`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`UNITED STATES DEPARTMENT OF COMMERCE
`United States Patent and Trademark Office
`Address: COMMISSIONER FOR PATENTS
`PO. Box 1450
`Alexandria1 Virginia 22313- 1450
`wwwnsptogov
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`APPLICATION NO.
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`
`
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` F ING DATE
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`FIRST NAMED INVENTOR
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`ATTORNEY DOCKET NO.
`
`
`
`
`CONF {MATION NO.
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`12/954,634
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`11/25/2010
`
`Benajamin HINDSON
`
`38938—705201
`
`5813
`
`21971
`
`7590
`
`10/03/2013
`
`WILSON, SONSINI, GOODRICH & ROSATI
`650 PAGE MILL ROAD
`PALO ALTO, CA 94304-1050
`
`EXAMINER
`
`OYEYEMI, OLAYINKA A
`
`ART UNIT
`
`1637
`
`MAIL DATE
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`10/03/2013
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`PAPER NUMBER
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`DELIVERY MODE
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`PAPER
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`Please find below and/or attached an Office communication concerning this application or proceeding.
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`The time period for reply, if any, is set in the attached communication.
`
`PTOL—90A (Rev. 04/07)
`
`

`

`
`
`Applicant(s)
`Application No.
` 12/954,634 HINDSON ET AL.
`
`Examiner
`Art Unit
`AIA (First Inventor to File)
`Office Action Summary
`
`1637OLAYINKA OYEYEMI [SENS
`
`-- The MAILING DA TE of this communication appears on the cover sheet with the correspondence address --
`Period for Reply
`
`
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`A SHORTENED STATUTORY PERIOD FOR REPLY IS SET TO EXPIRE 3 MONTH(S) OR THIRTY (30) DAYS,
`WHICHEVER IS LONGER, FROM THE MAILING DATE OF THIS COMMUNICATION.
`Extensions of time may be available under the provisions of 37 CFR 1.136(a).
`In no event however may a reply be timely filed
`after SIX () MONTHS from the mailing date of this communication.
`If NO period for reply is specified above, the maximum statutory period will apply and will expire SIX (6) MONTHS from the mailing date of this communication.
`-
`- Failure to reply within the set or extended period for reply will, by statute, cause the application to become ABANDONED (35 U.S.C. § 133).
`Any reply received by the Office later than three months after the mailing date of this communication, even if timely filed, may reduce any
`earned patent term adjustment. See 37 CFR 1 .704(b).
`
`Status
`
`1)IZI Responsive to communication(s) filed on 05/06/2013.
`El A declaration(s)/affidavit(s) under 37 CFR 1.130(b) was/were filed on
`
`2b)|:l This action is non-final.
`2a)|Z| This action is FINAL.
`3)I:I An election was made by the applicant in response to a restriction requirement set forth during the interview on
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`; the restriction requirement and election have been incorporated into this action.
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`4)|:| Since this application is in condition for allowance except for formal matters, prosecution as to the merits is
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`closed in accordance with the practice under Exparte Quay/e, 1935 CD. 11, 453 O.G. 213.
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`Disposition of Claims
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`5)IZI Claim(s) 1-39 59 75 89 and 101-105is/are pending in the application.
`5a) Of the above claim(s) 75 and 89 is/are withdrawn from consideration.
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`6)I:I Claim(s)
`is/are allowed.
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`7)|Z| Claim(s) 1 3-9 59 and 101- 105is/are rejected.
`8)|:I Claim(s)_ is/are objected to.
`
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`are subject to restriction and/or election requirement.
`9)I:I Claim((s)
`* If any claims have been determined allowable, you may be eligible to benefit from the Patent Prosecution Highway program at a
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`participating intellectual property office for the corresponding application. For more information, please see
`hit
`I/'/\WIIW.USOI.O. ovI’ atentS/init events/
`
`
`
`h/index.‘s or send an inquiry to PPI-iieedback{®usgtc.00v.
`
`Application Papers
`
`10)I:l The specification is objected to by the Examiner.
`11)I:l The drawing(s) filed on
`is/are: a)I:I accepted or b)I:I objected to by the Examiner.
`Applicant may not request that any objection to the drawing(s) be held in abeyance. See 37 CFR 1.85(a).
`
`Replacement drawing sheet(s) including the correction is required if the drawing(s) is objected to. See 37 CFR 1.121 (d).
`
`Priority under 35 U.S.C. § 119
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`12)I:| Acknowledgment is made of a claim for foreign priority under 35 U.S.C. § 119(a)-(d) or (f).
`Certified copies:
`
`a)I:l All
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`b)|:l Some * c)I:l None of the:
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`1.I:I Certified copies of the priority documents have been received.
`2.|:l Certified copies of the priority documents have been received in Application No.
`3.|:| Copies of the certified copies of the priority documents have been received in this National Stage
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`application from the International Bureau (PCT Rule 17.2(a)).
`* See the attached detailed Office action for a list of the certified copies not received.
`
`Attachment(s)
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`1) D Notice of References Cited (PTO-892)
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`3) D Interview Summary (PTO-413)
`
`Paper N°ISI/Ma" Date' —
`PTO/SB/08
`t
`t
`St
`I
`D'
`I'
`f
`2 IZI I
`
`)
`4) I:I Other:
`Isc osure
`n orma Ion
`)
`a emen (s) (
`Paper No(s)/Mai| Date 05/24/2013 05/24/2013.
`
`U.S. Patent and Trademark Office
`PTOL—326 (Rev. 08-13)
`
`Part of Paper No./Mai| Date 20130823
`
`Office Action Summary
`
`

`

`Application/Control Number: 12/954,634
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`Art Unit: 1637
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`Page 2
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`DETAILED ACTION
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`Status of the Applications, Amendments and/or Claims
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`1.
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`This action is written in response to applicant's correspondence submitted May 06, 2013. In the
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`paper of May 06, 2013, Applicants amended claims 1, 3-4, 39, 59 and 75 and added claims 103-105.
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`Claims 1-39, 59, 75, 89 and 101-105 are pending.
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`2.
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`All claim amendments and arguments from the paper of May 06, 2013 have been thoroughly
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`reviewed but were found insufficient to place the instantly examined claims in condition for allowance.
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`The following rejections are either newly presented, as necessitated by amendment, or are reiterated
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`from the previous Office Action. Any rejections not reiterated in this Office action have been withdrawn as
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`necessitated by applicant's amendments to the claims. This Office action is Final.
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`Status of the Claims
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`3.
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`Claims 1-39, 59, 75, 89 and 101-105 are pending. In the previous Office action, claims 75 and 89
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`were withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected
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`invention, there being no allowable generic or linking claim. Claims 1-39, 59 and 101-105 are under
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`examination.
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`Information Disclosure Statement
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`4.
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`The two information disclosure statements (IDS) submitted on 05/24/2013 were filed after the
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`mailing date of the Non-final Office Action of 12/05/2012. The submissions are in compliance with the
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`provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by
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`the examiner.
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`Objections
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`Response to Arguments
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`5.
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`Objection to the specification is withdrawn in view of amendments to remove the embedded
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`hyperlinks as stated in the previous Office action.
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`

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`Application/Control Number: 12/954,634
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`Page 3
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`Art Unit: 1637
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`Rejections
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`6.
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`The rejection of claims 3-4 under 35 U.S.C. § 112(b) stated in the previous Office action is
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`withdrawn based upon the amendment of claims 3-4 to recite “remain intact or stable” instead of
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`“substantially stable” which is indefinite. Applicant's argument (see pages 12-13 of the Remarks, V,
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`Rejection under 35 U.S.C. § 112, Second paragraph), that this rejection has been obviated by
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`amendment to claims 3-4 is persuasive.
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`7.
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`The rejection of claims 1-14, 17-26, 28-29, 31-35, 59 and 101-102 under 35 U.S.C. § 102(b),
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`stated in the previous Office action is moot in view of applicant’s amendment of claim 1 to recite the
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`limitation “wherein the amplifying occurs in said two or more partitions”. A new grounds of rejection
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`necessitated by claim amendment is presented below.
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`8.
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`The rejections of claims 15-16, 30, 36 and 37-38 under various 35 U.S.C. § 103(a) rejections, as
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`stated in the previous Office action are moot in view of applicant’s amendment of claim 1. A new grounds
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`of rejection necessitated by claim amendment is presented below. Applicant's argument (see page 16 of
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`the Remarks, VII, section A for claims 15-16 and 37-38 and page 16, Remarks, VII, section B for claim 36
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`and pages 17-18 of the Remarks, VII, section D for claim 30), that these rejections have been obviated by
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`the amendment to claim 1 are addressed in the Arguments section below.
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`9.
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`The rejection of claim 27 under 35 U.S.C. § 103(a), stated in the previous Office action is moot in
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`view of applicant’s amendment of claim 1. A new grounds of rejection necessitated by claim amendment
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`is presented below. Applicant's argument (see pages 16-17 of the Remarks, VII, section C), that this
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`rejection has been obviated by the amendment to claim 1 are addressed in the Arguments section below.
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`Arguments
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`Applicant's arguments have been fully considered but they are not persuasive for the reasons below:
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`10.
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`Regarding claims 1-14, 17-26, 28-29, 31-35, 59 and 101-102, applicant argue (pg 14, Remarks,
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`3rd para) against Brenner et al. Applicant argue that Brenner et al. do not teach or disclose “partitioning
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`one or more ligated products into two or more partitions" and "amplifying a region within said one or more
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`ligated products to obtain amplified products, wherein the amplifying occurs in said two or more
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`

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`Application/Control Number: 12/954,634
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`Art Unit: 1637
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`Page 4
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`partitions". The argument is not persuasive because Brenner teaches or discloses amplification in a
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`plurality of aliquots (i.e. partitions) and further disclose emulsion PCR (digital droplet PCR) for detecting
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`copy number on pg 10 [0063] and pg 13,para [0074].
`
`Regarding claim 39, applicant also argue (pg 15, Remarks, 3rd para) Brenner et al. do not teach
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`or disclose “partitioning one or more ligated products into two or more aqueous droplets within a
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`continuous oil phase," "amplifying a sequence within said one or more ligated products to obtain amplified
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`products, wherein the amplifying occurs in said two or more aqueous droplets," and "determining a
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`number of said two or more aqueous droplets that contain said amplified products," as recited in Claim
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`39. Again, this argument is not persuasive because Brenner teaches or discloses amplification in a
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`plurality of aliquots (i.e. partitions) and further disclose emulsion PCR (digital droplet PCR) for detecting
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`copy number on pg 10 [0063] and pg 13,para [0074].
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`Applicant argue (pg 15, Remarks, 3rd para) that while Brenner et al. teach clonal populations of
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`oligonucleotide tags are analyzed after the emulsion of Brenner is m, Brenner et al. do not disclose
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`amplification in two or more aqueous droplets and determining a number of said two or more aqueous
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`droplets that contain said amplified products." This argument is not persuasive because Brenner
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`teaches or discloses amplification in a plurality of aliquots (i.e. partitions) and further disclose emulsion
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`PCR (digital droplet PCR) for detecting copy number on pg 10 [0063] and pg 13, para [0074].
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`Furthermore, Beer et al. has been cited in the new grounds of rejection presented below to teach the how
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`to's for amplification in two or more partitions in view of Brenner et al. teaching on pg 10, para [0063] and
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`pg 13m para [0074].
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`11.
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`Regarding claim 59, applicant argue (on pg 15, Remarks, 3rd para of section C and pg 16,
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`Remarks, 1St para) that Brenner et al. do not teach or disclose "performing an amplification reaction within
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`said reaction volumes,". Brenner et al. instead teach that aliquots may be assayed using PCR, but
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`nothing in the cited disclosure of Brenner teaches or discloses "performing an amplification reaction within
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`said reaction volumes" or performing PCR in the aliquots. This argument is not persuasive in view of the
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`disclosure that method of counting target polynucleotides by Brenner et al. is amenable with emulsion
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`PCR (pg 10 [0063] and pg 13,para [0074]) for quantification of the target polynucleotides of each aliquot
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`Application/Control Number: 12/954,634
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`Art Unit: 1637
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`of two or more aliquots.
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`Page 5
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`12.
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`Applicant argue (pg 16, Remarks, section Vll, sections A-C) that claims 15-16, 27, 36-38 are not
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`obvious over Brenner for at least the reasons that claim 1
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`is not obvious and that Brenner does not teach
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`additional features of particular advantage and utility recited by the instant claims. This argument is not
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`persuasive for the same reasons presented above under paragraph 13 above addressing claim 1.
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`13.
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`Regarding claim 30, applicant argue (on pg 17, Remarks, section Vll, sections D) that Wang et
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`al. do not teach "amplifying a region within said one or more ligated products to obtain amplified products,
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`wherein the amplifying occurs in said two or more partitions," as newly recited in amended Claim 1 and
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`dependent Claim 30. Applicant further argue that Wang et al. do not teach or disclose "determining a
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`number of said partitions that contain said amplified products" and "calculating a copy number of said first
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`target polynucleotide based on said number of said partitions," as newly recited in Claim 1 and dependent
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`Claim 30. Applicant also argue Wang et al. do not teach or disclose "determining a number of said
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`partitions that contain said amplified products" and "calculating a copy number of said first target
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`polynucleotide based on said number of said partitions," as recited in Claim 1 and dependent Claim 30.
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`On the contrary, in Wang et al., reactions are pooled before determining whether a sample is
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`homozygous or heterozygous based on fluorescent intensity.
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`In response to Applicant's arguments against the references (i.e. “Wang”) individually, one cannot
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`show nonobviousness by attacking references individually where the rejections are based on
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`combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck &
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`Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). The Office asserts that Wang et al. in the
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`combination of references was cited only to teach the obviousness, advantages and expectation of
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`success of substituting the ligation probes that was taught by Brenner et al. with a molecular inversion
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`probe as taught by Wang et al. Wang et al. teach molecular inversion probe are suitable to use to
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`accurately and specifically measure allele copy number (abstract) and therefore it would have been
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`obvious to the ordinary skilled artisan to use an MIP probe as a functionally equivalent embodiment of the
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`ligation probes of Brenner et al. The skilled artisan would be might aware on how to make the substitution
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`in order to detect copy number variation when all of the cited references are consulted in addition to
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`

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`Application/Control Number: 12/954,634
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`Art Unit: 1637
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`Page 6
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`accessing knowledge in the art on how to ligate MIP probes to a target polynucleotide. Therefore,
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`applicant argument is considered non-persuasive.
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`New grounds of Rejections necessitated by claim amendment
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`Claim Rejections - 35 USC § 103
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`14.
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`The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness
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`rejections set forth in this Office action:
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`(a) A patent may not be obtained though the invention is not identically disclosed or described
`as set forth in section 102 of this title, if the differences between the subject matter sought to
`be patented and the prior art are such that the subject matter as a whole would have been
`obvious at the time the invention was made to a person having ordinary skill in the art to which
`said subject matter pertains. Patentability shall not be negatived by the manner in which the
`invention was made.
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`15.
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`This application currently names joint inventors. In considering patentability of the claims under
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`pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was
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`commonly owned at the time any inventions covered therein were made absent any evidence to the
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`contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention
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`dates of each claim that was not commonly owned at the time a later invention was made in order for the
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`examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e),
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`(f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
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`16.
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`Claims 1-39, 59, and 101-105 rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable
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`over Brenner et al. (US2007/0172873, published Jul 2007: previously cited) in view of Beer et al.,
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`(2007, Anal Chem 79: p8471-8475).
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`Regarding claim 1, Brenner et al. taught a method of detecting copy number of a target
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`polynucleotide within a population of genetic material by counting molecules in a sample via counting
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`sequences of oligonucleotide tags (see Brenner et al., page 1, [0005]) wherein the method comprises: a)
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`binding a first ligation probe to a first target polynucleotide and b) binding a second ligation probe to a
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`

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`Application/Control Number: 12/954,634
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`Art Unit: 1637
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`Page 7
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`second target polynucleotide (see page 1, [0005]: wherein Brenner et al. disclosed that molecules to be
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`counted in a sample are each labeled with a unique oligonucleotide tag and page 12, [0073] wherein
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`Brenner et al. disclosed that oligonucleotide probes specifically anneal to target polynucleotide); c)
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`subjecting said first and second ligation probes to a ligation reaction to obtain one or more ligated
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`products (page 12, [0073] wherein Brenner et al. disclosed a method that employs template driven
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`ligation [chemical ligation, enzymatic ligation with a ligase, or a ligation that includes a polymerase
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`extension followed by ligation by a ligase]); d) partitioning said one or more ligated products into two or
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`more partitions (see pages 1-2, [0009] wherein Brenner et al. disclosed the method for determining a
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`number of target molecules in a sample comprised step (a) i.e. providing molecule tag conjugates each
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`comprising an oligonucleotide tag such that substantially every different molecule of the sample is
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`attached to a different oligonucleotide tag, AND step (b) dividing the oligonucleotide tags of the molecule-
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`tag conjugates into aliquots by sorting the oligonucleotide tags according to the identity of a subunit within
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`a first or a successive position; AND step (c) repeating step (b) for at least one aliquot in each successive
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`application of step (b) until at least one aliquot has no oligonucleotide tags that can be separated into
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`aliquots, thereby determining the number of molecules in the sample to be in the range determined by a
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`first number equal to the size of the subset taken to a power equal to the lowest number of times step (b)
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`has been applied to produce an aliquot having no oligonucleotide tags less one and a second number
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`equal to the size of the subset taken to a power equal to the greatest number of times step (b) has been
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`applied to produce an aliquot having no oligonucleotide tags less one); Brenner et al. also taught on page
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`4, [0035]: a sequence-specific sorting process and referenced this sorting in Appendix 1 on pages 23-24.
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`e) amplifying a region within said one or more ligated products to obtain amplified products (page 1,
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`[0005]: Brenner et al. disclosed that tags are then amplified; see also page 12, [0073] wherein Brenner et
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`al. disclosed that after oligonucleotides probes were ligated, primers were added and ligation product was
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`amplified to form amplicon); f) determining a number of said partitions that contain said amplified
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`products; (Brenner et al. taught on page 4, [0035]: that after an initial sequence specific sorting, the
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`number of different tags in the population of fragments is determined by successively sorting of binary
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`tags into two separate aliquots using the process outlined in Appendix I on pages 23-24. Brenner et al.
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`

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`Application/Control Number: 12/954,634
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`Art Unit: 1637
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`Page 8
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`taught that aliquots are assayed using a PCR, which can be implemented with one or more controls or
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`internal standards for confirming the absence of fragments The sorting process continues until there is an
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`aliquot with no fragments detected. Such a process is outlined in FIG. 1B on sheet 2 of 37 of the
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`drawings); and g) calculating a copy number of said first target polynucleotide based on said number of
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`said partitions (Brenner et al. taught on page 4, [0035]: estimation and algorithm to determine the number
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`of molecule in the original mixture containing the target polynucleotides).
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`Regarding claims 2-4, Brenner et al. taught on page 4, [0035], a method wherein said first and
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`second target polynucleotides are not partitioned into said two or more partitions. Specifically, Brenner et
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`al. taught FIG. 1 B on sheet 2 of 37 of the drawings which shows that repeated sorting eventually results
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`in an aliquot with no fragments. Brenner et al. taught on page 4, [0035], sorting with PCR amplification
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`until repeated sorting eventually results in an aliquot with no fragments and therefore said two or more
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`partitions remain substantially intact.
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`Regarding claim 5, Brenner et al. taught a method wherein said first and second ligation probes
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`are each designed to bind to said first target polynucleotide (see page 12, [0073]). Brenner et al. taught
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`that first (6340) and second (6342) oligonucleotide probes specifically anneal to target polynucleotide
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`(6350).
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`Regarding claim 6, Brenner et al. taught on page 50, claim 17, a method wherein said
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`partitioning of said one or more ligated products into two or more partitions does not comprise partitioning
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`said first and second target polynucleotides. Specifically, Brenner et al. taught claim 17 wherein a
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`method of estimating a number of target polynucleotides in a mixture is recited. The method comprises
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`the steps of: labeling by sampling each target polynucleotide in the mixture so that substantially every
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`target polynucleotide has a unique oligonucleotide tag; amplifying the oligonucleotide tags of the labeled
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`target polynucleotides; and determining the number of different oligonucleotide tags in a sample of
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`amplified oligonucleotide tags by determining nucleotide sequences thereof, thereby estimating the
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`Art Unit: 1637
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`Page 9
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`number of target polynucleotides in the mixture. No division of oligonucleotide tag into aliquots or
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`successively sorting the oligonucleotide tags to form one or more separate mixtures is required for claim
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`17.
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`Regarding claim 7, Brenner et al. taught on page 4, [0035], a method wherein said two or more
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`partitions comprise an amplification reaction that is initiated from said one or more ligated products.
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`Regarding claim 8, Brenner et al. taught a method wherein said first ligation probe is designed to
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`bind to a polynucleotide sequence that is conserved between individuals within a species. Brenner et al.
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`taught probes specific for different single nucleotide polymorphism (SNP), each labeled with a unique
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`oligonucleotide tag (page 2, [0012]). In this instance, the specific SNP hybridizing probe binds the
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`nucleotides of the target polynucleotide sequence to which it is complementary to. The nucleotide
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`sequence of target polynucleotides having a SNP are conserved except at the single site of the
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`polynucleotide. Brenner et al. also taught the suitability of the specific probes for selected regions of
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`particular genes (page 19, [0109]). Brenner et al. taught probe are oligonucleotide complementary to a
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`target polynucleotide of interest such as a segment of genomic DNA, an RNA gene product.
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`Regarding claims 1, 59 and 9-10, Brenner et al. taught a method wherein said two or more
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`partitions are aqueous droplets present within a mixture of at least two immiscible fluids. Specifically,
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`Brenner et al. taught an exemplary embodiment of the invention employing emulsion-based
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`amplification and seguencing by synthesis to identify the oligonucleotide tags (Figs. 7A-7B and page
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`2, and Figs. 7A-7B legend, under Brief Description of the drawings; and also page 13, [0074]). Brenner et
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`al. taught on page 13, [0074] an aqueous phase solution containing the amplicon and amplification
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`reagents mixed with mineral oil, and beads derivatized with a primer oligonucleotide so that micro-
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`droplets of aqueous phase solution forms in the oil" i.e. continuous oil phase. Brenner et al. further taught
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`"the composition of these reagents is selected to maximize the formation of such microdroplets containing
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`a single bead and a single oligonucleotide tag from the amplicon. Once such an emulsion is formed,
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`Application/Control Number: 12/954,634
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`Art Unit: 1637
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`Page 10
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`conditions are selected for implementing an amplification reaction, such as PCR, after which the emulsion
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`is broken, the beads are collected, and the attached clonal populations of oligonucleotide tags are
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`analyzed, preferably by a sequencing by synthesis technique, such as pyrosequencing" (page 13,
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`[0074]).
`
`Regarding claims 11-12, Brenner et al. taught a method further comprising providing for each
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`target polynucleotide a plurality of nucleic acid probes (page 2, [0011]). Brenner et al. taught that such
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`plurality is a number in a range of 2 to 6 nucleotides (page 21, [0113]). Therefore Brenner et al. taught the
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`binding at least four ligation probes to said first target polynucleotide and to said second target
`
`polynucleotide.
`
`Regarding claim 13, Brenner et al. taught a method wherein a first ligation probe is designed to
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`bind to a first region within said first target polynucleotide and a second ligation probe is designed to bind
`
`to a second region within the first target polynucleotide, wherein said first and second regions do not have
`
`identical sequences. Brenner et al. taught in Fig. 6A, the first (6340) and second (6342) oligonucleotide
`
`probes specifically annealing to target polynucleotide (6350) by forming perfectly matched duplexes
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`between region (6301) and region (6301') of oligonucleotide probe (6340) and between region (6303) and
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`region (6303') of oligonucleotide probe (6342). First and second oligonucleotide probes (6340) and (6342)
`
`have primer binding site (6306) and (6308). Brenner et al. also taught on page 12, [0073] a separate
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`oligonucleotide ligation probe to fill gap (6304) within the first target polynucleotide and between the
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`binding regions of probes (6340) and (6342). Brenner et al. taught that after oligonucleotides (6340) and
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`(6342) are ligated (6314) to form ligation product (6316), primers (6309) and (6311) are added and
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`ligation product (6316) is amplified to form amplicon.
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`Regarding claim 14, Brenner et al. taught a method wherein said first target polynucleotide is not
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`identical to said second target polynucleotide (see page 18, [0102]). Specifically, Brenner et al. taught a
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`population comprising one or more target polynucleotides that are mixtures of different sequences.
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`Art Unit: 1637
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`Regarding claims 17-19, Brenner et al. taught that method for detecting genetic copy variation
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`that detects aneuploidies including chromosome 21 trisomy (page 1, [0004]). Brenner et al. taught a
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`labeled probe that target a first target polynucleotide of chromosome 21 at a selected locus (page 8,
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`[0054]). Brenner et al. further disclosed that the selected segment of chromosome is associated with fetal
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`aneuploidy (page 1, [0004], page 2, [0013], page 8, [0055]).
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`Regarding claims 20-21, Brenner et al. taught a method wherein the ligation reaction results in
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`ligation of a 5' region of said first ligation probe to a 3' region of said first ligation probe to obtain a circular
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`ligated product and also a ligation reaction results in ligation of a 5' region of said first ligation probe to a
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`3' region of said second ligation probe to obtain a linear ligated product comprising at least a portion of
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`said first and second ligation probes (page 12, [0073]). Brenner et al. particularly taught that "in the case
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`of the former type of ligation probe, ligation results in a ligation product that is a closed single stranded
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`DNA circle (page 12, [0073]). Brenner et al. also taught that in the case of a latter type of ligation probe,
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`ligation results in a ligation product that is a linear polynucleotide" (page 12, [0073]).
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`Regarding claims 22-25, Brenner et al. taught a method wherein said 5' region and said 3' region
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`of said first ligation probe are each designed to bind adjacent/neighboring sequences within said first
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`target polynucleotide (see Fig. 6A of sheet 17 of 37 of the drawings and page 12, [0073] wherein Brenner
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`et al. taught an embodiment in which after annealing to the same target polynucleotide, oligonucleotide
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`probes (6340) and (6342) are abutting so that gap or nick (6312) can be eliminated by ligation, e.g. by a
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`ligase. Brenner et al. taught that neighboring sequences are separated by at least one nucleotide (page
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`12, [0073]). Specifically, Brenner et al. disclosed that the gap between neighboring sequences can be
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`filled (6304) by ligating a separate oligonucleotide or it can be filled by extending a 3' end of one of the
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`oligonucleotide probes so that the ends abut, whereupon the ends are ligated. In one aspect, such gap is
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`in the range of from 1 to 40 nucleotides. In another aspect, it is in the range of from 1 to 2 nucleotides;
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`and in another aspect, it is one nucleotide. Therefore in the ligation reaction of Brenner et al. further
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`comprised a template-driven gap fill reaction to incorporate nucleotides in a region between said 5' region
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`and said 3' region of said first ligation probe.
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`Regarding claims 26 and 28, Brenner et al. taught a method wherein said first ligation probe
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`comprises a site cleavable by an enzyme (pages 12-13, [0073]) comprises a restriction site. Specifically,
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`Brenner et al. taught capture ligation products (6316) is then successively digested with restriction
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`endonucleases recognizing sites (6324) and (6326) to release (6354) oligonucleotide tags (6356) that can
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`be sequenced directly or concatenated (6358) to form concatemers (6359), which are then cloned and
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`sequenced (6360).
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`Regarding claim 29, Brenner et al. taught a method wherein said first ligation probe is a padlock
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`probe (page 12, [0073]). On page 12, [0073], Brenner et al. taught that "ligation probe" refers to padlock
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`probes or to probes comprising a pair of separate first and second oligonucleotide probes.
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`Regarding claim 31, Brenner et al. taught a method further comprising performing an enzymatic
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`reaction to remove linear polynucleotides. Brenner et al. taught on page 11, [0067] that polynucleotides
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`and fragments that do not circularize (4119) are destroyed by digesting (4116) them with one or more
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`nucleases, thereby removing a possible source of background signal.
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`Regarding claim 32, Brenner et al. taught a method further comprising performing an enzymatic
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`reaction to remove single-stranded polynucleotides. On page 3, [0032], Brenner et al disclosed that
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`nucleic acid probes of the invention associate with target nucleic acid by specific hybridization. Brenner et
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`al. taught that such specifically hybridized probes are then altered so that they may be isolated or
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`distinguished from non-specifically hybridized probes and further taught on page 12, [0073] after ligation,
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`the reaction mixture can be treated with 3' and 5' exonucleases to digest any unligated probes.
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`In the instance that linear polynucleotide are single stranded polynucleotides, Brenner et al. taught on
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`page 11, [0067] that polynucleotides and fragments that do not circularize (4119) are destroyed by
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`digesting (4116) them with one or more nucleases, thereby removing a possible source of background
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`signal.
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`Regarding claims 33-34, Brenner et al. taught a method wherein said first ligation probe is
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`conjugated to a first signaling agent and wherein said second ligation probe is conjugated to a second
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`signaling agent. Brenner et al. taught a method that counts molecules that are uniquely labeled with tags
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`wherein the probe binding target molecules have a different tag (page 3, [0030]). Brenner et al. taught an
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`example on page 17, [0099] wherein each polynucleotide was fluorescently labeled at the 5' end of the
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`metric tag side. On page 22, [0119], Brenner et al. taught a first signaling agent is a fluorescent marker of
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`a first color and a second signaling agent is a fluorescent marker of a second color since Brenner et al.
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`taught that different molecular species have different fluorescent labels having distinct emission spectra
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`and data is collected and recorded at multiple wavelengths.
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`Regarding claim 35, Brenner et al. taught a method wherein said determining a number of
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`partitions that contain said amplified products comprises detecting said first ligation probe with a first
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`signaling agent and detecting said second ligation probe with a second signaling agent (page 21, [0114]
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`and page 22, [0119]). Brenner et al. taught quantitative real time PCR measurement for making mult