pen-remiss {03-13;
`Approved for use through 0181.320“. OMB 0651-0032
`US. i‘atent and Trademark Office; Li .5 DE PkR‘lMENT OF COMMERCE
`tinder the Pa‘erwork Red uctiao Act. of £995 no arse-15 are re . uired m res-9nd to ; attention of Information unless it étsoia' _ a vslid [3MB control number
`
`ummr
`
`PATENT APPLICATION
`
`0i-0077’36U3
`
`Stephen Turner
`
`TRAN 5M I HAL
`:N:1‘.:<M:1'refi: Urinam lam our-em; aNALvrICM sensuous
`{Onlyfornewnmpmwsronaloppiiccflons under3?c‘£fi 1.53175» _ NEA ‘ filed Via EFS Web
`APPLiCATlON ELEMENTS
`Commissioner for Patents
`See MBEP chapter 600 concerning utility parent application contents.
`P-O- 30!! 1450
`Alexand ria, VA 22313-11350
`
`ADDRESS TO:
`
`
`
`
`E F“ .T".”’”‘i““' Fm“
`iPTO; $331? or equivalent]
`
`. j applicant asserts small entity status.
`See 3? CFQ 1.27
`_
`
`Applicant certifies mlcro entity status. See 3? CFR 1.29.
`Applicant must attach fOI m PTOHEEIISA or B or equivaienL
`
`{Total Pages _l£fi_____]
`. 3 Specification
`Both the claims and abstract must start on a new page.
`{See MPEP§ 608.01.."0}forfnfolmob'on on the preferred errongem ant}
`
`......=
`[Tom Sheets 24
`5. L; Drawingis} :35 U.S.C.113}
`{Toto} Page:
`. imentufs Oath or Oeclaratiun
`fincfudfng substitute statements under 3? CFR 1.154 and assignments
`r.
`-'
`5‘
`'
`"
`'
`'
`.r
`.ensn? as on can. ordec‘faratmfl uncle-'3; CFR Lei-ell
`Newly executed [original or copy}
`
`OR B Correspondence address below
`
`]
`1
`
`-
`
`'
`
`' a
`
`.
`
`.
`
`.
`
`
`ACCOMPANYING APPLICATiON PAPERS
`
`
`.
`
`_
`assignment 939*“
`{cover sheet 8: documentisJ}
`Pearl-.3 Bioscienoes of Cailfornia. Inc.
`-.
`.-
`—
`Name Of Assignee
`
`
`37 CFR 3.?3lc} Statement
`{when there is or: ossr‘gnee.‘
`
`Power of Attorney
`
`English Translation Document
`n; oppiicabie!
`Information Disclosure Statement
`[PTOISBJOS or PTO-1449}
`.
`.
`.
`D CDplES of Citahfll'IS attached
`PreiirrlinartcI Amendment
`Return Receipt Postcard
`I'MPEP § 503: [Should be specifically itemized]
`Certified Copy of 9rlartly Documenfls]
`iifforer'gn or‘r'or‘r'ty r's chimed]
`
`Nonpublication Request
`Under 35 LL35. 122{b}[2il3lil:'l- Applicant must attach form PTOr’SBBS
`0" equivaiem.
`
`
`
`
`
`
`
`
`
`If A copy from a prior appiicatson [3? CFR 1.63M}:
`
`. 4 Application Data Sheet
`* See no.9 below.
`
`See 3? CFR 1.?6 tPTOfAtA,’14 or equivalent]
`CD-RCIM or CD-R
`
`in duplicate, large tabie, or Computer Sirogram {Appendix}
`:l Landscape Table on CD
`9- Nucleotide am”or Amino Acid Sequence Submission
`{if applicable. items a. —c. are required}
`
`
`
`a. :l Computer Readable Form {CRF}
`{LC} Speclfication Sequence Listing on:
`i. 1:; contain orCD-R [2 ccpies}; or
`{ED Paper
`c. Cl Statements verifying identity of above copies
`*Note:
`(1} Benefit claims under 33' CPR 1.?8 and foreign priority claims under 1.55 must be included in an Application Data Sheet EADS}.
`{2} For applications filed under 35 U.S.C. 11}, the applicataon must Contain an ADS specifying the applicant if the applicant is an
`assignee, person to whom the inventor is under an obligation to assign, or person who otherwise shows sufficient proprietary
`interest in the matter. See 37 CFR 1.45m _l
`19. CORRESPONDENCE ADDRESS
`
`
`
`
`
`E The address associated with Customer Number: 5???0
`
`This collection Of inactivation is 'equired luv 37 (FR LEE}: The :nl'ornlation is require-d to obtain or retain a benefit bar the pubih: which is to file {and by the USP‘IC!
`to processj an application. Confidennality Is governed 5-;35 LLSL. 122 and 3? {FR 1.1'1 and 1.1-1. This collection Is estimated to take 12 minutes to complete,
`including gathering. preparing. and submitting the completed appiicetion form to the USPTO.T]me wiii var-f depending upon the individua! case. Any comments on
`the amount of time you require to camptote this form andg'orstlggest‘ons for reducing this burden, should be sent to the Chief Information Officer, US. Patent and
`Trademark Office, US. Department of Commerce. PO. Box 1450, Alexandria. VA 22 313-14 50-. DO NOT SEND FEES OR COMgLEI'ED FORMS T0 THEE ADDRESS. SENfi
`To: commissioner for Patents, REL Box 1456, Alexandria, VA 2313-1450.
`If you need ossrzmnce r'n compietmg the form, €an 1 -SBC‘-PTO-9.I 99 and seiect option 2.
`
`Oxford, Exh. 1013, p. 1
`
`Oxford, Exh. 1013, p. 1
`
`

`

`
`
`
`Applicant asserts small entity status. See 3? CFFL' 1.21
`H?“ Nam Ed l‘wenmr
`Stephen Turner
`Applicant certifies micro entity status. See 3? (FR 1.29.
`Examiner Name
`Not Yet Assigned
`Form FTUISBIESA or B or equivalent must either be enclosed or have
`Art U 'l.
`.
`been submitted previously.
`m
`“01 Yet Assiigned
`memes
`
`ism-so
`METHOD OF PAYMENT {check all that apply]
`
`D Check '1 Credit Card D Morley Order D None [I Other {please identify}:
`
`
`
`Deposit Account Name: PaCifiC BiDSCienLes
`Deposit Account Deposit Account Number: 5—H“)
`For the above-identified tie posit account, the Direct or is hereby authorized to (check ail that apply):
`
`erotsBJI-i {03-13;
`Approved for use through 018132014. OMB 0651-0032
`US. i‘aten‘t and trademark Office; US. DE PfiRTMEi‘JT OF COMMERCE
`under the Faaerwark Red action Act of .1995 no means are raauirad to res-and m a miieaicn of information unless it dis :a' _ a valid OMB contra! number
`
`Compiete if known
`Not YetAssigned
`
`FEE TRANSM ITTAL
`
`Application Number
`
`
`
`Undiscounted Fee [5|
`Fee Description
`BO
`Each claim over 20 iin ciuding Reissues]
`420
`Each independent claim over 3 {including Reissues]
`1'80
`Multipte depen tie nt claim 5
`Fee |§1
`Extra Claims
`Fee Paid fig}
`Totai Claims
`
`l’
`’“L
`0“”
`_
`-20 or H? =
`3
`x
`HP = highest number of total claims paid for. ifgicdter than 20.
`Indgg. Claims
`Extra Ciaims
`it
`-3 or HP-—
`_____
`5
`HP: highest number of independent claims paid forr if greater than 3
`3. APPLICATION SIZE FEE
`
`Charge feels} indicated below
`
` Charge any additional feeis} or underpayment of feeisi
`
`E Charge feeis} indicated below, except for the filing fee
`Credit anyr overpayment of feels)
`
`under 3? CFR1.16 and 1.1?
`information on til is form may become public. Credit card information should not be included on this form. Provide credit card
`WARNING:
`information and authorization an PTO-2038.
`FEE CALCULATION
`
`1. BASE HUNG, SEARCH, ANB EXAMWATION FEES {U = undiscountad fee; 5 = small entity fee; M = micro entityI fee}
`FI'.lNG FEES
`SEARCH FEES
`EXAMINHTFON FEES
`
`ME}
`Slét
`Uiél
`Nils!
`Salfii
`Ulfil
`MJOEFI
`Slit
`”{5}
`AginEaticin T322
`-80
`350
`3’20
`1513
`600
`110‘
`280
`Util 'itiir
`11
`230
`456
`30
`50
`120
`.5 5
`90
`189
`Design
`145
`2‘30
`SEE
`95
`19!)
`380
`1‘15
`‘30
`188
`Plant
`540
`1,080
`2,1 50
`150
`300
`660
`'J‘ 0
`146
`280
`Reiss Lie
`O
`D
`D
`0
`0
`G
`65
`130
`268
`Provisional
`* The 5149 small entity status filing fee For a :Itiiit'il apalication is further reduced to 370 for a small entity status appiicant who files the apaiitation via ESE-Web.
`2. EX'CESS CiAiM FEES
`
`Fees Paid {fill
`560” fit}
`
`Smali Entity Fee {:1 Micro Entig Fee [51
`40
`20
`210
`105
`39:}
`195
`
`Multigle De Eendent Claims
`Fee {fit
`Fee Paid {£1
`3-9”
`_
`
`Fee Paid {$1
`3 W
`
`if the specification and drawings exceed 100 sheets of paper {excluding electronically filed sequence or computer Iis tings under 3? CFR l.52{e}].
`the application size fee due is 5400 {$200 for smnil entity} {$100 for micro entity} for each additional 50 sheets or fraction thereof. See 35 {3.532.
`aliaiiliil‘il and 3? [FR 1. Ifiis}.
`
`4. OTHER rteisi
`Non-English specification, $130 fee {no small or micro entity discounti
`Non-electronic filing lee under 37 £58 1.1611} for a utility application, 5&00 fee (5200 small or micro entity}
`am e‘. (e.g., late fiiing surcharge}: Track 1 Prioniizaiion Fee and Process-ting Fee
`5U BMITFED BY
`
`«43.03
`
`signature
`
`Telephone 650-521-81 2?
`{David C, Scherer, Prim fftififgifiiggifiifiaaes
`Date December 19, 2015
`David C. Scherer. PM).
`Tl:ls collection of information is required by 37 (FR.‘ ”136 The information is required to obtain or retain a bane Fit by the public which is to fi'e {and bit Uie USPTO to
`process} ariappi'scation ConfidentialityI5 governedtr,r 35 U. S. C. 122 and 3? (FR 1 ‘14. This coliection'is estimated to take 30 minutes to com plete,: nclut‘iing
`gathering, greoaring. and submitting the comnletec' apniicaticn form to the us PTO. Time w:'iE vent depending upon the individual case. Anu comments on the
`amount of time you require to complete this form andfor suggestions for reducing this burden. shouid be sent to the Chief Iniorniatmn Officer, LLS. Patent and
`Trademark Office, LLS. Department oi Commerce. FD. Box 1450, Aiexandr‘a, VA 22313-141 50. DO NOT SENQ FEES OR CGMQLETED FORMS TD THES ADORESS. SEND
`TO: commissioner for Pater-ts, PD. Box 1450, Alexandria. wt 22313—1450.
`ifyou need assistance in campieting the form, cafi J-Sflfl-PTfl-QIQQ andseiect option 2.
`
`Oxford, Exh. 1013, p. 2
`
`Oxford, Exh. 1013, p. 2
`
`

`

`APPLICATION DATA SHEET
`
`APPLICATION MORMATION
`
`Application Type.
`Title::
`
`Attorney Docket Numb er: :
`Request for Early Publication?::
`Request For Non-Publication?::
`Total Drawing Sheets ::
`Small Entity? ::
`Petition included?::
`
`Licensed US Govt. Agency:
`Contract or Grant Numbersz:
`
`Secrecy Order in Parent App]ication?t:
`
`INVENTOR INFORMATION
`
`Inventor 1
`
`Primary Citizenship Country:
`Given Name:
`Middle Name:
`
`Family Name:
`City of Residence::
`State or Province of Residence:
`
`Country of Residence:
`Street of mailing address:
`City of mailing address:
`State or Province ofmailing address:
`Postal or Zip Code of mailing address::
`
`Inventor 2
`
`Primary Citizenship Country:
`Given Name:
`Middle Name:
`
`Family Name:
`City of Residence::
`State or Province of Residence:
`
`Country of Residence:
`Street of mailing address:
`City of mailing address:
`State or Province of mailing address::
`Postal or Zip Code of mailing address::
`
`Regular
`INTERMITTENT DETECTION DURING
`ANALYTICAL REACTIONS
`
`01-007706US
`
`Not Applicable
`No
`
`United States
`
`Stephen
`
`Turner
`Seattle
`
`W ashin gton
`United States
`
`4216 NE 113‘“ Street
`Seattle
`
`Washington
`98125
`
`United States
`J on
`
`Sorenson
`Alameda
`California
`United States
`1725 Nason Street
`Alameda
`California
`94501
`
`Initial 12!19!16
`
`Oxford, Exh. 1013, p. 3
`
`Oxford, Exh. 1013, p. 3
`
`

`

`Inventor 3
`
`Primary Citizenship Country:
`Given Name:
`Middle Name:
`
`Family Name:
`City of Residence:
`State or Province of Residence::
`
`Country of Residence::
`Street of mailing address::
`City of mailing address::
`State or Province of mailing address:
`Postal or Zip Code of mailing.)r address::
`
`Inventor 4
`
`Primary Citizenship Country:
`Given Name:
`
`Middle Name;
`
`Family Name:
`City of Residence::
`State or Province of Residence:
`
`COuntry of Residence::
`Street of mailing address:
`City of mailing address:
`State or Province of mailing address:
`Postal or Zip Code of mailing address::
`
`DOMESTIC PRIORITY INFORMATION
`
`United States
`Kenneth
`Mark
`Maxham
`
`Redwood City
`California
`United States
`241 Harrison Avenue
`
`Redwood City
`California
`94062
`
`United States
`John
`
`Eid
`San Francisco
`California
`United States
`
`52 Sheridan Street, #2
`San Francisco
`California
`94103
`
`Parent A heat] on Parent Filin- Date
`osmns
`11mm
`1280:“ 10
`
`6H099,696
`
`09f24f08
`
`12981029
`
`An application
`claiming the benefit
`under 35 USC 119(e)
`
`122’982‘029
`
`61/139,402
`
`12f19a’08
`
`
`
`
`
`An application
`claiming the benefit
`
`under 35 USC l 1903)
`Continuation-in-part
`of
`
`129821329
`
`12f4l3,226
`
`03/273’09
`
`Initial 12!19!16
`
`Oxford, Exh. 1013, p. 4
`
`Oxford, Exh. 1013, p. 4
`
`

`

`FOREIGN PRIORITY INFORMATION
`
`Priorit Claimed:
`
`Aulication number:
`
`Filin Date:
`
`APPLICANT INFORMATION
`
`Applicant is assignee’.’:
`Applicant Name:
`Street of Mailing Address:
`City of mailing address:
`State or Province of mailing address:
`Postal or Zip code of mailing address:
`
`Yes
`
`Pacific Biosciences of California, Inc.
`1380 Willow Road
`Menlo Park
`Cal i fomi a
`94025
`
`CORRESPOND ENC E INFORMATION
`
`Correspondence Address:
`
`Pacific Biosciences of California, Inc.
`[380 Willow Road
`
`Phone number:
`Fax number:
`
`E-Mail address:
`Customer Number:
`
`Menlo Park, CA 94025
`(650) 521-8127
`(650) 323-9420
`
`asthcirfischwn
`SW70
`
`REPRESENTATIVE INFORMATION
`
`Representative Name:
`Registration Number:
`
`David C. Scherer, PhD.
`56,993
`
`Si gnature: :
`
`{David C. Scherer, Ph.D.:I
`David C. Scherer, PhD.
`
`Initial IZKIQKIG
`
`Oxford, Exh. 1013, p. 5
`
`Oxford, Exh. 1013, p. 5
`
`

`

`PBI DOCKET NO: 0I-00 ??0fiUS
`
`lNTE-RMITTENT DETECTION DURING ANALYTICAL REACTIONS
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`[0001]
`
`This application is a continuation application of US. Patent Application No.
`
`ӣ708,603, filed May 11, 2015, which is a continuation application of US. Patent Application No.
`
`14t'091,961, filed November 27, 2013, now US. Patent No. 9,057,102, which is a continuation
`
`application of US. Patent Application No.
`
`l2z’982,029, filed December 30, 2010, now US. Patent
`
`No. 8,628,940, which (1) claims the benefit of US. Provisional Application No. 61f099,696, filed
`
`September 24, 2008; (2) claims the benefit of US. Provisional Application No. 61!] 39,402, filed
`
`December 19, 2008; and (3) is a continuation-in-part application of US. Patent Application No.
`
`12f413,226, filed March 27, 2009, now US. Patent No. 8,143,030, the full disclosures of all of
`
`which are incorporated herein by reference in their entireties for all purposes.
`
`[0002]
`
`This application is also related to U.S. Provisional Application No. 61/072,160, filed
`
`March 28, 2008, US. Patent Application No. 12883855, filed March 2?, 2009, now US. Patent
`
`No. 8,236,499, and US. Patent Application No. l2f4l3,258, filed March 27, 2009, now US. Patent
`
`No. 8,153,375, all ofwhich are incorporated herein by reference in their entireties for all purposes.
`
`STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
`
`[0003]
`
`Not Applicable.
`
`BACKGROUND OF THE INVENTION
`
`[0004]
`
`The use of optically detectable labeling groups, and particularly those groups having
`
`high quantum yields, e.g., fluorescent or chemiluminescent groups, is ubiquitous throughout the
`
`fields of analytical chemistry, biochemistry, and biology. In particular, by providing a highly visible
`
`signal associated with a given reaction, one can better monitor that reaction as well as any potential
`
`effectors of that reaction. Such analyses are the basic tools of life science research in genomics,
`
`diagnostics, pharmaceutical research, and related fields.
`
`[0005]
`
`Such analyses have generally been performed under conditions where the amounts of
`
`reactants are present far in excess of what is required for the reaction in question. The result of this
`
`excess is to provide ample detectability, as well as to compensate for any damage caused by the
`
`detection system and allow for signal detection with minimal impact on the reactants. For example,
`
`analyses based on fluorescent labeling groups generally require the use of an excitation radiation
`
`1
`
`Oxford, Exh. 1013, p. 6
`
`Oxford, Exh. 1013, p. 6
`
`

`

`PBI DOCKET NO: III-00 ??0filIS
`
`source directed at the reaction mixture to excite the fluorescent labeling group, which is then
`
`separately detectable. However, one drawback to the use of optically detectable labeling groups is
`
`that prolonged exposure of chemical and biochemical reactants to such light sources, alone, or when
`
`in the presence of other components, e.g., the fluorescent groups, can damage such reactants. The
`
`traditional solution to this drawback is to have the reactants present so far in excess that the number
`
`of undamaged reactant molecules far outnumbers the damaged reactant molecules, thus minimizing
`
`or negating the effects of the photo-induced damage.
`
`[0006]
`
`A variety of analytical techniques currently being explored deviate from the
`
`traditional techniques. In particular, many reactions are based on increasingly smaller amounts of
`
`reagents, e_g., in microfluidic or nanofluidic reaction vessels or channels, or in “single molecule“
`
`analyses. Such low reactant volumes are increasingly important in many high throughput
`
`applications, such as microarrays. The use of smaller reactant volumes offers challenges to the use
`
`of optical detection systems. When smaller reactant volumes are used, damage to reactants, such as
`
`from exposure to light somces for fluorescent detection, can become problematic and have a
`
`dramatic impact on the operation of a given analysis. In other cases, other reaction conditions may
`
`impact the processivity, rate, fidelity, or duration of the reaction, including salt or buffer conditions,
`
`pH, temperature, or even immobilization of reaction components within observable reaction
`
`regions.
`
`In many cases, the effects of these different reaction or environmental conditions can
`
`degrade the performance of the system over time. This can be particularly detrimental, for example,
`
`in real-time analysis of reactions that include fluorescent reagents that can expose multiple different
`
`reactions components to optical energy. In addition, smaller reactant volumes can lead to limitations
`
`in the amOunt of signal generated upon application of optical energy.
`
`[000?]
`
`Further, in the case of sequencing-by-synthesis applications, an additional challenge
`
`has been to develop ways to effectively sequence noncontiguous portions of a template nucleic acid
`
`on a single molecule. This challenge is exacerbated in template nucleic acids that contain highly
`
`repetitive sequence andfor are hundreds or thousands of nucleotides in length, such as certain
`
`genomic DNA fragments. The difficulty in generating such noncontiguous reads from a single
`
`template has hampered efforts to construct consensus sequences for long templates, for example, in
`
`genome sequencing projects.
`
`[0008]
`
`As such, methods and systems that result in enhanced reaction performance, such as
`
`an increase in processivity, rate, fidelity, or duration of a reaction of interest, would provide useful
`
`improvements to the methods and compositions currently available. For example, methods, devices,
`
`Oxford, Exh. 1013, p. 7
`
`Oxford, Exh. 1013, p. 7
`
`

`

`and systems that increase reaction performance by, e.g., mitigating to some extent photo-induced
`
`damage in a reaction ofinterest andi’or increasing various other performance metrics for the reaction
`
`would be particularly useful.
`
`PBI DOCKET NO:
`
`til-0f! ??flfilIS
`
`BRlEF SWNARY OF THE INVENTION
`
`[0009]
`
`In a general sense, the methods provided herein implement intermittent detection of
`
`analytical reactions as a means to collect reliable data From times during the reaction that are less or
`
`not able to be analyzed if detection is constant throughout the reaction. In particular, certain
`
`detection methods can cause damage to reaction components, and such intermittent detection allows
`
`the damage to be avoided or at least delayed, thereby facilitating detection of the reaction at later
`
`stages. For example, if a detection method causes a reduction in processivity ofa polymerase
`
`enzyme, then intermittent detection would allow data collection at noncontiguous regions of a
`
`template nucleic acid that extend farther from the initial binding site ofthe polymerase on the
`
`template than would be achievable under constant detection. Further, some detection methods have
`
`limits on how much data or for how long a time data may be generated in a single reaction, and
`
`intermittent detection of such a reaction can allow this data to be collected from various stages of a
`
`reaction, thereby increasing the flexibility of the investigator to spread out the data collection over
`
`multiple stages oFa reaction. In certain aspects, the present invention is particularly suitable to
`
`characterization of analytical reactions in real time, that is, during the course of the reaction. In
`
`certain aspects, the present invention is particularly suitable to characterization of single molecules
`
`or molecular complexes monitored in analytical reactions, for example, single enzymes,
`
`nucleotides, polynucleotides, and complexes thereof.
`
`[0010]
`
`In certain aspects, the present invention is directed to methods, devices, and systems
`
`for obtaining sequence data from discontiguous portions of single nucleic acid templates. The
`
`methods generally comprise pr0vi ding a monitorable sequencing reaction comprising a polymerase,
`
`template, and primer sequence, as well as the various types of nucleotides or nucleotide analogs that
`
`are to be incorporated by the polymerase enzyme in the template-directed primer extension reaction.
`
`Typically, at least one or more or all of the nucleotides or nucleotide analogs are embodied with a
`
`detectable property that permits their identification upon or following incorporation.
`
`In the context
`
`of the present invention, the sequence data for a first portion of a template nucleic acid is acquired
`
`during a first stage of the reaction under a first set of reaction conditions that includes at least one
`
`Oxford, Exh. 1013, p. 8
`
`Oxford, Exh. 1013, p. 8
`
`

`

`PBI DOCKET NO:
`
`Ill-0f! ??flfilIS
`
`reaction condition that results in degraded performance of the reaction, but that may contribute to
`
`the detectability of the nucleotides being incorporated. During a second stage of the reaction, the
`
`degradative influence is eliminated or reduced, which may result in an inability or a reduced ability
`
`to obtain sequence data from a second portion of the template nucleic acid, but where the second
`
`portion of the template nucleic acid is contiguous with the first portion. Subsequently, the reaction
`
`condition resulting in degraded performance is reinstated and sequence data is obtained for a third
`
`portion of the template nucleic acid during a third stage of the reaction, but where the third portion
`
`of the sequence is not contiguous with the first portion of the sequence, but is contiguous with the
`
`second portion. The elimination or reduction of the degradative influence during the second stage of
`
`the reaction may be accomplished by changing or shortening one or more reaction conditions
`
`underlying degradative reaction performance, e.g., by changing one or more reaction conditions
`
`(e.g., temperature, pH, exposure to radiation, physical manipulation, etc), and in particular may
`
`involve altering a reaction condition related to detection of one or more aspects or products of the
`
`reaction. However, in preferred embodiments, nucleotides or nucleotide analogs having the
`
`detectable property are present in the reaction mixture during all stages of the reaction, including
`
`stages in which the degradative influence is eliminated or reduced; as such, the reaction condition
`
`changed in stage two of such an embodiment w0uld not comprise removal or dilution of such
`
`detectable nucleotides or nucleotide analogs.
`
`[0011]
`
`In certain aspects, the present invention is generally directed to methods, devices,
`
`and systems for enhancing the performance of illuminated reactions. The term “illuminated
`
`reactions” as used herein refers to reactions which are exposed to an optical energy source. In
`
`certain preferred embodiments, illuminated reactions comprise one or more fluorescent or
`
`fluorogenic reactants. Typically, such illumination is provided in order to observe the generation
`
`ande’or consumption of reactants or products that possess a particular optical characteristic indicative
`
`of their presence, such as a shift in the absorbance spectrum andfor emission spectrum of the
`
`reaction mixture or its components. In some aspects, enhancing the performance of an illuminated
`
`reaction means increasing the processivity, rate, fidelity, andlor duration of the reaction. For
`
`example, enhancing the performance of an illuminated reaction can involve reducing or limiting the
`
`effects of photo-induced damage during the reaction. The term “photo-induced damage" refers
`
`generally to any direct or indirect impact of illumination on one or more reagents in a reaction
`
`resulting in a negative impact upon that reaction.
`
`Oxford, Exh. 1013, p. 9
`
`Oxford, Exh. 1013, p. 9
`
`

`

`PBI DOCKET NO:
`
`til-0ft ??flfill$
`
`[00l2]
`
`In certain aspects, methods of the invention useful for characterizing an analytical
`
`reaction comprise preparing a reaction mixture and initiating the analytical reaction therein,
`
`subjecting the reaction mixture to at least one detection period and at least one non-detecti on period
`
`during the course of the analytical reaction, collecting data during both the detection period(s) and
`
`the non-detection period(s), and combining the collected data to characterize the analytical reaction.
`
`In certain embodiments, the analytical reaction comprises an enzyme that exhibits an improvement
`
`in performance as compared to its performance in the analytical reaction under constant
`
`illumination, and such improvement may be related to various aspects of enzyme activity, e.g.,
`
`processivity, fidelity, rate, duration ofthe analytical reaction, and the like. In certain embodiments,
`
`stop or pause points are used to control the activity of the enzyme, and such stop or pause points
`
`may comprise elements such as large photolabile groups, strand-binding moieties, non-native bases,
`
`and others well known in the art. In certain preferred embodiments, the one or more detection
`
`periods are illuminated periods and the one or more non-detection periods are non-illuminated
`
`periods. In certain preferred embodiments, a plurality of analytical reactions disposed on a solid
`
`support are characterized, preferably in a coordinated fashion as described elsewhere herein.
`
`[0013]
`
`In certain preferred embodiments, the analytical reaction is a sequencing reaction
`
`that generates sequence reads from a single nucleic acid template during the detection period(s) but
`
`not during the non-detection period(s). For example, the analytical reaction can comprise at least
`
`two or more detection periods and can generate a plurality of noncontiguous reads from the single
`
`nucleic acid template. In some embodiments, the single nucleic acid template is at least 'I 00 bases in
`
`length andlor comprises multiple repeat sequences. In certain embodiments, the sequencing reaction
`
`comprises passage of the single nucleic acid template thrOugh a nanopore, and in other
`
`embodiments the sequencing reaction comprises primer extension by a polymerase enzyme.
`
`[0014]
`
`The analytical may optionally be a processive reaction monitored in real time, i.e.,
`
`during the course of the processive reaction. In preferred embodiments, such a processive reaction
`
`is carried out by a processive enzyme that can repetitively execute its catalytic function, thereby
`
`completing multiple sequential steps of the reaction. For example, a processive polymerization
`
`reaction can comprise a polymerase enzyme repetitively incorporating multiple nucleotides or
`
`nucleotide analogs, as long as such are available to the polymerase within the reaction mixture, e.g.,
`
`without stalling on the template nucleic acid. Such a processive polymerization reaction can be
`
`prevented by incorporation of nucleotides or nucleotide analogs that contain groups that block
`
`additional incorporation events, eg, certain labeling groups or other chemical modifications.
`
`Oxford, Exh. 1013, p. 10
`
`Oxford, Exh. 1013, p. 10
`
`

`

`PBI DOCKET NO:
`
`til-0ft ??flfilIS
`
`[WIS]
`
`In certain preferred embodiments, the analytical reaction comprises at least one
`
`component comprising a detectable label, e.g., a fluorescently labeled nucleotide. In certain
`
`embodiments, the labeled component is present throughout the course of the analytical reaction, i.e.,
`
`during both the detection and the non-detection periods. The method may further comprise an
`
`optical system to collect the data during the detection period, but optionally not to collect the data
`
`during the non-detection period.
`
`[0016]
`
`In certain aspects, methods of the invention comprise providing a substrate having a
`
`reaction mixture disposed thereon and illuminating the reaction mixture on the substrate with an
`
`excitation illumination for multiple, noncontiguous periods during the course of the reaction,
`
`thereby subjecting the reaction mixture to intermittent excitation illumination. In some
`
`embodiments, the reaction mixture comprises first reactant and a second reactant, wherein an
`
`amount of photo-induced damage to the first reactant occurs as a result of interaction between the
`
`first reactant and the second reactant under excitation illumination. In certain embodiments, the
`
`method further comprises monitoring a reaction between the first and second reactants during
`
`illumination and collecting the data generated therefrom. In some embodiments, the reaction is a
`
`primer extension reaction andfor the first reactant is a polymerase enzyme. In certain embodiments,
`
`the second reactant is a t‘luorogenic or fluorescent molecule.
`
`[0017]
`
`In yet another aspect, the methods are useful for mitigating photo-induced damage in
`
`an illuminated reaction by subjecting the illuminated reaction to intermittent illumination rather
`
`than constant illumination. For example, certain methods of the invention monitor a reaction
`
`mixture comprising at least one enzyme and a fluorescent or fluorogenic substrate for the enzyme,
`
`wherein interaction of the enzyme and the Substrate under excitation illumination can result in
`
`altered activity of the enzyme, eg if such excitation illumination is present over an extended period
`
`of time. Such methods can comprise directing intermittent excitation illumination at a first
`
`observation region for a first period that is less than a photo-induced damage threshold period under
`
`the intermittent illumination conditions, but that is greater than a photo-induced damage threshold
`
`period under constant illumination conditions. As such, certain aspects of the invention lengthen a
`
`photo-induced damage threshold period for an analytical reaction through intermittent inactivation
`
`of the excitation illumination source since the photo-induced damage threshold period under
`
`intermittent illumination is longer than the photo-induced damage threshold period under constant
`
`illumination.
`
`Oxford, Exh. 1013, p. 11
`
`Oxford, Exh. 1013, p. 11
`
`

`

`PBI DOCKET NO:
`
`til-00 ??0filIS
`
`[00l8]
`
`In a related aspect, the invention also provides methods of performing an enzyme
`
`reaction, comprising providing an enzyme within a first observation region, contacting the enzyme
`
`with a fluorescent or fluorogenic substrate for the enzyme, and directing an excitation radiation at
`
`and detecting signals from the first observation region for a period that is less than a photo-induced
`
`damage threshold period under intermittent illumination conditions, but that is greater than a photo-
`
`induced damage threshold period under constant illumination conditions.
`
`[0019]
`
`In further aspects, the invention provides methods of monitoring a primer extension
`
`reaction, comprising providing a polymerase enzyme within a first observation region, contacting
`
`the polymerase with at least a first fluorescent or H uorogenic nucleotide analog, and monitoring a
`
`fluorescent signal emitted from the first observation region in response to illumination with
`
`excitation radiation for a period that is less than a photo-induced damage threshold period under
`
`intermittent illumination conditions, but that is greater than a photo-induced damage threshold
`
`period under constant illumination conditions.
`
`[0020]
`
`In addition, the invention provides methods for generating a plurality of
`
`non contiguous sequence reads from a single nucleic acid template molecule, Such methods
`
`generally comprise preparing a reaction mixture comprising the template molecule, a polymerase
`
`enzyme, and a set of differentially labeled nucleotides or nucleotide analogs, wherein the set
`
`comprises at least one type of nucleotide or nucleotide analog for each of the natural nucleobases
`
`(A, T, C, and G). The polymerization reaction is initiated, the polymerase begins processive
`
`incorporation of the labeled nucleotides or nucleotide analogs into a nascent nucleic acid strand, and
`
`during such incorporation the reaction is monitored by optical means to detect incorporation events,
`
`thereby generating a first sequence read. In a subsequent step, the labeled nucleotides or analogs are
`
`replaced with unlabeled nucleotides or nucleotide analogs and the polymerization is allowed to
`
`proceed without detecting incorporation events. Subsequently, the unlabeled nucleotides or analogs
`
`are replaced with labeled nucleotides or nucleotide analogs and the polymerization is allowed to
`
`proceed once again with real time detection of incorporation events, thereby generating a second
`
`sequence read that is noncontiguous to the first sequence read. The substitution of labeled for
`
`unlabeled, and unlabeled for labeled, nucleotides and nucleotide analogs can be repeated multiple
`
`times to generate a plurality of noncontiguous sequence reads, each of the plurality generated during
`
`a period when the labeled nucleotides or nucleotide analogs are being incorporated into the nascent
`
`strand and such incorporation is being detected in real time.
`
`Oxford, Exh. 1013, p. 12
`
`Oxford, Exh. 1013, p. 12
`
`

`

`PBI DOCK