`
`111111 111111
`
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`1111111111111111111111111111111111111111111111111111111111111111111111111111 1111111111111111111111111111111111111111111111111111111111111111111111111111
`
`us 20160017392Al us 20160017392Al
`
`
`(19) United States (19) United States
`
`(12) Patent Application Publication (12) Patent Application Publication
`
`Arnold et al. Arnold et al.
`
`
`(10) Pub. No.: US 2016/0017392 Al (10) Pub. No.: US 2016/0017392 Al
`
`Jan. 21, 2016 Jan. 21, 2016
`
`(43) Pub. Date: (43) Pub. Date:
`
`
`(54) METHODS FOR AMPLIFICATION OF (54) METHODS FOR AMPLIFICATION OF
`
`NUCLEIC ACIDS ON SOLID SUPPORT NUCLEIC ACIDS ON SOLID SUPPORT
`
`
`(71) Applicants: Lyle J. ARNOLD, Poway, CA (US); (71) Applicants: Lyle J. ARNOLD, Poway, CA (US);
`
`Norman C. NELSON, San Diego, CA Norman C. NELSON, San Diego, CA
`
`(US) (US)
`
`
`
`(72) (72)
`
`
`Inventors: Lyle J. Arnold, Poway, CA (US); Inventors: Lyle J. Arnold, Poway, CA (US);
`
`Norman C. Nelson, San Diego, CA Norman C. Nelson, San Diego, CA
`
`(US) (US)
`
`
`
`(21) Appl. No.: (21) Appl. No.:
`
`
`
`141773,362 141773,362
`
`
`
`(22) PCTFiled: (22) PCTFiled:
`
`
`
`Mar. 14, 2014 Mar. 14, 2014
`
`
`
`(86) PCTNo.: (86) PCTNo.:
`
`
`
`PCT/US2014/029817 PCT/US2014/029817
`
`
`§ 371 (c)(1), § 371 (c)(1),
`
`(2) Date: (2) Date:
`
`
`
`Sep.7,2015 Sep.7,2015
`
`
`
`Related U.S. Application Data Related U.S. Application Data
`
`
`(60) Provisional application No. 611781,356, filed on Mar. (60) Provisional application No. 611781,356, filed on Mar.
`
`14,2013. 14,2013.
`
`
`
`Publication Classification Publication Classification
`
`
`
`(51) (51)
`
`
`Int. Cl. Int. Cl.
`
`C12P 19/34 C12P 19/34
`
`
`
`(2006.01) (2006.01)
`
`
`(52) U.S. Cl. (52) U.S. Cl.
`
`CPC ...................................... C12P 19/34 (2013.01) CPC ...................................... C12P 19/34 (2013.01)
`
`(57) (57)
`
`ABSTRACT ABSTRACT
`
`The present invention provides methods for amplifying a The present invention provides methods for amplifying a
`
`nucleic acid from a sample containing a mixture of nucleic nucleic acid from a sample containing a mixture of nucleic
`
`acids utilizing a solid support. Methods are provided utilizing acids utilizing a solid support. Methods are provided utilizing
`
`user-defined primer oligonucleotides for directional amplifi(cid:173)user-defined primer oligonucleotides for directional amplifi(cid:173)
`
`cation that assists in further manipulation of the target nucleic cation that assists in further manipulation of the target nucleic
`
`acid, such as sequencing. Methods are also provided utilizing acid, such as sequencing. Methods are also provided utilizing
`
`blocker and displacer oligonucleotides for generating ampli(cid:173)blocker and displacer oligonucleotides for generating ampli(cid:173)
`
`fied target nucleic acids of defined length. One of these meth(cid:173)fied target nucleic acids of defined length. One of these meth(cid:173)
`
`ods provides a first oligonucleotide and a second oligonucle(cid:173)ods provides a first oligonucleotide and a second oligonucle(cid:173)
`
`otide affixed to a solid support or separate solid supports. The otide affixed to a solid support or separate solid supports. The
`
`first oligonucleotide is blocked to prevent extension from the first oligonucleotide is blocked to prevent extension from the
`
`3'-terminus and has a sequence complementary to a first por(cid:173)3'-terminus and has a sequence complementary to a first por(cid:173)
`
`tion of a target nucleic acid. The second oligonucleotide has tion of a target nucleic acid. The second oligonucleotide has
`
`a sequence that is identical to a second portion of the target a sequence that is identical to a second portion of the target
`
`nucleic acid. In this method, a sample is applied to the solid nucleic acid. In this method, a sample is applied to the solid
`
`support and the target nucleic acid within the sample binds support and the target nucleic acid within the sample binds
`
`said first oligonucleotide. The solid support is then washed to said first oligonucleotide. The solid support is then washed to
`
`remove unbound nucleic acids. A primer sequence containing remove unbound nucleic acids. A primer sequence containing
`
`a target binding region and a polymerase promoter sequence a target binding region and a polymerase promoter sequence
`
`is then annealed to the bonnd target nucleic acid and extended is then annealed to the bonnd target nucleic acid and extended
`
`producing a first duplex nucleic acid. The target sequence is producing a first duplex nucleic acid. The target sequence is
`
`then removed leaving a first nucleic acid that can now bind the then removed leaving a first nucleic acid that can now bind the
`
`second oligonucleotide. The second oligonucleotide is second oligonucleotide. The second oligonucleotide is
`
`extended to produce a second duplex nucleic acid that con(cid:173)extended to produce a second duplex nucleic acid that con(cid:173)
`
`tains a second nucleic acid. The second nucleic acid is then tains a second nucleic acid. The second nucleic acid is then
`
`amplified by adding a polymerase. amplified by adding a polymerase.
`
`Enzo Exhibit 2119
`BD v. Enzo
`Case IPR2017-00181
`
`Exhibit 2119 Page 1
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 1 of 15
`
`US 2016/0017392 Al
`
`FIGURE lA
`
`SOLID PHASE T7 AMPLIFICATION
`OF NUCLEIC ACID TARGETS
`
`./ CAPTURE OLIGOMER WITH
`3' CAP TO PREVENT EXTEN SION
`~--~; ~_/./--
`¥"""' ,t:,.. .... ---
`
`~ ~~~.-SOLID PHASE BOUND
`
`PRIMER
`
`{J.-- CAPTUR"E TARGET
`NUCLEIC ACID
`
`/ FOR RNA,
`¥/-' POSSIBLE POLY A TAIL
`,IV''''
`
`BIND PRIMER CONTAINING A TAIL
`WITH A T7 OR OTHER PROMOTER
`SEQUENCE
`
`- -•• - - - - - - - - - - -
`
`~~~---- T7 OR OTHER
`
`PROMOTER SEQUENCE
`
`1/)11
`
`U EXTEND PR!MER USING
`
`A POLYMERASE
`
`I I DENATURE, STRAND DISPLACE
`V OR CLEAVE WITH RNASE H,
`DEPENDING ON TARGET
`
`~.~~~~~/ ... --...
`
`"'-'-------- FIRST EXTENSION
`PRODUCT
`
`Exhibit 2119 Page 2
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 2 of 15
`
`US 2016/0017392 Al
`
`FIGURE lA CONT.
`
`----------------------~
`
`+
`
`/
`
`RECAPTURE FIRST EXTENS10N
`PRODUCT ON SU PPORT
`BOUND PRIMER
`
`~--_/
`
`{} EXTEND USING A POLYMERASE
`
`----f~.--FORMSDOUBLE
`~
`TTfTT
`
`STRANDED T7 OR
`OTHER PROMOTER
`
`I I AMPLIFY USING T7 OR OTHER
`V
`PROMOTER POLYMERASE
`
`+
`+
`. " I ! - - - - - - - - - - - -+ - . - - - - - - - - -
`
`..
`.. - - - - - - - - - - - - - - - - - - - - - -
`
`} FORMS A LARGE NUMBER
`OF SINGLE STRANDED RNA
`Af\.'lPLlCONS
`
`...
`...
`
`DETECT PRODUCTS USI~~G
`PROBES, DYES OR OTHER
`CONVENTIONAL METHODS
`
`Exhibit 2119 Page 3
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 3 of 15
`
`US 2016/0017392 Al
`
`FIGURE lB
`
`41(
`
`FURTHER AMPLIFICATION OF PRODUCTS
`FROM THE REACTION SHOWN IN FIGURE
`. . . , ; : - - - - - -+ - - - - - - 1 AMPUFICATION
`+
`l PRODUCTS FROM~ __ -.,
`..
`r THE REACTION
`... ______ + _____ . ...1 SHOWN IN FIGURE
`..
`• ..
`-tJ RECAPTURE ON PRlrli4ER
`
`SOUND SUPPORT
`
`II EXTEND
`'l...J'.
`
`REPEAT - - - - . J
`
`·nh·
`
`/ / / T
`
`CLEAVE WITH RNASE H,
`DENATURE OR DISPLACE
`
`HYBRIDIZE WITH PRIMER
`CONTAfNINGA TAtL WITHAT7 OR
`OTHER PROMOTER SEQUENCE
`
`~A'"-~~ Tl OR OTHER
`,------------....
`I
`I / 7 T
`
`PROMOTER
`SEQUENCE
`
`. . -
`
`EXTEND
`
`AMPUFYUS!NG T7 OR OTHER
`PROMOTER POLYMERASE
`
`Exhibit 2119 Page 4
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 4 of 15
`
`US 2016/0017392 Al
`
`FIGURE 2
`
`SOLID PHASE T7 AMPLIfiCATION USING isoClisoG
`CASSETTE TO FIX IN PLACE RANDOM PRIMING EVENTS
`
`~- CAPTURE OLIGOMER
`.--~-7 _. __ //--- WITH 3' CAP
`""' ...... ---
`17-:,
`--------.... I
`/ /7 / T7"7-T "--"--- SOLID PHASE BOUND
`
`~,...-
`
`PRIMER
`
`CAPTURE TARGET
`NUCLEIC ACID
`
`CD--------------------~~
`
`"//)/1
`
`I I BIND PRIMER WITH 3' RANDOt,,1 SEGMENT,
`V
`AN isoClisoG CASSETTE, AND A 5' T7 OR
`OTHER PROMOTER SEQUENCE
`. J.:.-...s.,;--~ .. "- T7 SEQUENCE
`.... --.. --Ie 1 ..... _<..
`~. -.~m~~..f~i~~¥lE~GMENT
`
`~. \
`
`"
`
`-"",.
`'-'---~ RANDOM SEQUENCE
`(TYPICALLY 6-9 BASES
`'''\
`\
`!N LENGTH)
`
`...
`
`/7/71
`
`® ......... ---------{( >{
`
`TARGET SEQUENCE
`
`I I DENATURE, STRAND DISPLACE
`V OR CLEAVE WITH RNASE H
`
`@ ... - - - - - - - - - - - - - l ( /-.~~~ .. " FIRST EXTENS!ON
`
`PRODUCT
`
`Exhibit 2119 Page 5
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 5 of 15
`
`US 2016/0017392 Al
`
`FIGURE 2 CONT.
`
`/
`•• ------------------4( }J
`+
`
`I I RECAPTURE FIRST EXTENSION
`V
`PRODUCT ON SUPPORT
`BOUND PRiMER
`
`I I • EXTEND USING POLYMERASE
`V
`., INCLUDE isoCTP ANDIOR isoGTP
`
`® ,'*:-I-I;-;.-.. -----------i( tl /' COrv1PLEMENTOF
`
`ORIGINAL CASSETTE
`
`... { ~_/ .. /
`
`I I AMPLIFY USING T? OR OTHER PROMOTER
`V
`POLYMERASE: INCLUDE isoCTP
`ANDIOR isoGTP
`
`CD
`
`041: .-------~----~( ~
`.-------------~( r(cid:173)
`+
`.. : ..
`+
`~--------+----~( ~
`....
`.---------.----~( ~
`•
`•
`I I REBIND TO PRIMER ON
`V SOLID SUPPORT
`
`® 71 ) ... :-;-, .. - - - - - - - i t )---
`
`Exhibit 2119 Page 6
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 6 of 15
`
`US 2016/0017392 Al
`
`FIGURE 2 CONT.
`
`'"
`
`) -
`
`I I .. EXTEND USING POLYMERASE
`V,. iNCLUDE iSOCTP AND/OR isoGTP
`
`GD
`
`•• ---------------4( r-
`r--
`
`-(
`
`I
`/ ill/ 7
`
`I I DENATURE, STRAND DISPLACE
`V OR CLEAVE WITH RNASE H
`
`r - - - - - - - - - - - - - - - - - - - { ( >-------
`
`I
`///77
`
`®
`I
`. 11777
`
`BIND PRIrI,,1ER WITH isoC/isoG
`CASSETTE SEQUENCE AND 1'7 TAIL
`
`EXTEND WITH POLYMERASE
`
`@
`
`""1-. -------{~ 0
`
`I
`7---;1----,11--,7~/-r-'
`
`'
`
`/
`
`I i AMPLIFY USING 1'7 OR OTHER PROMOTER
`V
`POLYMERASE: iNCLUDE 1soCTP
`AND/ORisoGTP
`, }--
`" -( } -
`
`..
`•
`
`II!
`
`oj
`
`+
`+
`+
`
`,. .. ,.
`JJ
`REPEAT TO STEP (1)
`
`( } -
`-{ } -
`
`'~:-'
`
`Exhibit 2119 Page 7
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 7 of 15
`
`US 2016/0017392 Al
`
`FIGURE 3
`
`METHOD FOR DIRECTIONALLY AMPLIFYING mRNA OR
`SINGLE STRANDED DNA FOR SEQUENCINE
`
`NUCLEIC AGIO CO~frAI~~!NG SAMPLE
`{FFPE, TISSUE, OTHER BIOLOGrCAL SAtv1PLE)
`
`-JJ -ISOLATE.NUCLEIC ACID
`_.+_«:-._-- mRNA OR SINGLE
`FRAGM ENT
`_________________________ ~
`STRANDED DNA
`
`I I CAPTU RE ON SUPPORT
`V' BOUND OLIGONUCLEOTIDE
`fi\ -------------------------~ 3'
`\V~5'
`
`HYBRIDIZE WITH OLIGOMER CONTAINING 3'
`BINDING REGION RANDOMER, A BARCODE, AND
`A TAIL SEQUENCE WHERE THE TAll IS USED IN
`SEQUENCING REACTIONS
`
`L ..... ,,_5'r~",·~TAIL SEQUENCE
`.... --- ~
`2..,,,,~",~ BARCODE (OPTIONAL)
`....
`\
`~'-.-. TARGET BINDING SEQUENCE
`
`® ~5'
`177l1l7
`
`II .. WASH
`V' .EXTEND
`
`------------------x~
`CD -.-------------------------~
`
`DENATURE, STRAND DISPLACE OR TREAT
`WITH RNASE H DEPENDING ON TARGET
`
`QD ... ----------------------~
`
`Exhibit 2119 Page 8
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 8 of 15
`
`US 2016/0017392 Al
`
`FIGURE 3 CONT.
`
`·------1 (
`
`CAPTURE TAIL SEQUENCE USING
`OUGO ON A SOLID SUPPORT
`
`/ / 4-'<'!:..~,, __ CAPPED TO PREVENT
`® • ... - - - - - - - - - - t l r 1/
`EXTENSION
`
`~
`!
`!
`!
`
`7////
`{j,. PRIME WITH OLiGOS WITH A RANDOM
`SECOND TAIL
`3' TARGET BINDING SEQUENCEAND A
`SEQUENCE (OPTIONAL) SECOND OPTIONAL TAIL USED IN SEQUENCING
`\
`REACTIONS
`5~ \,~~
`® >uu", __ ~,.u", 3'
`
`./
`_ /
`
`F'"-.... -
`
`f
`RAN DOMER
`SEQUENCE
`
`~J -\l\lASH
`.. > .. EXTEND \;\/ITH A POLYMERASE
`",DENATURE
`
`"
`
`t)"
`
`3'
`5'
`/3'
`-~
`(' ~ u_'S>----,uTAIL 1 SEQUENCE
`I:
`CD "~-----+-----j. (...-
`CORRESPONDS TO
`•
`OR!G!NAL 3' END OF
`\
`TARGET
`"--- BARCODE FOR SAMPLE
`lNDEXINGIlDENTI PICATION
`
`.. PCRAMPUFY USING TAIL
`SEQUENCiNG FOR PRIMING
`.. CONDUCT SEQUENCING USING
`TAILJADAPTER SEQUENCES
`
`Exhibit 2119 Page 9
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 9 of 15
`
`US 2016/0017392 Al
`
`FI GURE 3 CONT.
`
`OPTIONS:
`.. IN STEP r2i THE PRII\.1ING SEQUENCE MAY BE A DUPLEX OR
`A HAIRPi~W!TH AN OPTtONAL LlGAT!ON STEP TO ATTACH
`THE PRIMER DIRECTLY TO THE 3' END OF THE TARGET
`SEQUENCE
`
`• I / I
`
`r
`TARGET
`SEQUENCE
`
`Exhibit 2119 Page 10
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 10 of 15
`
`US 2016/0017392 Al
`
`FIGURE 4
`
`PR1
`TARGET
`5'-------------------------------------------3'
`
`+
`
`5'
`3'
`BLOCKER
`OUGO
`
`+
`
`5'
`5'
`3'
`BLOCKER
`OLIGO
`
`3'
`
`FIRST
`
`~MER
`SOUD
`SUPPORT
`
`1 ANNEAL
`
`+
`
`5'
`3'
`D!SPLACER
`OLIGO
`
`TARGET",
`J
`5'
`3'
`D!SPLACER
`OLIGO
`
`1 WASH
`
`EXTEND
`
`TARGET '"
`5'
`J
`5'~~'d FIRST ~~5'
`3'
`BLOCKER
`) . PRIMER
`D!SPLACER
`J 77-
`OLIGO
`FIRST
`-;
`OLIGO
`NUCLEIC
`ACID
`
`1 DISPLACEMENT
`
`TARGET 3'
`5'
`3' 5·~~~~~· 5'
`BLOCKER·
`SECOND NUCLE!Cj
`DISPLACER
`OUGO
`ACID
`OLIGO
`+
`
`3'~~"-,,'~-[RST
`PRIMER
`
`!
`
`FIRST
`NUCLEIC
`AC!D
`
`Exhibit 2119 Page 11
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 11 of 15
`
`US 2016/0017392 Al
`
`FIGURE 4 CONT.
`
`PR1
`3' ~r-..r~~/',./'~-~
`
`!
`
`FIRST
`NUCLEIC
`ACID
`
`+
`
`SOLID
`SUPPORT
`
`3'
`
`5'
`SECOND
`PRIMER
`
`SECOND
`PRIMER
`\
`
`5'
`
`EXTEND
`/~ __ THIRD
`NUCLEIC
`
`(
`
`3',,-....... -....... ~--..r-~-.~: /-FIRST.
`
`J ANNEAL
`J~ _____________ L; ACID
`)
`-r/ PRIMER
`T7 I; --
`
`FIRST
`NUCLEIC
`ACID
`
`SOUD
`SUPPORT
`
`I REMOVE
`t
`
`SECOND
`PRIMER
`
`THIRD
`- NUCLEIC
`l~ ACID
`-
`
`3'~~ ......... -
`
`-
`
`FIRST
`
`(~P'~~-4ER 5'--------------'-- -----:
`, , ,
`,TTfTT
`SOLID
`SUPPORT
`
`FIRST
`NUCLEiC
`ACID
`
`+
`
`+ 5'
`5'
`3'
`BLOCKER
`OLIGO
`
`TARGET 3' +
`5'
`3'
`DISPLACER
`OLIGO
`
`3<'--"""-""'-~~5'
`DISPLAGER
`OUGO
`
`SECOND _J
`NUCLEIC
`ACID
`
`.,..
`
`3'
`
`FIRST
`
`~ER +
`
`3'
`5'
`SECOND
`PRIMER
`
`Exhibit 2119 Page 12
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 12 of 15
`
`US 2016/0017392 Al
`
`FIGURE 5
`
`CONTINUATION OF CONCEPT
`
`IN ANOTHER ASPECT. THE {+} 1t .. ~MOBILIZATION OLIGOMER IS ALSO BOUND
`TO THE SUPPORT. BRIDGE AMPLIFICATION CAN THEN BE PERFORMED:
`
`SECOND
`IMMOBIL
`OLIGO (+)~"'"
`
`FIRST DNA THEN BINDS TO THE
`SECOND IMM081L. OLIGO
`
`/FIRST CAPTURE
`,A.~",/ OLIGO H (OR IMMOBiLIZATION)
`
`/
`
`I SECOND IN.1MOB1L .. OLIGO
`
`IS THEN EXTENDED
`
`--~ ..
`SECOND/( .... -""
`eDNA. ~,,,-"SECOND eDNA
`IMMOBIL.
`i .
`i 1
`OLIGO (+)-~:
`,
`I
`
`.-.. ;
`
`~-
`
`{
`FIRST
`IMfv10BIl.
`OLIGO (-)
`
`1 D. E.;NATU. REAND .. REP. EAT.- BY
`
`BINDING TO OTHER, UNOCCUPIED
`IMMOBI.UZATION OUGOS
`
`Exhibit 2119 Page 13
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 13 of 15
`
`US 2016/0017392 Al
`
`FIGURE 6
`
`IMMOBILIZATION ANDAMPUFICATION ~ PCR
`
`TARGET
`
`I ~ ®
`~
`l//Ir\ .
`
`F1RST IMMOBIL.
`OUGO
`
`4- FIRST IMMOBILIZATION OLIGO
`BINDS TO TARGET (SINGLE(cid:173)
`STRANDED DNA OR RNA)
`
`+FIRST PRIMER CAN ALSO BE
`ANNEALED DURING TARGET
`CAPTURE STEP
`
`t ·WASH
`
`'" ANNEA. L FIRST PRIMER~
`"EXTEND
`
`eDNA
`FIRST PRIMER
`,1
`~~~~
`*"
`~
`,
`I'-------';\=--. ----<(0)
`TAR GET
`1/); I \ -
`FIRST IMMOBIL
`OLIGO
`
` SEPARATE STRANDS (E.G., HEAT)
`
`.IF RNA CAN DIGEST (E.G., RNASE H)
`. - BIND eDNA (-J TO SECOND IMMOBILIZATION OLIGO
`• EXTEND
`
`1·
`
`eDNA
`
`FIRST PR! MER
`
`~~~~~~­
`+ ~---------SECOND-~DNA--- +
`TrfTT\
`SECOND IMMOBIL.
`OliGO
`
`ALSO, + STRAND IF DNAANDlOR NOT DEGRADED
`WILL REBIND TO FIRST IMMOBlL OLIGO AND/OR
`WILL BE PRIMED WITH THE FIRST PRIMER,
`EXTEND AND ~,;lAKE MORE TEMPLATE. THAT 1$,
`THE FIRST ROUND OF PCRWILLOCCUR
`I -SEPARATE STRANDS
`t "'REPEAT
`
`Exhibit 2119 Page 14
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 14 of 15
`
`US 2016/0017392 Al
`
`FIGURE 7
`
`3'
`
`/ I
`
`TARGET--..
`3'
`
`..
`5'--3' 5' (+)
`'
`"--FIRST":'-' '"
`IMMOBIL. BLOCKER H
`I
`OLIGO (-)
`
`~---.
`
`B!ND TARGET TO FIRST
`IMMOBILIZATION OLIGOMER BIND
`BLOCKER TO TARGET ICAN BE
`CONCURRENT W/BINDiNG OF
`TARGET TO FIRST tMMOBIL
`OLIGO OR SUBSEQUENT)
`
`1 EXTEND (WILL STOP AT BLOCKER)
`
`IF TARGET IS RNA, DIGEST
`W/RNASE H; IF TARGET IS DNA,
`DENATURE tN/HEAT THESE ARE
`EXAMPLES, GOAL-REiIJ10VE
`TARGET STRAND FROM
`IMMOBtL!ZED eDNA
`
`1 ADD (+}PRIMERWfT7 PROMOTER
`
`+ PR!r:v1ER CAN BE BLOCKED
`ON THE 3'- END (SHO'VVN)
`OR NOT.
`
`5'
`3'~ ~~""~ T7 PROMOTER
`.----------.~~~~'~ ~~,
`3'
`~,( +) PRIMER
`-L,
`"',
`"
`~"FIRST
`IMMOBIL.cDNA
`,""; --,--,.-J7-r-/-,-.
`OLIGO (.)
`
`1 EXTEND eDNA
`
`T7~ (+)
`+ PRI1l..1E~< (~)
`
`IF (+) PRIMER IS NOT BLOCKED,
`IT WILL EXTEND, TOO.
`
`~;;0~"-
`/IJ
`IMMOBIL. eDNA
`OLiGOH
`
`I ADD T7 RNA POLYMERASE
`• +RNA IS GENERATED (MULTIPLE COPIES)
`
`Exhibit 2119 Page 15
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 15 of 15
`
`US 2016/0017392 Al
`
`FIGURE 7 CONT.
`
`~~~~~+RN.A
`
`~~~~~+RNA
`
`~~~~~~~~~~r+RNA
`
`• ..
`"
`I THESE +RNA STRANDS WILL BIND TO THE FIRST
`IMMOB1UZATION OLIGOMER (UNOCCUPIED)
`•
`
`~~ .............. ~~/-./~~- +RNA
`
`l7I7t;- FIRST IMMOBIL OLIGO (-)
`I EXTEND FIRST IMfvtOBIL. OLIGO, GENERATING
`t MOREcDNA
`
`. ,. ,
`
`.
`
`~-,cDNA ~ ------------L-----------
`
`I . .... '-~~-" FIRST IMMOBIL OLIGO
`
`I REMOVE THE RNA STRAND (E.G"VViRNASE H)
`+ BIND + PRIMER AND START THE CYCLE AGAIN
`~+PRIMER
`777~~IR~~I~i'6l~t.·oLlGO
`
`Exhibit 2119 Page 16
`
`

`

`US 2016/0017392 Al
`
`Jan. 21, 2016
`
`1
`
`METHODS FOR AMPLIFICATION OF
`NUCLEIC ACIDS ON SOLID SUPPORT
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application is a non-provisional patent appli(cid:173)
`cation of provisional patent application Ser. No. 611781,356
`filed Mar. 14, 2013 and claims the benefit of the filing date of
`PCTlUS2014/029817 filed 14 Mar. 2014 under 35 U.S.c.
`§371 from which the PCT application claims priority.
`
`STATEMENT REGARDING FEDERALLY
`SPONSORED RESEARCH OR DEVELOPMENT
`
`[0002] None
`
`INCORPORATION-BY-REFERENCE OF
`MATERIAL SUBMITTED ON COMPACT DISC
`
`[0003] None
`
`BACKGROUND OF THE INVENTION
`
`(1) Field of the Invention
`[0004]
`[0005] The present invention relates to methods of purifi(cid:173)
`cation, immobilization and amplification of nucleic acids.
`Specifically, nucleic acid amplification using a solid support.
`(2) Description of Related Art
`[0006]
`[0007] There are a variety of methods for the purification
`and amplification of nucleic acids known to those skilled in
`the art. However, these methods can be slow, tedious, expen(cid:173)
`sive and difficult to automate and manufacture. They also lack
`the performance in regards to sensitivity, specificity, preci(cid:173)
`sion' accuracy and other features that would otherwise allow
`them to be used successfully in a number of diverse applica(cid:173)
`tions. These methods are often complicated, requiring isola(cid:173)
`tion of nucleic acid targets from samples, preparing the tar(cid:173)
`gets
`for amplification, performing amplification and
`detecting the amplification product. In some applications, this
`may also include preparing templates for sequencing includ(cid:173)
`ing next generation sequencing and sequencing the target
`nucleic acids. In many of these methods one or more purifi(cid:173)
`cation steps may also be required. All of these steps increase
`performance time, cost, complexity and labor. Providing
`methods with versatility as to specificity, sensitivity, selectiv(cid:173)
`ity, precision and accuracy is important. As are methods that
`are rapid, efficient and easy to use with minimal assay steps
`and reagents for readily obtaining a desired isolated and
`amplified product from a raw sample. These products may be
`amplified nucleic acids or set of amplified nucleic acids (i.e.,
`multiplexing, including high level multiplexing) that may be
`utilized as templates, free in solution or on solid supports, for
`immediate sequencing and next generation sequencing. In
`addition, methods for directional amplification that preserve
`strand orientation provide proper aligmnent of sequences
`during mapping and more accurate quantitative determina(cid:173)
`tions (e.g. of gene expression levels). In addition, these meth(cid:173)
`ods can be utilized with a wide variety of nucleic acid ampli(cid:173)
`fication methods that are known in the art. In addition these
`methods can be utilized with tag sequences. The present
`invention provides methods for purifYing, immobilizing and
`amplifying specific nucleic acids from a sample that over(cid:173)
`come many of the current limitations in the art.
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0008] The present invention provides methods of immo(cid:173)
`bilizing and amplifYing nucleic acids on solid support. More
`specifically, it is a method of immobilizing and amplifYing a
`desired nucleic acid or a number of nucleic acids (multiplex(cid:173)
`ing) from a sample containing a mixture of nucleic acids.
`[0009]
`In one aspect of the present invention, the method
`provides first and second oligonucleotides affixed to a solid
`support or alternatively separate solid supports. The first oli(cid:173)
`gonucleotide is blocked to prevent extension from the 3'-ter(cid:173)
`minus and has a sequence complementary to a first portion of
`a target nucleic acid. The second oligonucleotide has a
`sequence that is identical to a second portion of the target
`nucleic acid. A sample is applied to the solid support and
`target sequences in the sample bind the first oligonucleotide.
`The solid support is then washed to remove unbound nucleic
`acids and other components from the sample. A primer con(cid:173)
`taining a target-binding region and a tag region comprising
`polymerase promoter sequence is annealed to the bound tar(cid:173)
`get nucleic acid. The primer is extended by polymerase to
`produce a first duplex nucleic acid. The target sequence is
`removed from the first duplex nucleic acid to produce an
`unbound first nucleic acid. The first nucleic acid is annealed to
`the second oligonucleotide and the second oligonucleotide is
`extended by polymerase to produce a second duplex nucleic
`acid containing a first nucleic acid and a second nucleic acid.
`Multiple copies of a third nucleic acid are then generated by
`adding polymerase specific to the promoter sequence to the
`solid support thereby amplifying the target nucleic acid.
`[0010]
`In a second embodiment the primer may be com(cid:173)
`bined with the sample and solid support such that the primer
`hybridizes to the target and the target hybridizes to the first
`oligonucleotide concurrently. The solid support is then
`washed to remove unbound nucleic acids and primer as well
`as other components from the sample.
`In a third embodiment, the method may further com(cid:173)
`[0011]
`prise steps that allow for additional amplification of the
`desired target nucleic acid. In these steps, the multiple copies
`of the third nucleic acid bind to the second oligonucleotide on
`the solid support. The second oligonucleotide is extended by
`polymerase to produce a third duplex nucleic acid. The third
`nucleic acid is removed from the third duplex nucleic acid to
`produce a fourth nucleic acid bound to the solid support. The
`primer sequence is annealed to the fourth nucleic acid and
`both the primer and the fourth nucleic acid are extended by
`polymerase to produce a fourth-duplex nucleic acid contain(cid:173)
`ing a fifth nucleic acid and additional second nucleic acid. The
`fourth duplex nucleic acid is incubated with a polymerase
`being specific to a promoter on the fourth duplex nucleic acid
`to produce additional multiple copies of the third nucleic acid.
`According to this embodiment the third nucleic acid may then
`be amplified further.
`[0012]
`In a fourth embodiment, the primer comprises a tag
`sequence containing one or more non-natural nucleotides,
`such as isocytosine (isoC) and/or isoguanine (isoG) to
`increase specificity for binding the target nucleic acid and
`decreases amplification of other nucleic acids.
`[0013]
`In a fifth embodiment, four different second oligo(cid:173)
`nucleotides may be bound to the solid support each having a
`different nucleotide at the 3'-terminus and sequence that
`binds the first nucleic acid at a specific single nucleotide
`polymorphism (SNP) site. In this configuration, amplifica(cid:173)
`tion may be utilized to identify all four possible SNPs in a
`single assay.
`
`Exhibit 2119 Page 17
`
`

`

`US 2016/0017392 Al
`
`Jan. 21, 2016
`
`2
`
`[0014]
`In a second aspect, directional amplification of a
`fragmented nucleic acid target is provided for subsequent
`manipulation, such as sequencing. In one embodiment, a
`sample and a first primer are applied to a solid support. The
`first primer comprises a random sequence of about 6 to about
`9 nucleotides on the 3 I-terminus and a first tag sequence on the
`5'-terminus, which is used to determine the orientation of the
`fragmented nucleic acid target. First and second oligonucle(cid:173)
`otides bound to solid support are provided that may be bound
`to the same solid support or alternatively on different solid
`supports. The first oligonucleotide is optionally blocked to
`prevent potential exonuclease digestion, for example, at the
`5'-terminus and has a sequence complementary to a portion of
`the fragmented nucleic acid target. Alternatively, the first
`oligonucleotide comprises a sequence (such as for example, a
`random or semi-random sequence) that allows for immobili(cid:173)
`zation of a number of fragments from the fragmented nucleic
`acid target. The second oligonucleotide sequence is comple(cid:173)
`mentary to at least a portion of said tag sequence of the first
`primer.
`[0015] The first primer is annealed to the fragmented
`nucleic acid target and the bound fragmented nucleic acid
`target is annealed to the first oligonucleotide. The solid sup(cid:173)
`port is washed to remove unbound sample and first primer.
`The first primer sequence is extended by polymerase to pro(cid:173)
`duce a first duplex nucleic acid containing the fragmented
`nucleic acid target and a first nucleic acid.
`[0016] The fragmented nucleic acid target is removed from
`the first duplex nucleic acid to produce a first nucleic acid.
`The first nucleic acid is annealed to the second oligonucle(cid:173)
`otide and a second primer is added to the solid support. The
`second primer comprises a random sequence of about 6 to
`about 9 nucleotides on the 3'-terminus and a second tag
`sequence on the 5'-terminus, which mayor may not contain
`elements the same as or similar to those found in the first tag
`sequence of the first primer. The second primer is annealed to
`the first nucleic acid and extended by polymerase to produce
`a second duplex containing the first nucleic acid and a second
`nucleic acid having a first tag sequence on the 3'-teminus and
`a second tag sequence on the 5'-terminus.
`[0017] This second nucleic acid is further amplified to pro(cid:173)
`duce a target nucleic acid containing tag sequences that deter(cid:173)
`mine the orientation of the nucleic acid target.
`[0018]
`In one embodiment of this aspect, the first primer
`may further comprise a barcode sequence as part of the tag
`sequence to further assist in identification and orientation.
`[0019]
`In third aspect, a method is provided wherein the
`first primer having a sequence is complementary to a first
`portion of the target nucleic acid is immobilized on a solid
`support. In one embodiment, displacer and blocker oligo(cid:173)
`nucleotides are utilized. The displacer oligonucleotide
`hybridizes to the nucleic acid target at a location in the target
`sequence that is 3' of another bound oligonucleotide or oli(cid:173)
`gonucleotides on the same nucleic acid target. When the
`displacer is extended with polymerase, the growing extension
`product encounters the other bound oligonucleotide( s) and
`displaces it/them, along with any extension product(s) initi(cid:173)
`ated by the bound oligonucleotide( s), from the target nucleic
`acid strand. In this embodiment, the displacer oligonucleotide
`has a sequence complementary to a second portion of the
`target nucleic acid that is located to the 3' side of the first
`portion. The blocker has an oligonucleotide sequence
`complementary to a third portion of the target nucleic acid
`sequence that is located to the 5' side of the first portion. These
`
`oligonucleotides and the target nucleic acid are applied to the
`solid support. The displacer and blocker oligonucleotides are
`annealed to the target nucleic acid and the annealed target is
`immobilized onto the solid support by hybridization to the
`first primer.
`[0020] The solid support is washed to remove unbound
`nucleic acids and other components of the sample. The first
`primer is extended by polymerase. Extension terminates at
`the blocker oligonucleotide to produce a first duplex nucleic
`acid of defined length. The displacer oligonucleotide is also
`extended by polymerase to displace the target strand from the
`first duplex nucleic acid. Extension terminates at the blocker
`oligonucleotide to produce a second duplex nucleic acid con(cid:173)
`taining a second nucleic acid of defined length unbound to the
`solid support.
`[0021] A second primer is annealed to the first nucleic acid
`and extended by polymerase to produce a third duplex con(cid:173)
`taining the first nucleic acid and a third nucleic acid. The
`second and third duplex nucleic acids are then dissociated,
`e.g., heat denaturation or other appropriate means known in
`the art. The second primer is annealed to the first nucleic acid
`and extended to produce more of the third duplex nucleic
`acid.
`[0022] The third nucleic acid is immobilized onto the solid
`support by hybridization to unoccupied first primer. The first
`primer is extended by polymerase to produce more third
`duplex nucleic acid. The original target nucleic acid, with
`displacer and blocker oligonucleotides bound thereto, is
`annealed to unoccupied first primer on the solid support. The
`first primer is extended by polymerase to produce more first
`duplex nucleic acid. The displacer oligonucleotide is also
`extended, displacing the target nucleic acid from the first
`duplex nucleic acid. Extension terminates at the blocker oli(cid:173)
`gonucleotide to produce a second duplex nucleic acid con(cid:173)
`taining a second nucleic acid of defined length that is not
`bound to the support.
`[0023] The second primer is annealed to the second nucleic
`acid by hybridization and is extended by polymerase to pro(cid:173)
`duce a fourth duplex nucleic acid containing the second
`nucleic acid and a fourth nucleic acid. All duplexes are then
`denatured, and the cycle is repeated as desired to amplify the
`target nucleic acid.
`[0024]
`In one embodiment of this aspect, the nucleic acid
`target is double stranded and both strands are immobilized
`and amplified. A primer and optionally a displacer and/or a
`blocker are prepared for each strand according to the method
`described above. The primer for each target nucleic acid
`strand is affixed to a solid support if a single support is utilized
`or one primer on each support if two supports are utilized.
`Each target strand is immobilized by hybridization to the
`primer having a complementary sequence for that target
`nucleic acid strand. The primer is extended by polymerase to
`produce a cDNA and the target nucleic acid strands are then
`removed. Removal may be by displacement if a displacer is
`used, by heat denaturation if a displacer is not used, or by
`some other appropriate method known in the art. At this point,
`the primer designed for one of the target nucleic acid strands
`can serve as the second primer for the cDNA produced from
`the other target nucleic acid strand, and vice versa. The
`cDNAs bind to their respective second primers (i.e., the first
`primers of the opposite target nucleic acid strands), the sec(cid:173)
`ond primers are extended by polymerase and the resulting
`duplexes are disassociated. This process is then repeated as
`desired thereby amplifying the target nucleic acid.
`
`Exhibit 2119 Page 18
`
`

`

`US 2016/0017392 Al
`
`Jan. 21, 2016
`
`3
`
`[0025] Other aspects of the invention are found throughout
`the specification.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0026] FIG. 1: (A) is a schematic diagram of one method of
`the present invention and (B) shows additional steps that may
`be incorporated into the method in FIG. 1A.
`[0027] FIG. 2: is a schematic diagram of another method of
`the present invention utilizing a primer containing non-natu(cid:173)
`ral nucleotides to increase selectivity and decrease amplifi(cid:173)
`cation of undesired nucleic acid sequences.
`[0028] FIG. 3: is a schematic diagram of another method of
`the present invention utilizing a primer with a tag sequence to
`establish the orientation of the target oligonucleotide after
`amplification.
`[0029] FIG. 4: is a schematic diagram of another method of
`the present invention utilizing blocker and displacer to pre(cid:173)
`pare amplified nucleic acid target of defined length.
`[0030] FIG. 5: is a schematic diagram of another method of
`the present invention utilizing first and second immobiliza(cid:173)
`tion oligonucleotides that bind the first and second strand
`cDNAs of the target nucleic acid in a bridge configuration.
`[0031] FIG. 6: is a schematic diagram of another method of
`the present invention that incorporates PCR.
`[0032] FIG. 7: is a schematic diagram of another method of
`the present invention wherein the first immobilization oligo(cid:173)
`nucleotide and blocker oligonucleoti