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`(Printftype}|David C. Scherer, Ph.D. {attorney/Agent|VO,993
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`if you needassittence im completing the farm, call 1-800-FTO-2199 aad ssiect antion 2,
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`Oxford, Exh. 1013, p. 1
`
`Oxford, Exh. 1013, p. 1
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`300
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`SUBMITFFED BY
`Signature [David C. Scherer, Ph.Ds|jstomevingens 96,993 Telephone 650-521-8127
`
`
`David C. Scherer, Ph.D.
`bate December 19, 2016
`This collection of «ahosmatian is requived by 37 CFR LAG. Thre information & requlred to obtain or retain a benefit by the pubkhe whichis to file yand by Gre USPTO to
`process} a0 appication, Confidentiality is governed by 35 U.S.C, b22 aod 37 CFR 1 14, This cotection is estimated te take 3G minutes to complete, inclucting
`gathering, prenaring, and submitting the completed anpiication form ta the USPTO. Time wilt vary depending upon the individual case, Any comments on the
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`Oxford, Exh. 1013, p. 2
`
`Oxford, Exh. 1013, p. 2
`
`

`

`APPLICATION DATA SHEET
`
`APPLICATION INFORMATION
`
`Application Type::
`Title::
`
`Attorney Docket Number::
`Request for Early Publication?::
`Request for Non-Publication?::
`Total Drawing Sheets ::
`Small Entity? ::
`Petition included?::
`Licensed US Govt. Agency::
`Contract or Grant Numbers::
`Secrecy Order in Parent Application?::
`
`INVENTOR INFORMATION
`
`Inventor]
`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:
`
`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
`No
`No
`24
`
`No
`No
`No
`Not Applicable
`No
`
`United States
`Stephen
`
`Turmer
`Seattle
`Washington
`United States
`4216 NE 113"Street
`Seattle
`Washington
`98125
`
`United States
`Jon
`
`Sorenson
`Alameda
`California
`United States
`17245 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 address::
`
`Inventor 4
`PnmaryCitizenship 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::
`
`United States
`Kenneth
`Mark
`Maxham
`Redwoad City
`Califormia
`United States
`241 Harrison Avenue
`Redwood City
`Califormia
`94062
`
`United States
`John
`
`Eid
`San Francisco
`California
`United States
`52 Sheridan Street, #2
`San Francisco
`California
`94103
`
`DOMESTIC PRIORITY INFORMATION
`
`Parent Application::|Parent Filing Date
`05/11/15
`11/27/13
`12/30/10
`09/24/08
`
`12/982,029
`
`An application
`claiming the benefit
`under 35 USC 119(e)
`
`61/099,696
`
`
`
`
`
`12/982,029
`
`An application
`claiming the benefit
`
`under 35 USC 119(e)
`
`
`12/982,029 Continuation-in-part|12/413,226 03/27/09
`of
`
`61/139,402
`
`12/19/08
`
`Initial 12/19/16
`
`Oxford, Exh. 1013, p. 4
`
`Oxford, Exh. 1013, p. 4
`
`

`

`FOREIGN PRIORITY INFORMATION
`
` Priority Claimed:
`
`PT
`
`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
`California
`94025
`
`CORRESPONDENCE INFORMATION
`
`Correspondence Address::
`
`Phone number::
`Fax number::
`E-Mail address::
`Customer Number::
`
`Pacific Biosciences of California, Inc.
`1380 Willow Road
`Menlo Park, CA 94025
`(650) 21-8127
`(650) 323-9420
`dscherer@pachcom
`57770
`
`REPRESENTATIVE INFORMATION
`
`Representative Name::
`Registration Number;
`
`David C. Scherer, Ph.D.
`56,993
`
`Signature::
`
`/David C. Scherer, Ph.D./
`David C. Scherer, Ph.D.
`
`Initial 12/19/16
`
`Oxford, Exh. 1013, p. 5
`
`Oxford, Exh. 1013, p. 5
`
`

`

`PBI DOCKET NO: O1-O07706US
`
`INTERMITTENT DETECTION DURING ANALYTICAL REACTIONS
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`[0001]
`
`This application is a continuation application of U.S. Patent Application No,
`
`14/708,603, filed May 11, 2015, which is a continuation application of U.S. Patent Application No.
`
`14/091,961, filed November 27, 2013, now U.S. Patent No. 9,057,102, which is a continuation
`
`application of U.S. Patent Application No, 12/982,029, filed December 30, 2010, now LS. Patent
`
`No.8,628,940, which (1) claims the benefit of U.S. Provisional Application No. 61/099,696,filed
`
`September 24, 2008; (2) claims the benefit of U.S. Provisional Application No. 61/139,402,filed
`
`December 19, 2008: and (3) is a continuation-in-part application of U.S. Patent Application No.
`
`12/413,226, filed March 27, 2009, now US. Patent No. 8,143,030, the full disclosures ofall of
`
`which are incorporated herein by reference in their entireties for all purposes.
`
`(0002)
`
`This applicationis also related te U.S. Provisional Application No. 61/072,160, filed
`
`March 28, 2008, U.S. Patent Application No. 12/383,855, filed March 27, 2009, now U.S. Patent
`
`No. 8,236,499, and US. Patent Application No. 12/413,258, filed March 27, 2009, now US. Patent
`
`No. 8,153,375, all of which are incorporated herein by reference in their entireties for all purposes.
`
`STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
`
`[0003]
`
`Not Applicable.
`
`BACKGROUND OF THEINVENTION
`
`[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 ofanalytical 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
`
`l
`
`Oxford, Exh. 1013, p. 6
`
`Oxford, Exh. 1013, p. 6
`
`

`

`PBI DOCKET NO: O1-O07706US
`
`source directed at the reaction mixture to excite the fluorescent labeling group, which 1s then
`
`separately detectable. However, one drawbackto the use of optically detectable labeling groupsis
`
`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 lowreactant volumesare increasingly important in many high throughput
`
`applications, such as microarrays. The use of smaller reactant volumes offers challenges to the use
`
`ofoptical detection systems. When smaller reactant volumes are used, damageto reactants, such as
`
`from exposure to light sources 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.
`
`[0007]
`
`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 and/or 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.
`
`[00038]
`
`As such, methods and systemsthat 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
`
`damagein a reaction ofinterest and/or increasing various other performance metrics for the reaction
`
`would be particularly useful.
`
`PBI DOCKET NO: O1-O07706US
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0009]
`
`In a general sense, the methods provided herein implement intermittent detection of
`
`analytical reactions as a meansto 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 of a 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 of the 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 maybe 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 of a reaction. In certain aspects, the present invention is particularly suitable to
`
`characterization of analytical reactionsin 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 methads, devices, and systems
`
`for obtaining sequence data from discontiguous portions of single nucleic acid templates. The
`
`methods generally comprise providing 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 1s acquired
`
`during a first stage of the reaction undera first set of reaction conditions that includes at least one
`
`Oxford, Exh. 1013, p. 8
`
`Oxford, Exh. 1013, p. 8
`
`

`

`PBI DOCKET NO: O1-O07706US
`
`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 influenceis 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 performanceis 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
`
`ofthe sequence 1s not contiguous with the first portion of the sequence, but is contiguous with the
`
`second portion. The elimination or reduction ofthe 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 mote 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 ofthe 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 would not comprise removal or dilution of such
`
`detectable nucleotides or nucleotide analogs.
`
`[0011]
`
`Tn 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
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`fluorogenic reactants. Typically, such illumination is provided in order to observe the generation
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`and/or consumption of reactants or products that possess a particular optical characteristic indicative
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`of their presence, such as a shift in the absorbance spectrum and/or emission spectrum ofthe
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`reaction mixture or its components. In some aspects, enhancing the performance of an illuminated
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`reaction means increasing the processivity, rate, fidelity, and/or duration of the reaction. For
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`example, enhancing the performance ofan illuminated reaction can involve reducing or limiting the
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`effects of photo-induced damage during the reaction. The term “photo-induced damage”refers
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`generally to any direct or indirect impact of illumination on one or more reagents in a reaction
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`resulting in a negative impact uponthat reaction.
`
`Oxford, Exh. 1013, p. 9
`
`Oxford, Exh. 1013, p. 9
`
`

`

`PBI DOCKET NO: O1-O07706US
`
`[0012]
`
`In certain aspects, metheds of the invention useful for characterizing an analytical
`
`reaction comprise preparing a reaction mixture and initiating the analytical reaction therein,
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`subjecting the reaction mixture to at least one detection period and at least one non-detection period
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`during the course of the analytical reaction, collecting data during both the detection period(s) and
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`the non-detection period{s), and combining the collected data to characterize the analytical reaction.
`
`In certain embodiments, the analytical reaction comprises an enzymethat exhibits an improvement
`
`in performance as compared to its performance in the analytical reaction under constant
`
`illumination, and such improvement maybe related to various aspects of enzyme activity, e.g.,
`
`processivity, fidelity, rate, duration of the analytical reaction, and the like. In certain embodiments,
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`stop or pause points are used to control the activity of the enzyme, and such stop or pause points
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`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
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`periods are uminated periods and the one or mere non-detection periods are non-illuminated
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`periods. In certain preferred embodiments,a plurality of analytical reactions disposed on a solid
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`support are characterized, preferably in a coordinated fashion as described elsewhere herein.
`
`[0013]
`
`In certain preferred embodiments, the analytical reaction is a sequencing reaction
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`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 compriseat least
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`two or more detection periods and can generate a plurality of noncontiguous reads from the single
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`nucleic acid template. In some embodiments, the single nucleic acid template is at least 100 bases in
`
`length and/or comprises multiple repeat sequences. In certain embodiments, the sequencing reaction
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`comprises passage ofthe single nucleic acid template through a nanopore, and in other
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`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.,
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`during the course of the processive reaction. In preferred embodiments, such a processive reaction
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`is carried out by a processive enzymethat can repetitively execute its catalytic function, thereby
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`completing multiple sequential steps of the reaction. For example, a processive polymerization
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`reaction can comprise a polymerase enzyme repetitively incorporating multiple nucleotides or
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`nucleotide analogs, as long as such are available to the polymerase within the reaction mixture, ¢.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, e.g., cerlain labeling groups or other chemical modifications.
`
`Oxford, Exh. 1013, p. 10
`
`Oxford, Exh. 1013, p. 10
`
`

`

`PBI DOCKET NO: O1-O07706US
`
`[0015]
`
`In certain preferred embodiments, the analytical reaction comprisesat least one
`
`component comprising a detectable label, ¢.g., a fluorescently labeled nucleotide. In certain
`
`embodiments, the labeled componentis present throughout the course of the analytical reaction,i.e.,
`
`during both the detection and the non-detection periods. The method mayfurther 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
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`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
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`embodiments, the reaction mixture comprises first reactant and a second reactant, wherein an
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`amount of phote-induced damageto the first reactant occurs as a result of interaction between the
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`first reactant and the second reactant under excitation illumination. In certain embediments, the
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`method further comprises monitoring a reaction between the first and second reactants during
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`illumination and collecting the data generated therefrom. In some embodiments, the reaction is a
`
`primer extension reaction and/or the first reactant 1s a polymerase enzyme. In certain embodiments,
`
`the second reactant is a fluorogenic 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 enzymeand a fluorescent or flucrogenic substrate for the enzyme,
`
`wherein interaction of the enzyme and the substrate under excitation illumination can result in
`
`altered activity of the enzyme, e.g 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 damagethreshold 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
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`photo-induced damage threshold peried for an analytical reaction through intermittent inactivation
`
`of the excitation illumination source since the photo-induced damage threshold period under
`
`intermittentillumination 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: O1-O07706US
`
`[0018]
`
`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 fluorogenic nucleotide analog, and monitoring a
`
`fluorescent signal emitted from the first observation region in responseto illumination with
`
`excitation radiation for a petiod that is Jess than a photo-induced damage threshold period under
`
`intermittent illumination conditions, but that is greater than a photo-induced damage threshold
`
`petiod under constantiJlumination conditions,
`
`[0020]
`
`In addition, the invention provides methods for generating a plurality of
`
`noncontiguous 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 beyins processive
`
`incorporation of the labeled nucleotides or nuclectide 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 nuclectide analogs and the polymerizationis 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 1s noncontiguous to the first sequence read. The substitution of labeled for
`
`unlabeled, and unlabeled for labeled, nuclectides and nucleotide analogs can be repeated multiple
`
`times to generate a plurality of noncontiguous sequence reads, each of the plurality generated during
`
`a pericd whenthe labeled nucleotides or nucleotide analogs are being incorporated into the nascent
`
`stran