`Page 3
`
`FOREIGN PATENT DOCUMENTS
`
`8810315 12/1988 WIPO ..................................... 435/6.
`8901050 2/1989 WIPO .
`29014439 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—1l
`rotavirus gene 8”, Nucleic Acids Research, 10:7075,,
`1982.
`
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`tRNA1Tyr genes using DNA fragments produced by
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`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.
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`
`nucleic acids by a multienzyme reaction modeled after
`retroviral replication”, Proc. Natl. Acad. Sci. USA,
`87:l874, 1990.
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`dated Jul. 12, 1991.
`Kwoh et al., Proc. Natl. Acad. Sci. USA 8621175, 1989..
`Krug and Berger, Proc. Natl. Acad. Sci USA 86:3539,
`1989.
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`Journal of Biological Chemistry,
`
`al.,
`Oyama et
`264:18808, 1989.
`Grachev et al., Nuc. Acids Res. 12 (22) 1984, p. 8501.
`Milligan et al., Nuc. Acids Res. l5(2l) 1987, p. 8783.
`Gubler, Guide to Molecular Cloning Techniques; Aca-
`demic Press, NY, N.Y. 1987, p. 330.
`Krug et al., Guide to Molecular Cloning Techniques;
`Academic Press, N. Y., N. Y., (1987) pp. 316-325.
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`ence Guide, (1988) Bethesda Research Laboratories,
`Bethesda, Md., p. 37.
`Cox et al., J. Med. Microbiol., vol. 35 (1991), pp.
`284—290.
`
`J. S. Tyagi et al., Trop. Med. Parasitol. 41 (1990), pp.
`294-296.
`
`Ariosa Exhibit 1020, pg. 3
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 3
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 1 of 26
`
`5,399,491
`
`5'
`"I09;
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`
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`
`Ariosa Exhibit 1020, pg. 4
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 4
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 2 of 26
`
`5,399,491
`
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`
`Ariosa Exhibit 1020, pg. 5
`|PR2013—OO276
`'
`
`Ariosa Exhibit 1020, pg. 5
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 3 of 26
`
`5,399,491
`
`.
`
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`
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`
`Ariosa Exhibit 1020, pg. 6
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 6
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 4 of 26
`
`5,399,491
`
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`Ariosa Exhibit 1020, pg. 7
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 7
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 5 of 26
`
`5,399,491
`
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`Ariosa Exhibit 1020, pg. 8
`|PR2013—OO276
`
`3.
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`Ariosa Exhibit 1020, pg. 8
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 6 of 26
`
`5,399,491
`
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`
`Ariosa Exhibit 1020, pg. 9
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 9
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 7 of 26
`
`5,399,491
`
`.
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`P2
`
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`
`Ariosa Exhibit 1020, pg. 10
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 10
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 8 of 26
`
`5,399,491
`
`5'
`
`'~
`
`"W0
`w FIRST PRIMER /SPLICE TEIII’LATE I+)
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`
`Ariosa Exhibit 1020, pg. 11
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 11
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 9 of 26
`
`5,399,491
`
`5.
`FIRST PRINERI+)
`I E RNA TARGET I—)
`IONASYNTHESIS
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`/L76. //.9
`
`Ariosa Exhibit 1020, pg. 12
`|PR2013—OO276
`
`3.
`
`4-
`
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`
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`
`Ariosa Exhibit 1020, pg. 12
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 10 of 26
`
`5,399,491
`
`'
`
`FIRST PRIMER (+I
`5'
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`
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`
`ANNEAI. PRIMER
`
`F/6.
`
`//.
`
`Ariosa Exhibit 1020, pg. 13
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 13
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 11 of 26
`
`5,399,491
`
`/:/C; /;,_
`
`I ‘ :IRRRR5:°
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`
`Ariosa Exhibit 1020, pg. 14
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 14
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 12 of 26
`
`5,399,491
`
`I.
`
`5 E FIRST PRINER/SPLICE TEMPLATE (+I
`
`RNA TARGET I-)
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`/F76.
`
`//.
`
`Ariosa Exhibit 1020, pg. ‘I5
`|PR2013—OO276
`
`
`
`PRO I—I
`RNA SYNTHESIS
`
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`5' FIRST PRIMER EXTENSION PRODUCT (+I
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`DNA SYNTHESIS
`
`RNA PRODUCT I-I
`
`Ariosa Exhibit 1020, pg. 15
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 13 of 26
`
`5,399,491
`
`TEST PRINER (+)
`5.
`I. E RNA TARGET (-I
`IONA SYNTHESIS
`
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`
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`
`Ariosa Exhibit 1020, pg. 16
`|PR2013—OO276
`
`RNA PRODUCT I-I
`
`(‘I-I
`
`Ariosa Exhibit 1020, pg. 16
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 14 of 26
`
`5,399,491
`
`1
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`-
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`FIRST PRIMER (+)
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`
`Ariosa Exhibit 1020, pg. 17
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 17
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 15 of 26
`
`5,399,491
`
`/-76.
`
`/0.
`
`5.
`‘WI
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`
`
`Ariosa Exhibit 1020, pg. 18
`|PR2013—00276
`
`Ariosa Exhibit 1020, pg. 18
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 16 of 26
`
`5,399,491
`
`|
`
`'
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`2
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`
`F76. 20.
`
`Ariosa Exhibit 1020, pg. 19
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 19
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 17 of 25
`
`5,399,491
`
`H6. Eb.
`
`I
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`
`RNA SYNTHESIS
`
`Ariosa Exhibit 1020, pg. 20
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 20
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 18 of 26
`
`5,399,491
`
`-
`
`5
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`
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`
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`5.
`
`5.
`
`7-
`
`
`
`FIRST smcs TEMPLATE-PRIMER (+)
`PR0 ‘+’
`"-fi___.._ FIRST SPLICE TEMPLATE-PRIMER
`
`pm (1)
`
`EXTENSION Rmoucr (->
`
`DNA smmssas
`
`msr SPUCE IEMRLAIR—RRm:R <+)
`"*0 ""
`5-
`3- ' RRsr SPLICE IEMPLATE-PRMER
`
`EXTENSW PROMET H
`
`RNA SYNTFESIS
`
`Ariosa Exhibit 1020, pg. 21
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 21
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 19 of 26
`
`5,399,491
`
`‘
`
`'
`
`2
`
`'
`
`3-
`
`5'
`
`DNA TARGET I+I
`FIRST PRIIER I-I
`
`lam snmisus
`
`5' DNA TARGET I+I
`FIIST PRIER EXTENSION PRODUCT I—)
`Isemme snmos
`5'T INA TARGET (+I
`—.——.—.
`FIRST PRIIIER-EXTENSION PRODUCT I-I
`
`/-76. 24.
`
`'
`
`5
`
`'
`
`FIRST PRIMER EXTENSION PRDDUCT I-I
`
`FRST PRINER EXIENSDN PRDDLKIT I-I
`
`imam mask
`(1.)
`5!
`4 % SEMI FRIIERISPLICE TEWLATE (+)
`lam SYIITIESIS
`5'm SECOND PRIER EXTENSION PRODUCT I+)
`lam smmssas
`5_ 5' RNA PRDIXICT II»)
`
`IN-I000 DDPIE5
`
`60 TD AUTDCATALYTIC AIIPLIFICATIDN METIIDD
`
`7
`
`~
`
`3-
`
`9.
`
`ANIEAL PRIMER
`
`5-
`
`RNA PRODUCT <+)
`ms: PRIIER I-)
`
`DNA smuesus
`
`5'
`
`RNA Pmoucr (+)
` o ms: mm EXTENSDN mowcw I-I
`
`oscsuoe RNA
`
`FIRST PRIMER EXTENSION PRODUCT I-I
`
`ANIHL PRIMER
`
`
`
`'0 § SEWND
`-
`FIRST Hm EXTENSKIN PRIXIUCT (-I
`loom snmrsns
`
`
`
`
`sscom mm sxrsusnn moucr In-I
`
`FIRST PRIAER EXTENSION PRIXAJCT I-I
`
`RNA SYNTHESIS
`
`Ariosa Exhibit 1020, pg. 22
`|PR2013—OO276
`
`PRO (+)
`
`H
`
`5» 9'” “"
`
`Ariosa Exhibit 1020, pg. 22
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar.»21, 1995
`
`Sheet 20 of 26
`
`5,399,491
`
`1
`
`"
`
`2
`
`'
`
`3.
`
`5'W DNA TARGET (+)
`
`FIRST PRIMER I-)
`
`DNA SYNTHESIS
`
`5‘ ' °"‘ W‘ ""
`KSEPARATESTRANDS
`
`FIRST PRIMER EXTENSIRI
`
`5'S DNA TARGET (+)
`
`PRODUCT I-I
`
`9 FIRST PRIMER EXTENSION PROIIJCT I-—)
`
`ANNEAL PRIMER
`
`'
`
`FIRST PRIMER EXTENSDN PRODUCT (—)
`
`9709)
`5'
`4. E SPLEE TEIPLATE (+)
`lam SYNIPESIS
`5-
`smce TEMPLATE (+)
`5- 9:-—. msr mum EXTENSION mooum I-I
`lam svmsus
`no-
`5' cI£xIEs§1 RNA PRODUCT (+)
`
`6.
`
`-I
`
`'
`
`ANNEAI. PRIIER
`
`GO ID AIJTOCATAUTIO AMPLFICATDN IETHOD
`
`5‘ RNA FROIXICT (+)
`IONA SYNTHESIS
`
`FIRST PRIMER I-I
`
`lREIKIVE RNA FRM FRST
`
`PRIAEREXTENSICN PROIAXIT
`
`
`
`
`9. S FRST PRIER EXTENSION PRODUCT I—)
`
`lmm FRIIIER
`smcs IEIIPIAIE (+)
`msw PRIMER EXIENSKIN mower (-I
`
`5*
`
`3
`
`.0
`
`H
`
`'
`
`‘
`
`PRO (+)
`
`I
`
`IWASYNTIESIS
`
`
`
`RNASYIITIESIS
`
`smcs TEMPLATE In
`msr Pm sxmasuou mom I-)
`
`Ariosa Exhibit 1020, pg. 23
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 23
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 21 of 26
`
`5,399,491
`
`L E FIRST PRIMER (+)
`DNA TARGET H
`
`DNA SYNTHESIS
`
`3
`
`'
`
`'
`
`5'
`P309)
`2. msr mm EXTENSION PRODJCT (+)
`LSEPERATE DNA STRANDS
`
`DNA TARGET I—I
`
`PRO (+)
`S‘2 FIRST PRKR EXTERSIIYI PRODUCT (+)
`lAIRIEAL PRIER
`5' FIRST PRIMER EXTENSION PRODUCT (+)
`seooun PRIIIER I-I
`lama SYNTHESIS
`5 5 FIRST PRIIIEREXTENSION PIODOCT (+)
`LRIIA SYNTHESIS
`
`FROM
`
`SHJOID PIIIIER EXTENSDII PRIIIUCT (—)
`
`IO0—IO0O COPIES
`5' 8:15:31 RNA PRODUCT (+)
`
`GO TO AUTOCATAUTTC AMPLIFICATION IETHOD
`
`ANNEAL PRIMER
`
`5' RNA PROTIET (+)
`lONA SYNTHESIS
`
`SECOND PRMER (—)
`
`5
`
`RNA PRODUCT (+)
`SECOND PRIIER EXTENSION PRODUCT (—)
`
`lnecmne RNA
`
`2 SECOND PRIMER EXTENSION PRODIKST (-I
`
`SECOIII PRIMER EXTENSION PRIDUCT I-I
`
`
`
`3.
`
`4-
`
`‘
`
`6.
`
`7
`
`'
`
`8
`
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`
`'
`
`LANNEAL PRIIER
`,
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`SmFlTSTPRlAER/SPLICETEMPLATE(+)
`lDNA SYNTHESIS
`
`,
`
`PRO (+)
`
`w_
`
`5
`
`FRST PRIIER EXTENSION PRODJCT (+)
`SECOID PRIER EXTENSION PRIJUCT (-)
`
`RNA SYNTHESIS
`
`Ariosa Exhibit 1020, pg. 24 '
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 24
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 22 of 26
`
`5,399,491
`
`
`
`AMPUFICATION SYSTEM - AUTOCATALYTIC CYCLE
`
`100-1000 GOHES
`I, m: RNA PRODUCT (—)
`
`mm mm
`
`2
`
`FRST PRIMER <+>
`5-
`' ) RNA mooucx (—)
`
`DNA svunvzsus
`
`ANNEAL PRIMER
`
`5
`
`'
`
`5' FRST PRIMER EXTENSION PRODUCT (+)
`PR0 H SECOND PRIMER/SPLICE TEMPLATE (-)
`
`DNA SYNTHESIS
`
`
`
`
`
`
`FRST PRKR EXTENSKN PR(IlX)T (+)
`
`SECOWPRIMER/SPLICE TEIPLATH-)
`
`
`
`PRO (-)
`
`RNA SYNFHESIS
`
`P76. 40.
`
`Ariosa Exhibit 1020, pg. 25
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 25
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 23 of 26
`
`5,399,491
`
`!
`
`RNA TARGET WITH 5' END OF TARGET & TARGET REGION IOENTICAL
`5'
`FIRST PRIMER (+)
`' I RNA TARGET (—)
`IIIIIA SYNTHESIS
`FIRST PRIMER EXTENSION PROOIICT I+)
`5'
`' s RNA TARGET (—)
`IRNose HIIGRADATKIN If RNA
`FIRST PRINER EXTENSION PRODUCT (+)
`5-
`3. - RNA TARGET I-I
`
`2
`
`IANIEAL PRIMER
`5-
`FIRST PRIMER EXTENSION PRODUCT (+)
`4- s SECOND PRMER/SPLICE TEMPLATE I-I
`P1909
`
`mu SYNTIESIS
`
`5-
`
`5- c secono PRIER EXTENSION PRODUCT(-)
`IIIIII SYNTHESIS
`
`FIRST PRIMER EXTENSION PRODUCT I+)
`
`IOO-IOOO COPIES
`6. En RNA PRODUCT I-I
`
`lANNEAI. PRIMER
`FIRST PRIMER I+)
` E$ RNA PRODUCT (-)
`IIIIIA SYNTHESIS
`5‘ FIRST PRIMER EXTENSION PRODUCT I+I
`IRNose H DEGRADATIONIfRNA
`.
`5‘
`FRST PRIAER EXTENSION PRIDUCT (+I
`' ' RNA PImIIcI (-)
`
`5-
`
`3,
`
`.
`
`7
`
`'
`
`9
`
`RNA PRODUCT I-I
`
`
`
`ANNEAL PRIMER
`
`,0
`
`msr PRIMER EXTENSION PRODUCT (+)
`5-
`- o SECOND PRIMER/SPLICETEIPLATE I-I
`
`PRO I-I
`
`
`
`PROI-I
`
`RNA SYNTHESIS
`
`
`
`FIRST FRIER EXTENSDN PRIDOCT (+)
`SECOND PRIMERISPLICE TEIPLATE I-I
`
`Ariosa Exhibit 1020, pg. 26
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 26
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 24 of 26
`
`5,399,491
`
`_
`
`AMPLIFICATION SYSTEM-GENERAL AETNOD
`RNA TARGET IITH TM-TARGET SEGENES 5'10 TARGETREGNNT
`IITIATDN |£CHAN|SN I
`
`1
`
`PM(
`
`msr PRIMER (+>
`
`RNA mam-)
`
`INA SYNTHESIS
`
`5 Q”
`1
`5'
`570(+1
`
`
`lllllllllllllllllllllllllllllllll
`xu.1§§11Q‘§11§111\‘§§\
`
`
`
`FIRST PRTMER EXTENSDN PRGJUCT (+)
`
`RNA TARGET (-)
`
`
`
`lmase H o£cRAnAmA or RNA
`
`5'2 FIRST PRmER Exmsaon mmuct (+)
`
`”‘° ""
`
`5«
`
`'
`
`mo <+)
`
`5'
`
`lANNEAL mm
`FIRST mmm EXTENSION PRODUCT (+)
`W secom mmea (-)
`lamsmuesns
`
`Has! PHMER EXTENSION PRODUCT (+)
`sscom PRIMER EXTENSKN mooucn-)
`
`lam smnssns
`5'm' RNA PRODUCT (+)
`LANNEAL PRIMER
`5' RNAPRWUCT (+)
`
`8 . “""°“°°“°‘ ‘*’
`lRNuse H IIGRADATION (I RNA
`5' SECOND PRIMER EXTENSDN PRODUCT (-)
`LANNEAL PRIMER
`
`,
`
`'
`
`3.
`
`4-
`
`5
`
`'
`
`6.
`
`7
`
`'
`
`'
`
`9.
`
`
`
`secouo mmsm-1
`
`mA smmssxs
`
`SE60?!) PRIDER EXTENSKN PRODUCT (—)
`
`'
`
`H
`
`'
`
`5-
`
`Pm ""
`
`H6. 46.
`
`sscono PRIMER EXTENSTON mooucr (-)
`
`loomsmnssns
`
`RNA smusszs
`
`
`
`msmm/svucs TEMPLATE (+)
`
`sscouo mum EXTENSION PRODUCTH
`
`Ariosa Exhibit 1020, pg. 27
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 27
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 25 of 26
`
`5,399,491
`
`RNA TARCET VIITII IDN—TAR(£T SEOIENCES 5'TO TARGET REGION INITIATION IECIIANISN II
`
`'
`
`RNA macs (—)
`
`‘
`
`5-
`Pm)
`, E FIRST PRIMER (+)
`lam SYNTHESIS
`
`lRNose HOECRAOATKNIf RNA
`
`(+)
`
`IllllllllllllIllllllllllllllllll
`tQ‘§\\§'\\.1“1§.\‘§\\\\‘
`
`
`
`
`FIRST PRIMER EXTENSION PRODUCT (+)
`
`RNA TARGET I-I
`
`PRO (+)
`
`4-
`
`5 ' sacono PRIMER (-)
`IRMA SYNTHESIS
`
`‘ FIRST PRIIER EXTENSION PRODUCT (+)
`
`
`
`5|
`
`
`
`5«
`RNA mooucr (+)
`8- sscouo PRIMER EXTENSION mowcr (—I
`lmaose H oscnmmou or RNA
`9- - seoouu PRIMER EXTENSION PROIIICT I-I
`lANNEAI. PRIMER
`FRST PRIMER/SPLICE TEMPLATE (+)
`Pm (+)
`5-
`- o secoun PRIMER EXTENSION PROOIXETI-I
`lam SYNTHESIS
`
`
`,0
`
`H
`
`5-
`
`P” “’
`
`FIRST PRIMER/SPLICE TEMPLATE (+)
`
`SECOND PRIIER EXTENSION PRODUCT (-I
`
`
`
`RNA SYNTHESIS
`
`Ariosa Exhibit 1020, pg. 28
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 28
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Mar. 21, 1995
`
`Sheet 26 of 26
`
`5,399,491
`
`/-76. 5.
`
`Ariosa Exhibit 1020, pg. 29
`|PR2013—OO276
`
`Ariosa Exhibit 1020, pg. 29
`IPR2013-00276
`
`

`
`1
`
`5,399,491
`
`NUCLEIC ACID SEQUENCE AMPLIFICATION
`METHODS
`
`