UNITED STATES PATENT AND TRADEMARK OFFICE
`
`UNITED STATES DEPARTMENT OF COMMERCE
`United States Patent and Trademark Office
`Address: COMIVHSSIONER FOR PATENTS
`PO. Box 1450
`Alexandria1 Virginia 22313-1450
`www.uspto.gov
`
`w
`
`'I AND1%9
`
`
`
` \
`
`
`
`
`
`12/954,634
`
`11/25/2010
`
`Benajamin HINDSON
`
`QL1364
`
`5813
`
`(11/19/2017 —KOLISCH HARTWELL, pc. m
`7590
`23581
`200 PACIFIC BUILDING
`OYEYEML OLAYINKAA
`520 SW YAMHILL STREET
`PORTLAND, OR 97204
`
`PAPER NUMBER
`
`1637
`
`NOTIFICATION DATE
`
`DELIVERY MODE
`
`01/19/2017
`
`ELECTRONIC
`
`Please find below and/or attached an Office communication concerning this application or proceeding.
`
`The time period for reply, if any, is set in the attached communication.
`
`Notice of the Office communication was sent electronically on above—indicated "Notification Date" to the
`following e—mail address(es):
`
`docketing @khpatent.c0m
`veronica @khpatent.c0m
`
`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
`
`A SHORTENED STATUTORY PERIOD FOR REPLY IS SET TO EXPIRE g MONTHS FROM THE MAILING DATE OF
`THIS COMMUNICATION.
`Extensions of time may be available under the provisions of 37 CFR1. 136( a).
`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).
`
`In no event, however, may a reply be timely filed
`
`Status
`
`1)IZI Responsive to communication(s) filed on 06/06/2016.
`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
`
`; the restriction requirement and election have been incorporated into this action.
`
`4)|:| Since this application is in condition for allowance except for formal matters, prosecution as to the merits is
`
`closed in accordance with the practice under Exparte Quay/e, 1935 CD. 11, 453 O.G. 213.
`
`Disposition of Claims*
`
`5)IZI Claim(s) 13 4 681014-16181922—3133-353738 and 101 is/are pending in the application.
`5a) Of the above claim(s)
`is/are withdrawn from consideration.
`
`6)I:I Claim(s)
`is/are allowed.
`
`7)|Z| Claim(s) 134 681014-16 18- 19 2231 33-35 37-38 101 is/are rejected.
`8)|:I Claim(s)_ is/are objected to.
`
`
`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
`
`participating intellectual property office for the corresponding application. For more information, please see
`hit
`I/'/\WIIW.USOI.O. ovI’ atentS/init events/
`
`
`
`hI/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
`
`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
`
`b)|:l Some” c)I:l None of the:
`
`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
`
`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)
`
`
`
`3) D Interview Summary (PTO-413)
`1) E Notice of References Cited (PTO-892)
`Paper No(s)/Mai| Date.
`.
`.
`4) I:I Other'
`2) I] InformatIon DIsclosure Statement(s) (PTO/SB/08a and/or PTO/SB/08b)
`Paper No(s)/Mai| Date
`US. Patent and Trademark Office
`PTOL—326 (Rev. 11-13)
`
`Office Action Summary
`
`Part of Paper No./Mai| Date 20160727
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 2
`
`DETAILED ACTION
`
`Notice of Pre-AIA or AIA Status
`
`1.
`
`The present application is being examined under the pre-AlA first to invent provisions.
`
`Status of the Applications, Amendments and/or Claims
`
`2.
`
`This action is written in response to Applicant's correspondence submitted June 06, 2016. In the
`
`paper of June 06, 2016, Applicant amended claims 1, 4, 10, 15-16, 22-23, 25, 35 and 101 and canceled
`
`claims 2, 5, 7, 11-13, 17, 20-21, 32, 36, 39, 59, 75, 89, 102 and 105.
`
`3.
`
`4.
`
`Claims 1, 3-4, 6, 8, 10, 14-16, 18-19, 22-31, 33-35, 37-38 and 101 are pending.
`
`All claim amendments and arguments from the paper of June 06,2016 have been thoroughly
`
`reviewed but were found insufficient to place the instantly examined claims in condition for allowance.
`
`The following rejections are either newly presented, or are reiterated from the previous Office Action. Any
`
`rejections not reiterated in this Office Action have been withdrawn. This Office Action is Final.
`
`Status of the Claims
`
`5.
`
`Claims 1, 3-4, 6, 8, 10, 14-16, 18-19, 22-31, 33-35, 37-38 and 101 are pending. Claims 2, 5, 7,
`
`11-13, 17, 20-21, 32, 36, 39, 59, 75, 89, 102 and 105 are canceled. Claims 1, 3-4, 6, 8, 10, 14-16, 18-19,
`
`22-31, 33-35, 37-38 and 101 are currently under examination.
`
`Objections
`
`Response to Arguments
`
`6.
`
`The objection made to claim 105 for missing a concluding period is moot based on the
`
`cancellation of claim 105.
`
`7.
`
`The objections made to claims 6, 32, 39 and 105 under 37 CFR 1.75 as being a substantial
`
`duplicate of claims 2, 31, 1 and 102 are moot based on the cancellations of claims 6, 32, 39 and 105.
`
`Rejections
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 3
`
`8.
`
`The rejections of claims 2, 5, 7, 11-13, 17, 20-21, 32, 36, 39, 59, 102 and 105 under 35 U.S.C.§
`
`112(b) are moot based on the cancellation of these claims.
`
`9.
`
`The rejection of claims 1, 3-4, 6, 8, 10, 14-16, 18-19, 22-31, 33-35, 37-38 and 101 under 35
`
`U.S.C. § 112(b), is withdrawn based on the amendments made to claim 1.
`
`10.
`
`The rejections of claims 5, 7, 11-13, 21 and 36 under 35 U.S.C. § 112(d) are moot based on the
`
`cancellation of these claims.
`
`11.
`
`The rejection of claims 2, 7, 17,20 and 39 under 35 U.S.C. § 103(a) as being unpatentable over
`
`Wang et al. (2007, Genome Biol. 8(11): 1-14: hereafter “Wang et al.”) in view of Beer et al. (2007, Anal
`
`Chem 79(22):8471-5: hereafter “Beer et al.”) is moot based on the cancellation of these claims.
`
`12.
`
`The rejection of claims 1, 3-4, 6, 8, 10, 14-16, 18-19, 22, 26, 31, 35, 37 and 101 under 35 U.S.C.
`
`§ 103(a) as being unpatentable over Wang et al. in view of Beer et al. is withdrawn based on
`
`amendment to claim 1 to recite “partitioning of circular ligation products, ...wherein said two or more
`
`partitions are aqueous droplets”.
`
`13.
`
`The rejection of claims 11-12, 21 and 32 under 35 U.S.C. § 103(a) as being unpatentable over
`
`Wang et al. in view of Beer et al., further in view of Hardenbol et al. (2003, Nat Biotechnol. 21 (6):673-8:
`
`hereafter “Hardenbol et al.”) is moot based on the cancellation of these claims.
`
`14.
`
`The rejection of claims 23-25, 27-29 and 31 under 35 U.S.C. § 103(a) as being unpatentable over
`
`Wang et al. in view of Beer et al., further in view of Hardenbol et al. is withdrawn based on amendment
`
`to claim 1 to recite “partitioning of circular ligation products, ...wherein said two or more partitions are
`
`aqueous droplets”.
`
`15.
`
`The rejection of claims 5, 11-13 and 32 under pre-AIA 35 U.S.C. 103(a) as being unpatentable
`
`over Wang et al. in view of Beer et al. further in view of Ji et al. (2006, Cancer Res. 66(16):7910-9:
`
`hereafter “Ji et al.”) is moot based on the cancellation of these claims.
`
`16.
`
`The rejection of claims 23-25, 27-28 and 30 under pre-AIA 35 U.S.C. 103(a) as being
`
`unpatentable over Wang et al. in view of Beer et al., further in view of Ji et al. is withdrawn based on
`
`amendment to claim 1 to recite “partitioning of circular ligation products, ...wherein said two or more
`
`partitions are aqueous droplets”.
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 4
`
`17.
`
`The rejection of claims 5, 11-12, 21 and 36 are rejected under pre-AIA 35 U.S.C. 103(a) as being
`
`unpatentable over Wang et al. in view of Beer et al., further in view of Hardenbol et al. and Church et al.
`
`(US2008/0269098: hereafter “Church et al.”) is moot based on the cancellation of these claims.
`
`18.
`
`The rejection of claims 23-25, 28, 33-34 and 38 are rejected under pre-AIA 35 U.S.C. 103(a) as
`
`being unpatentable over Wang et al. in view of Beer et al. as applied to claim 1 above, further in view of
`
`Hardenbol et al. and Church et al. is withdrawn based on amendment to claim 1 to recite “partitioning of
`
`circular ligation products, ...wherein said two or more partitions are aqueous droplets”.
`
`19.
`
`The rejection of claims 59, 102 and 105 under pre-AIA 35 U.S.C. 103(a) as being unpatentable
`
`over Wang et al. in View of Ge et al. (2006, Clinica Chimica Acta 369(1): 82-88: hereafter “Ge et al.”) and
`
`Beer et al. is moot based on the cancellation of these claims.
`
`Rejections and Arguments
`
`Response to Arguments
`
`20.
`
`Applicant’s arguments filed 06/06/2016 have been fully considered but are moot because the
`
`arguments do not apply to any of the references being used in the current rejection.
`
`Claim Rejections - 35 USC § 112
`
`21.
`
`The following is a quotation of 35 U.S.C. 112(b):
`(b) CONCLUSION—The specification shall conclude with one or more claims particularly
`pointing out and distinctly claiming the subject matter which the inventor or a joint inventor
`regards as the invention.
`
`The following is a quotation of 35 U.S.C. 112 (pre-AIA), second paragraph:
`The specification shall conclude with one or more claims particularly pointing out and distinctly
`claiming the subject matter which the applicant regards as his invention.
`
`22.
`
`Claims 33-34 and 38 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA), second
`
`paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter
`
`which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
`
`Claims 33-34 recite that the “first ligation probe is conjugated to a first signaling agent” and that
`
`the “second ligation probe is conjugated to a second signaling agent”. Claim 38 recites “first and second
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 5
`
`ligation probes are conjugated to a signaling agent with the same signaling color”. Claims 33-34 and 38
`
`are indefinite as it is unclear whether a probe conjugated to a signaling agent (e.g. a padlock or MIP
`
`ligatable probe) can be further amplified within the emulsion such that the amplification products would
`
`provide an accurate measure of the copy number of either the first or the second target polynucleotides
`
`present in the sample. The amplification product produced from amplifying the labeled circularized probes
`
`would comprise the amplified target nucleotide sequence but would n_ot comprise the signaling agent of
`
`the original circularized probe. It is unclear whether Applicant intends the circularizable first and second
`
`ligation probes to be conjugated to different/same signaling agents or whether Applicant intends to claim
`
`additional detection probes that are conjugated to a first and a second signaling agent, wherein the
`
`signaling agents are different and the detection probes are specific for either the first or the second
`
`circular ligation probe provided in the aqueous droplet. Clarification or amendment of these claims is
`
`required.
`
`23.
`
`The following is a quotation of the appropriate paragraphs of pre-AlA 35 U.S.C. 102 that form the
`
`Claim Rejections - 35 USC § 102
`
`basis for the rejections under this section made in this Office action:
`
`A person shall be entitled to a patent unless —
`
`(e) the invention was described in (1) an application for patent, published under section
`122(b), by another filed in the United States before the invention by the applicant for patent or
`(2) a patent granted on an application for patent by another filed in the United States before
`the invention by the applicant for patent, except that an international application filed under
`the treaty defined in section 351 (a) shall have the effects for purposes of this subsection of an
`application filed in the United States only if the international application designated the United
`States and was published under Article 21(2) of such treaty in the English language.
`
`24.
`
`Claims 1, 3-4, 6, 10, 14, 22, 29-30, 31 and 101 are rejected under pre-AIA 35 U.S.C. 102(e) as
`
`being anticipated by Macevicz et al. (filed May 12, 2009, U82011/0020793).
`
`Regarding claims 1, 3-4 and 22, Macevicz et al. teach their method as being useful for detection
`
`of gene copy number polymorphisms or allelic imbalance (i.e. detecting copy number of a target
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 6
`
`polynucleotide within a population of genetic material: see para [0003]).
`
`Macevicz et al. teach providing a first and second ligation probes for binding a first target
`
`polynucleotide or for binding a second target polynucleotide respectively) (see para [0010] wherein
`
`Macevicz et al. disclose target sequences 100 consisting sequence A and sequence B (i.e. corresponding
`
`to the instant first and second target polynucleotides and probe A 120 corresponding to the instant first
`
`ligation probe and probe B 122 corresponding to the instant second ligation probe; see also para [0021]).
`
`The method of Macevicz et al. comprises the steps of:
`
`a) annealing/binding a first ligation probe to a first target polynucleotide (para [0010]);
`
`b) annealing/binding a second ligation probe to a second target polynucleotide (para [0010]);
`
`c) subjecting said first and second ligation probes to a ligation reaction to obtain a circular first
`
`ligated product and a circular second ligated product (para [0010]: Macevicz et al. disclose reaction
`
`mixture containing circular DNAs);
`
`d) partitioning said circular first ligated product and a circular second ligated product into two or
`
`more partitions wherein said two or more partitions are aqueous droplets (para [0010]: Macevicz et al.
`
`disclose forming an emulsion such that micelles of the emulsion on average contain one or fewer probes
`
`using a reaction mixture comprising primers (106), other amplification components (128) and signal
`
`generating components e.g. molecular beacons (104);
`
`e) amplifying a region within said one or more ligated products to obtain amplified products,
`
`wherein the amplifying occurs in said two or more partitions (see Fig. 1A wherein Macevicz et al. shows
`
`an amplification of a sequence of the single stranded circular DNA performed within each partition/micelle
`
`and see also para [0010]);
`
`f) detecting said partitions to determine a number of said partitions that contain said amplified
`
`products (see para [0010] wherein Macevicz et al. disclose analysis of unique optical signal (130) of Fig.
`
`1A within each micelle); and
`
`g) calculating a copy number of said first target polynucleotide based on said number of said
`
`partitions (para [0010] wherein Macevicz et al. teach determining numbers of micelles generating each of
`
`the different optical signals thereby providing a digital microscopic readout of the ratio of the different
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 7
`
`target polynucleotides present in the original sample and also the abstract and para [0016]).
`
`Regarding claim 3, Macevicz et al. teach during said amplification process said two or more
`
`partitions remain intact or stable since the ePCR step of Fig. 1A shows an intact micelle bearing
`
`amplified/multiple copies of a single stranded circular DNA.
`
`Regarding claim 4, Macevicz et al. teach during said determining of step (f), said two or more
`
`partitions remain intact or stable since the ePCR step of Fig. 1A and arrow 129 of Fig. 1A shows an intact
`
`micelle bearing amplified/multiple copies of a single stranded circular DNA that are each bound to a
`
`molecular beacon probe.
`
`Regarding claim 6, Macevicz et al. teach the partitioning of step (d) does not comprise
`
`partitioning said first and second target polynucleotides since Macevicz et al. teach the use of
`
`exonucleases for removing unligated non-circularized probes and linear target polynucleotide(s) prior to
`
`amplification (para [0010]).
`
`Regarding claims 31 and 101, Macevicz et al. teach the method of claim 1 further comprising
`
`performing an enzymatic reaction to remove said first and second target polynucleotides, wherein said
`
`first and second target polynucleotides are linear polynucleotides or single-stranded polynucleotides and
`
`wherein said enzymatic reaction is performed prior to the amplification step (see para [0010] wherein
`
`Macevicz et al. teach that the reaction mixture containing circular DNA is treated with an exonuclease so
`
`that all non-circularized polynucleotides are destroyed).
`
`Regarding claim 10, Macevicz et al. teach micelles in oil (Figs. 3A-30 and para [0013]) thereby
`
`teaching aqueous droplets enclosed by a continuous oil phase.
`
`Regarding claim 14, Macevicz et al. teach said first and said second target polynucleotides are
`
`not identical (para [0033]).
`
`Regarding claim 22, Macevicz et al. teach the 5' region and the 3' region of the first ligation probe
`
`are each designed to bind adjacent sequences within said first target polynucleotide (see Fig 1A and para
`
`[0010] wherein Macevicz et al. teach the ends of the probes are ligated together to form a single stranded
`
`circular DNA (102)).
`
`Regarding claims 29-30, Macevicz et al. teach that the ligation probe is a padlock probe or
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`molecular inversion probe (para [0021j-[0022] and claim 8).
`
`Page 8
`
`Accordingly the instant claims 1, 3-4, 6, 10, 14, 22, 29-30, 31 and 101 are anticipated by
`
`Macevicz et al.
`
`Claim Rejections - 35 USC § 103
`
`25.
`
`The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness
`
`rejections set forth in this Office action:
`
`(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.
`
`26.
`
`This application currently names joint inventors. In considering patentability of the claims under
`
`pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was
`
`commonly owned at the time any inventions covered therein were made absent any evidence to the
`
`contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention
`
`dates of each claim that was not commonly owned at the time a later invention was made in order for the
`
`examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e),
`
`(f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
`
`27.
`
`Claims 15-16, 18-19, 35 and 37 are rejected under pre-AIA 35 U.S.C. 103(a) as being
`
`unpatentable over Macevicz et al. (filed May 12, 2009, U82011/0020793) in view of Wang et al.
`
`(2007, Genome Biol. 8(11): 1-14: previously cited).
`
`The teachings of Macevicz et al. are set forth above.
`
`Regarding claims 15 and 37, Macevicz et al. do NOT teach a first target polynucleotide that is a
`
`test chromosome and a second target polynucleotide that is a reference chromosome.
`
`Regarding claim 16, Macevicz et al. do NOT teach a test chromosome selected from the group
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 9
`
`consisting of: chromosome 21, chromosome 13, chromosome 18, and an X chromosome. Regarding
`
`claim 18, Macevicz et al. do NOT teach a first target polynucleotide is a segment of a chromosome.
`
`Regarding claim 19, Macevicz et al. do NOT teach a segment of a chromosome that is associated with
`
`fetal aneuploidy.
`
`Regarding claim 35, Macevicz et al. teach determining of step (f) comprising detecting a first
`
`ligation probe with a first signaling agent and detecting a second ligation probe with a second signaling
`
`agent (para [0010]-[0011], [0013] and claim 1:wherein Macevicz disclose micelles as having different
`
`optical signals from signal generating component specific for a tag of each probe/target polynucleotide).
`
`Wang et al.
`
`Wang et al. teach that it was conventional at the time of the instant invention to assess the ratio
`
`for a first target polynucleotide to a second and different reference polynucleotide so as to identify any
`
`copy number aberration between the two polynucleotides.
`
`Regarding claims 15-16, Wang et al. teach a first target polynucleotide (test chromosome:
`
`chromosome X) is not identical to said second target polynucleotide (reference chromosome:
`
`chromosome 2) (pg 5, right col, 1St para below Fig. 2).
`
`Regarding claim 18, Wang et al. teach a first target polynucleotide that is a segment of a
`
`chromosome (pg 4, Fig. 1:wherein a segment of chromosome X (an autosomal marker) is measured;
`
`also pf 5, right col, 1St para: a control region of chromosome 2 measured and pg 3, left col, 2nd para).
`
`Regarding claim 19, Wang et al. teach said first target polynucleotide is chromosome X (pg 2,
`
`right col, “Detection of aberrations”: wherein Wang et al. disclose measurement for a cell line carrying one
`
`copy of chromosome X (male) and for cells carrying the chromosomal aberration (fetal aneuploidy) of
`
`three copies of chromosome X (trisomy), four copies of chromosome X (tetrasomy) or 5 copies of
`
`chromosome X (pentasomy) (see pg 3, left col, 4th para and pg 4, Fig. 1 and pg 5, left col, section entitled
`
`“Accuracy of copy number estimation” etc.).
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 10
`
`Regarding claims 35 and 37, Wang et al. teach said first ligation probe (a first MIP) is conjugated
`
`to a first signaling agent and wherein said second ligation probe (a second MIP) is conjugated to a
`
`second signaling agent (since pg 4, Fig. 1 each a different colors for all markers with each chromosome
`
`uniquely colored; pg 10, all text of right col).
`
`It would have been obvious to a person of ordinary skill in the art at the time of the invention
`
`wanting to detect the ratio/relative amounts of a test polynucleotide/test chromosome segment to a
`
`reference polynucleotide/reference chromosome segment to perform the method of Macevicz et al. by
`
`providing a first ligation probe to bind the test polynucleotide/test chromosome segment under a condition
`
`that promotes the circularization of the first ligation probe, providing a second ligation probe to bind the
`
`reference polynucleotide/reference chromosome segment under a condition that promotes the
`
`circularization of the second ligation probe, partitioning the circularized first and second ligation probes
`
`into aqueous droplets, detecting the first and second ligation probes within the aqueous droplets and
`
`determining the ratio of the test polynucleotide/test chromosome segment to the reference
`
`polynucleotide/reference chromosome segment following the detection of the probes within the droplets.
`
`Wang et al. teach that it was routine/conventional at the time of the invention to assess copy
`
`number aberration of chromosomes by determining the ratio of a test polynucleotide/test chromosome
`
`segment to a reference polynucleotide/reference chromosome segment, thereby predicting patient
`
`response and/or prognosis from the analysis of chromosomal copy number aberration. Further, the
`
`motivation to detect a ratio of the test polynucleotide/test chromosome segment to the reference
`
`polynucleotide/reference chromosome segment in the manner as taught by Macevicz et al. comes from
`
`Macevicz et al. who teach their method as being useful for determining relative amounts of a plurality of
`
`target polynucleotides in a sample (Macevicz et al., claim 1) or for a determining ratio of polynucleotides
`
`by detecting a color readout/tag corresponding to either a first ligation probe or a second ligation probe in
`
`each droplet/micelle.
`
`It would also have been obvious for the ordinary skilled artisan to provide a first ligation probe to
`
`assess the relative amount of test chromosome 21, 13, 18 or X to a reference chromosome for example
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 11
`
`the reference chromosome 2 is taught by Wang et al. as fetal aneuploidy has been diagnosed by
`
`determination of copy number aberration of these test chromosomes relative to a reference chromosome.
`
`In view of the combined teachings and suggestions of all of the cited prior art references, the
`
`instant claims 15-16, 18-19, 35 and 37 are prima facie obvious.
`
`28.
`
`Claims 23-25 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over
`
`Macevicz et al. (filed May 12, 2009, U82011/0020793) in view of Lizardi et al. (1998, US 5,845,033).
`
`Regarding claims 23-25, Macievicz et al. do NOT teach said 5' region and said 3' region of
`
`said first ligation probe are each designed to bind neighboring sequences within said first target
`
`polynucleotide, wherein said neighboring sequences are separated by at least one nucleotide and
`
`wherein said ligation reaction further comprises a template-driven gap fill reaction to incorporate
`
`nucleotides in a region between said 5' region and said 3' region of said first ligation probe.
`
`Regarding claims 23-25, Lizardi et al. (1998) teach the binding of a 5' region and a 3' region of a
`
`first ligation probe to neighboring sequences within a first target polynucleotide (see Lizardi et al. (1998),
`
`Figs. 1-2 and col 6, ln 5-10 and col 6, ln 12—46), wherein said neighboring sequences are separated by at
`
`least one nucleotide (Lizardi et al. (1998), Figs. 1-2 and col 6, ln 5-10 and col 6, ln 12—46) and wherein
`
`said ligation reaction further comprises a template-driven gap fill reaction to incorporate nucleotides in a
`
`region between said 5' region and said 3' region of said first ligation probe (Lizardi et al. (1998), Figs. 1
`
`and col 6, ln 5-10 and col 6, ln 12—46).
`
`It would have been obvious to a person of ordinary skill in the art at the time of the invention to
`
`apply teachings from Lizardi et al. of providing open circular templates that would be amplified to the
`
`method of Macevicz et al., specifically within the droplet/micelles of Macevicz et al. since Lizardi et al.
`
`particularly teach or suggest such templates provide a high detection specificity and sensitivity for specific
`
`target nucleic acids since circularization of the ligation probes occurs in proportion to the amount of target
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 12
`
`sequence present in the sample (Lizardi et al., col 2, In 50-67) and since amplification of such circularized
`
`ligation probes via a rolling circle amplification enhances the detection of the amount of target sequence
`
`present in the sample due to the greater order of magnitudes of this replication compared to PCR (Lizardi
`
`et al., col 2, ln 50-67).
`
`Further, Lizardi et al. teach two advantages for providing their circularizable ligation probes, for
`
`circularization of these ligation probes and for amplification of their probes following a ligation event.
`
`These advantages include quantitative, consistent amplification and detection of a target nucleic acid
`
`sequence since amplification takes place not in cycles but in a continuous replication mode (Lizardi et al.,
`
`col 3, ln 24-35). Lizardi et al. also teach the suitability of providing the circularizable ligation probes for
`
`multiplex detection (Fig. 10 and col 4, ln 19-36: wherein different target nucleic sequences are detected
`
`with different ligation probes that may be differently labeled; see also col 11, In 55-65, col 22, In 1-7, col
`
`22, In 20-67); particularly teaching that multiplex detection with their probes yields an assay wherein the
`
`true ratio of the target sequences in a sample are accurately reflected (Lizardi et al., col 1, ln 42-44 and
`
`col 2, In 16-45).
`
`One of ordinary skill in the art would have implemented the emulsion PCR amplification of ligation
`
`probes of Lizardi et al. in a manner as taught by Macevicz et al. as Macevicz et al. teach that emulsion
`
`amplification reaction of circularizable ligation probes are particularly suitable for determining the relative
`
`amounts of a plurality of target polynucleotides (Macevicz et al., US2011/0020793, para [0003]).
`
`One of ordinary skill in the art would have been motivated to provide a ligation probe that bind
`
`neighboring sequences separated by at least one nucleotide of a first target polynucleotide in a manner
`
`as taught by Lizardi et al. since the specificity and accuracy of ligation assay is derived from the template
`
`driven gap fill reaction that is activated which serves to incorporate correct nucleotides to the ligation
`
`probe for a subsequent circularization of the ligation probe.
`
`In view of the combined teachings and suggestions of all of the cited prior art references, the
`
`instant claims 23-25 are prima facie obvious.
`
`29.
`
`Claims 26-28 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 13
`
`Macevicz et al. (filed May 12, 2009, U82011/0020793), Lizardi et al. (1998, US 5,854,033) and Lizardi
`
`et al. (2001, US 6,316,229).
`
`Regarding claims 26-28, Macievicz et al. teach the circularizable ligation probes “MIP” may
`
`comprises a site cleavable by an enzyme and wherein said site cleavable by an enzyme (e.g. “uracil, N
`
`glycosylase”) comprises a restriction site (para [0021]—[0022]) and wherein the cleavable site is one or
`
`uracils (para [0022]). However, in para [0021]—[OO22], Macievicz et al. provide linearized MIP ligation
`
`probes into droplets (following cleavage with uracil N glycosylase), rather than the circularized ligation
`
`probes.
`
`Regarding claims 26 and 28, Lizardi et al. (1998) teach an open circle probe (“padlock probe))
`
`hybridized to a target sequence and further ligated (Lizardi et al. (1998), col. 3, Fig. 3 and col 5, ln 21-67
`
`and col 6, In 1-46). Lizardi et al. (1998) teach probes suitable for determining the ratio of the amount of
`
`different target sequences present in a sample (Lizardi et al. (1998) col 2, In 24-38, col 2, ln 54-67 and col
`
`3, In 1-35).
`
`Regarding claims 26 and 28, Lizardi et al. (2001) further teach that open circle circularizable
`
`ligation probes (e.g. “padlock probes”) which Lizardi et al. (1998 and 2001) teach are suitable for
`
`accurately determining copy number (Lizardi et al. (2001), col 88, In 60-67), may further comprise a site
`
`cleavable by an enzyme (Lizardi et al., 2001, col, 3, ln 32-46 and col 44, In 25-28); wherein the enzyme is
`
`a restriction enzyme (Lizardi et al. (2001), col 44, In 25-28).
`
`Thus, in view of the teachings and suggestions of Macievicz et al. and Lizardi et al. (1999 and
`
`2001), it would have been obvious to a person of ordinary skill in the art at the time of the invention to
`
`provide a site cleavable by an enzyme to a circularizable ligation probes to be ligated, for the purpose of
`
`accurately determining copy number of a first or a second target polynucleotide that is present in a
`
`sample. The cited references teach that the prior art contemplated that the site is cleavable by a
`
`

`

`Application/Control Number: 12/954,634
`
`Art Unit: 1637
`
`Page 14
`
`restriction enzyme or by an enzyme such as uracil N glycosylase in which case, the cleavable site of the
`
`ligation probe would comprise one or more uracils.
`
`In view of the combined teachings and suggestio