(12) United States Patent
`US 6,329,150 B1
`(10) Patent N0.:
`
`Lizardi et al.
`(45) Date of Patent:
`*Dec. 11, 2001
`
`USOO6329150B1
`
`(54) UNIMOLECULAR SEGMENT
`AMPLIFICATION AND SEQUENCING
`
`(75)
`
`Inventors: Paul M. Lizardi, Cuernavaca (MX);
`Michael Caplan, Woodbridge, CT (US)
`(73) Assignee: Yale University, New Haven, CT (US)
`( * ) Notice:
`Subject. to any disclaimer, the term of this
`patent ls extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`21121111;patent is subject to a terminal dis-
`
`(21) Appl. No.: 09/602,428
`
`(22)
`
`Filed:
`
`63
`
`)
`
`(
`
`Jun. 23: 2000
`Related U'S' Application Data
`N
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`f
`1.
`.
`N 08754681 fil d
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`C
`210Iggy:tlgélwoPg)Flngfit6101123 :95 Which is :1 coitigflatigl
`in—part of application N0. 08/563,912, filed on Nov. 21,
`1995, now Pat. No. 5,854,033
`Provisional application No. 60/016,677, filed on May 1,
`1996-
`Int. Cl.7 .............................. C12Q 1/68; C12P 19/34;
`C07H 21/02; C07H 21/04; C12N 15/00
`............................ 435/6; 435/91.2; 536/231;
`536/243; 935/76; 935/77; 935/78
`(58) Field of Search ..................... 435/6, 91.2; 536/231,
`536Q43; 935/76, 77, 78
`
`(60)
`
`(51)
`
`(52) US. Cl.
`
`(56)
`
`References Cited
`
`.
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`
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`Primary Examiner—Ethan Whisenant
`(74) Attorney) Agenh 0r Firm—Needle & Rosenberg, PC.
`(57)
`ABSTRACT
`
`Disclosed are compositions and a method for amplification
`0f and mumplex daemon 0f m01eCU1eS Of interest iHVolVing
`rolling circle replication. The method is useful for simulta-
`neously detecting multiple specific nucleic acids in a sample
`with high specificity and sensitivity. The method also has an
`inherently low level of background signal. A preferred form
`of the method consists of an association operation, an
`amplifi‘iation OPCFatiOF1> and a deuxtlon Operation The
`assoc1atlon operatlon mvolves assoc1atlon of one or more
`specially designed probe molecules, either wholly or partly
`nucleic acid, to target molecules of interest. This operation
`associates the probe molecules to a target molecules present
`in a sample. The amplification operation is rolling circle
`replication of circular nucleic acid molecules, termed ampli-
`fication target circles, that are either a part of, or hybridized
`to,
`the probe molecules. A single round of amplification
`“Sing “’11ng Cirde replication results in a large amplifica'
`tion of the amplification target circles. Following rolling
`circle replication, the amplified sequences are detected using
`combinatorial multicolor coding probes that allow separate,
`simultaneous, and quantitative detection of multiple differ-
`ent amplified target circles representing multiple different
`target molecules. Since the amplified product is directly
`proportional to the amount of target sequence present in a
`sample’ quantltatlve measurf’mems rehably.represem the
`amount Of a target sequence 1“ a sample~ Mal“ advantages
`of this method are that a large number of distinct target
`molecules can be detected simultaneously, and that differ-
`ences in the amounts of the various target molecules in a
`sample can be accurately quantified. It is also advantageous
`that the DNA replication step is isothermal, and that signals
`are strictly quantitative because the amplification reaction is
`linear and is catalyzed by a highly processive enzyme.
`
`43 Claims, 43 Drawing Sheets
`
`Ariosa Exhibit 1031, pg. 1
`|PR2013—00276
`
`Ariosa Exhibit 1031, pg. 1
`IPR2013-00276
`
`

`

`US 6,329,150 B1
`
`Page 2
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`Ariosa Exhibit 1031, pg. 2
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 2
`IPR2013-00276
`
`

`

`US 6,329,150 B1
`
`Page 3
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`Ariosa Exhibit 1031, pg. 3
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 3
`IPR2013-00276
`
`

`

`US 6,329,150 B1
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`IPR2013-00276
`
`

`

`US 6,329,150 B1
`Page 5
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`* cited by examiner
`
`Ariosa Exhibit 1031, pg. 5
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 5
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 1 0f 43
`
`US 6,329,150 B1
`
`SINGLE— STRANDED TARGET
`
`LEFT PROBE/P RIGHT PROB
`
`
`5‘- PHOSPHATE AT THE END
`OF THE RIGHT PROBE
`
`OPEN CIRCLE PROBE
`
`F/G.
`
`7
`
`SINGLE— STRANDED TARGET
`
`P P
`
`LEFT PROBE \RTGHT PROBE
`
`GAP OLIGONUCLEOTIDE
`
`WITH 5'- PHOSPHATE
`
`OPEN CIRCLE PROBE
`
`FIG. 2
`
`Ariosa Exhibit 1031, pg. 6
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 6
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 2 0f 43
`
`US 6,329,150 B1
`
`SINGLE—STRANDED TARGET
`
`
`
`ROLLING CIRCLE
`REPLICATION
`PRIMER
`
`LIGATED OPEN CIRCLE PROBE
`
`FIG. 3
`
`ROLLING CIRCLE AMPLIFICATION
`
`SINGLE—STRANDED
`TARGET
`
`
`
`LIGATED
`OPEN
`CIRCLE
`
`TANDEM
`SEQUENCE
`DNA (TS — DNA)
`
`F/G. 4
`
`Ariosa Exhibit 1031, pg. 7
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 7
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 3 0f 43
`
`US 6,329,150 B1
`
`OPEN CIRCLE PROBE
`
`
`
`II
`
`‘
`
`[1"
`
`TARGET PROBE (LEFT AND RIGHT
`TARGET PROBES)
`
`II
`
`II
`
`PROMOTER
`
`PRIMER COMPLEMENT
`
`TARGETS)
`
`DETECTION TAGS (OR SECONDARY
`
`£110
`
`GAP OLIGONUCLEOTIDE
`
`F/G. 5
`
`Ariosa Exhibit 1031, pg. 8
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 8
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 4 0f 43
`
`US 6,329,150 B1
`
`ADDRESS PROBE HYBRIDIZING TO TS—DNA PORTION
`BRIDGING GAP OLIGONUCLEOTIDE AND TARGET PROBE ENDS
`
`5'—CCTT—
`
`-3'
`
`GAP
`(MJGONUCLEOHDE
`
`5'—TTTTTTTTTTTTTTTGTATTCCTTGCCTG —3'
`
`ADDRESS
`PROBE
`
`HYBRIDIZATION OF TS-DNA AND ADDRESS PROBE
`
`3' ~ACAGACGACGGGAGACllkTAAl-CGWAICISAGGT:CCTAGACGAGT -g'
`8
`NA
`I
`II
`/GTATTCT CCTG
`—
`ADDRESS PROBE
`5'—TTTTTTTTTTTTTTT/
`
`F/G. 6
`
`DETECTION PROBES WITH
`FLUORESCENT LABELS
`
`
`
`
`a =GAP OLIGONUCLEOTIDE
`STATE
`STATE
`
`
`
`
`COMPLEMENT
`DETECTOR
`DETECTOR
`
`
`
`=PRIMER SEQUENCE
`
`
`
`=SEQUENCE TAGS FOR
`SECONDARY DETECTION
`
`= PROMOTER COMPLEMENT
`
`F/G.
`
`7
`
`Ariosa Exhibit 1031, pg. 9
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 9
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 5 0f 43
`
`US 6,329,150 B1
`
`LM-RCA FOLLOWED BY TRANSCRIPTION
`
`TARGET
`
` PROMOTER
`
`OCP
`
`F/G‘. 8
`
`UNLIGATED OPEN CIRCLE
`PROBES HYBRIDIZE T0 T8—
`
`DNA GENERATING MULTIPLE
`SITES OF TRANSCRIPTIONAL
`INITIATION
`
`
`
`ROLLING
`CIRCLE
`REPLICATION
`
`UNLIGATED OPEN
`CIRCLE PROBE
`
`
`
`
`0..
`l...'0
`UNLIGATED OPEN
`""1"...
`
`CIRCLE PROBE
`PROMOTER
`
`"""'
`
`
`
`T
`
`ROLLING CIRCLE
`REPLICATION
`PRIMER
`
`Ariosa Exhibit 1031, pg. 10
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 10
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 6 0f 43
`
`US 6,329,150 B1
`
`REPORTER ANTIBODIES
`
`5'————————3'
`
`A
`
`1962 )V5 45 BASES
`
`'——————— '
`
`3
`
`3
`DIFFERENI
`DNA
`
`TAGS
`
`DNA 2
`
`IqGB )Q‘5m3 DNA3
`
`ASSAY
`
`)KN
`ANALYTE—p %
`\ /
`ll
`IMMOBILIZED
`_
`IMMUNO COMPLEX
`
`f
`LM—RCA OR M /V
`LM_RCT
`rf/i/
`USING 3 SETS
`OF PROBES
`/V"
`
`/‘/
`
`AMPLIFIED
`DNA OR RNA
`CORRESPONDING
`T0 DNA TAG
`SEQUENCE
`THAT WAS
`BOUND
`
`F/G.
`
`9
`
`Ariosa Exhibit 1031, pg. 11
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 11
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 7 0f 43
`
`US 6,329,150 B1
`
`DETECHON EXAMPLE
`
`F/G.
`
`IO
`
`,
`
`/\//\/
`/\//\/
`//V'/1>/
`
`AMPLIFIED DNA
`OR RNA
`#2
`
`FLUORESCENT
`
`MOETY‘
`
`————————————>-
`HYBRHMZAHON
`WITH THREE
`SPECIFIC DETECTION PROBES
`wrn41NFFERENT
`LABELS
`
`#2
`
`DETECHON EXAMPLE
`
`/avrv ,1/
`
`//t;i_/j/
`
`AMPLNHED DNA
`0R RNA
`
`#2
`
`HYBRHNZATwN
`
`...____—_—’
`
`DETECTOR
`SURFACE
`
`1
`
`C)
`
`AMMJFED RNA
`BINDS TO DOT #2
`BY HYBRIDIZATION
`
`Ariosa Exhibit 1031, pg. 12
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 12
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 8 0f 43
`
`US 6,329,150 B1
`
`F/ G.
`
`7 70
`
`TARGET
`
`LIGATION
`
`|lllllillllllllll
`
`0 c P
`
`ROLLING CIRCLE
`REPLICATION
`
`
`
`TARGET
`
`
`
`P2
`
`
`
`SECONDARY DNA STRAND
`DISPLACEMENT PRIMER (P2)
`
`IS PRESENT AT HIGH
`CONCENTRATION AND
`HYBRIDIZES RAPIDLY T0
`T8— DNA.
`
`
`
`Ariosa Exhibit 1031, pg. 13
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 13
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 9 0f 43
`
`US 6,329,150 B1
`
`F/G‘.
`
`lib
`
`TARGET
`
`OPPOSITE
`STRAND
`
`l
`
`5
`
`Aw"
`
`: E'
`
`oofillni"“
`
`:
`:0
`
`POLYMERIZATION AND STRAND
`DISPLACEMENT BY
`W DNA
`POLYMERASE IN A
`MEL—ml;
`ZATION REACTION.
`
`.y
`e"
`,3
`.o’
`
`.
`
`5
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`I
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`
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`t
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`.
`{0"
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`.~
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`I
`
`n'
`I
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`'o
`’v
`9o
`'0.
`
`'0
`
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`
`"""n 3‘
`
`5|
`
`Ariosa Exhibit 1031, pg. 14
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 14
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 10 0f 43
`
`US 6,329,150 B1
`
`F/G.
`
`72
`
`GAP PROBE
`FIRST OPEN __
`TARGET
`CIRCLE PROBE
`
`
`
`SECONDARY
`PRIMER
`
`SECONDARY OPEN
`CIRCLE PROBE
`
`TARGET
`
`
`
`ROLLING CIRCLE
`REACTION DOES NOT
`LOOSEN THE FIRST
`OPEN CIRCLE PROBE
`
`
`
`Ariosa Exhibit 1031, pg. 15
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 15
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 11 0f 43
`
`US 6,329,150 B1
`
`STRAND DISPLACEMENT CASCADE REACTION
`
`P‘
`
`ROLLING
`GRCLE
`
`P2
`
`P2
`
`
`
`TS-DNA-4
`Ts—DNA-4
`
`TS—DNA-2
`
`TS-DNA—1
`
`
`13 ['0
`
`P2
`
`P2
`
`F76:
`
`73
`
`Ariosa Exhibit 1031, pg. 16
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 16
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 12 0f 43
`
`US 6,329,150 B1
`
`OPPOSITE STRAND AMPLIFICATION
`
`RELEASED SET OF TSDNA—2
`
`RELEASED SET OF
`
`TS - DNA — 2
`
`%\
`
`/
`
`O TS‘DNA
`(\r’C
`
`TS - DNA—2
`
`RELEASED
`SET OF
`
`RELEASED
`SET OF
`TS-DNA—2
`
`F/G.
`
`l4
`
`Ariosa Exhibit 1031, pg. 17
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 17
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 13 0f 43
`
`US 6,329,150 B1
`
`F/G‘.
`
`l5
`
`TARGET SEQUENCE
`
` SPACE
`
`
`LEFT TARGET
`W PROBE PORTION
`
`SPACER
`PORTION
`
`
`
`RIGHT TARGE
`PROBE
`PORTION
`
`guRTEMRERROGATION
`I
`MATCHING \
`
`0GP
`
`INTERROGATION
`PRIMER
`/ COMPLEMENTARY
`
`TO OCP
`
`
`SEQUENCE
`
`Ariosa Exhibit 1031, pg. 18
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 18
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 14 0f 43
`
`US 6,329,150 B1
`
`Somaticmu’ro’rion
`
`F/GI66
`
`Homozygous
`
`
`
`F/G.16,4
`
`HeTerozygous
`
`
`
`F/G./65
`
`|—®Q<[
`
`$69690
`
`Ariosa Exhibit 1031, pg. 19
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 19
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`SheetlS 0f43
`
`US 6,329,150 B1
`
`~_:________________________________________
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`AmmwfluowfiosqomvmoqmsvmmHaflm
`
`Ariosa Exhibit 1031, pg. 20
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 20
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 16 0f 43
`
`US 6,329,150 B1
`
`E g L
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`Ariosa Exhibit 1031, pg. 21
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 21
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 17 0f 43
`
`US 6,329,150 B1
`
`Slide5
`
`Figure19
`
`
`
`Ariosa Exhibit 1031, pg. 22
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 22
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 18 0f 43
`
`US 6,329,150 B1
`
`Slide5
`
`Figure20
`
`
`
`Ariosa Exhibit 1031, pg. 23
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 23
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet 19 0f 43
`
`US 6,329,150 B1
`
`«Hmmusmflm
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`HUBUBUHUHwflHUUB¢UUB¢UU¢<HUH¢U¢UUHUHm30%
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`
`UH¢U¢B¢U¢G¢HBU<U¢U¢U¢U¢U¢U¢U<U<U¢UBdwwflHUUflHUUBHdUdBUHUU<U¢B
`
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`
`HmUflHm
`
`Ariosa Exhibit 1031, pg. 24
`|PR2013-00276
`
`Ariosa Exhibit 1031, pg. 24
`IPR2013-00276
`
`

`

`US. Patent
`
`Dec. 11,2001
`
`Sheet20 0f43
`
`US 6,329,150 B1
`
`mammusmflm
`
`
`
`
`
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