US. Application No. 12/954,634
`Response to Restriction Requirement mailed September 13, 2012
`Electronically filed November 5, 2012
`
`AMENDMENTS TO THE CLAIMS:
`
`This listing of claims will replace all prior versions, and listings, of claims in this
`
`application.
`
`Listing of claims:
`
`1.
`
`(Original) A method of detecting copy number of a target polynucleotide within a
`
`population of genetic material comprising:
`
`a. binding a first ligation probe to a first target polynucleotide;
`
`b. binding a second ligation probe to a second target polynucleotide;
`
`c.
`
`subjecting said first and second ligation probes to a ligation reaction to obtain
`
`one or more ligated products;
`
`(1. partitioning said one or more ligated products into two or more partitions;
`
`e. amplifying a region within said one or more ligated products to obtain amplified
`
`products;
`
`f. determining a number of said partitions that contain said amplified products; and
`
`g. calculating a copy number of said first target polynucleotide based on said
`
`number of said partitions.
`
`2.
`
`(Currently amended) The method of claim 1, wherein said first and second target
`
`pebnueleetidepolflucleotides [[is]]m not partitioned into said two or more partitions.
`
`3.
`
`(Currently amended) The method of claim 1, wherein during [[the]]@ amplification
`
`process said two or more partitions remain substantially intact.
`
`4.
`
`(Original) The method of claim 1, wherein during said determining of step (i), said two or
`
`more partitions remain substantially intact.
`
`5.
`
`(Currently amended) The method of claim 1, wherein said first and second ligation probes
`
`are each designed to bind to said first target polynucleotide;
`
`2
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`Atty. Docket No. 38938-705201
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`

`

`US. Application No. 12/954,634
`Response to Restriction Requirement mailed September 13, 2012
`Electronically filed November 5, 2012
`
`6.
`
`(Currently amended) The method of claim 1, wherein said partitioning of step ((1) does not
`
`comprise partitioning said first and second target polflucleotidespel—ynueleetiele—meleeule.
`
`7.
`
`(Currently amended) The method of claim 1, wherein said two or more partitions comprise
`
`an amplification reaction that is initiated from said one or more ligated preduetproducts.
`
`8.
`
`(Original) The method of claim 1, wherein said first ligation probe is designed to bind to a
`
`polynucleotide sequence that is conserved between individuals within a species.
`
`9.
`
`(Currently amended) The method of claim 1, wherein said two or more partitions are
`
`aqueous droplets present within a mixture of at least two immiscible fluids.
`
`10.
`
`(Currently amended) The method of claim [[10]]2, wherein a continuous oil phase
`
`comprises said aqueous droplets.
`
`11.
`
`(Original) The method of claim 1, fiarther comprising binding at least four ligation probes
`
`to said first target polynucleotide.
`
`12.
`
`(Original) The method of claim 1, fiarther comprising binding at least four ligation probes
`
`to said first target polynucleotide and at least four ligation probes to said second target
`
`polynucleotide.
`
`13.
`
`(Original) The method of claim 1, wherein said first ligation probe is designed to bind to a
`
`first region within said first target polynucleotide and said second ligation probe is designed to
`
`bind to a second region within said first target polynucleotide, wherein said first and second
`
`regions do not have identical sequences.
`
`14.
`
`(Currently amended) The method of claim 1, wherein said first target polynucleotide is not
`
`identical to said second target polynucleotide,
`
`15.
`
`(Currently amended) The method of claim [[15]]fi, wherein said first target
`
`polynucleotide is a test chromosome and said second target polynucleotide is a reference
`
`chromosome.
`
`3
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`Atty. Docket No. 38938-705201
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`

`

`US. Application No. 12/954,634
`Response to Restriction Requirement mailed September 13, 2012
`Electronically filed November 5, 2012
`
`16.
`
`(Currently amended) The method of claim [[16]]1_5, wherein said test chromosome is
`
`selected from the group consisting of: chromosome 21, chromosome 13, chromosome 18, and
`
`[[the]]@ X chromosome.
`
`17.
`
`(Currently amended) The method of claim 1, wherein said first target polynucleotide is a
`
`chromosome selected from the group consisting of ehremesemechromosomes 1, 2, 3, 4, 5, 6, 7, 8,
`
`9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, and Y.
`
`18.
`
`(Original) The method of claim 1, wherein said first target polynucleotide is a segment of a
`
`chromosome.
`
`19.
`
`(Currently amended) The method of claim [[19]]fi, wherein said segment of a
`
`chromosome is associated with fetal aneuploidy.
`
`20.
`
`(Currently amended) The method of claim 1, wherein said ligation reaction results in
`
`[[the]] ligation of a 5’ region of said first ligation probe to a 3’ region of said first ligation probe to
`
`obtain a circular ligated product.
`
`21.
`
`(Currently amended) The method of claim 1, wherein said ligation reaction results in [[the
`
`]]ligation of [[the]]a 5’ region of said first ligation probe to [[the]]a 3’ region of said second
`
`ligation probe to obtain a linear ligated product comprising at least a portion of said first and
`
`second ligation probes.
`
`22.
`
`(Currently amended) The method of claim [[21]]@, wherein said 5 ’ region and said 3’
`
`region of said first ligation probe are each designed to bind adjacent sequences within said first
`
`target polynucleotide.
`
`23.
`
`(Currently amended) The method of claim [[21]]@, wherein said 5’ region and said 3’
`
`region of said first ligation probe are each designed to bind neighboring sequences within said first
`
`target polynucleotide,
`
`24.
`
`(Currently amended) The method of claim [[24]]Q, wherein said neighboring sequences
`
`are separated by at least one nucleotide.
`
`4
`
`Atty. Docket No. 38938-705201
`
`

`

`US. Application No. 12/954,634
`Response to Restriction Requirement mailed September 13, 2012
`Electronically filed November 5, 2012
`
`25.
`
`(Currently amended) The method of claim [[25]]fi, wherein said ligation reaction further
`
`comprises a template-driven gap fill reaction to incorporate nucleotides in [[the]]a region between
`
`said 5’ region and said 3’ region of said first ligation probe.
`
`26.
`
`(Original) The method of claim 1, wherein said first ligation probe comprises a site
`
`cleavable by an enzyme.
`
`27.
`
`(Currently amended) The method of claim [[27]]E, wherein said site cleavable by an
`
`enzyme comprises one or more uracils.
`
`28.
`
`(Currently amended) The method of claim [[27]]&, wherein said site cleavable by an
`
`enzyme comprises a restriction site.
`
`29.
`
`(Original) The method of claim 1, wherein said first ligation probe is a padlock probe.
`
`30.
`
`(Original) The method of claim 1, wherein said first ligation probe is a molecular inversion
`
`probe.
`
`31.
`
`(Original) The method of claim 1, fiarther comprising performing an enzymatic reaction to
`
`remove linear polynucleotides.
`
`32.
`
`(Original) The method of claim 1, fiarther comprising performing an enzymatic reaction to
`
`remove single-stranded polynucleotides.
`
`33.
`
`(Currently amended) The method of claim 1, wherein said first ligation probe is
`
`conjugated to a first signaling agent and wherein said second ligation probe is conjugated toa
`
`second signaling agent.
`
`34.
`
`(Currently amended) The method of claim [[34]]fi, wherein said first signaling agent is a
`
`fluorescent marker of a first color and said second signaling agent is a fluorescent marker of a
`
`second color.
`
`5
`
`Atty. Docket No. 38938-705201
`
`

`

`US. Application No. 12/954,634
`Response to Restriction Requirement mailed September 13, 2012
`Electronically filed November 5, 2012
`
`35.
`
`(Currently amended) The method of claim [[35]]1, wherein said determining of step (f)
`
`comprises detecting said first ligation probe with a first signaling agent and detecting said second
`
`ligation probe with a second signaling agent.
`
`36.
`
`(Currently amended) The method of claim [[3 6]]1, wherein said first ligation probe
`
`comprises a plurality of first ligation probes, wherein each probe of said plurality of first ligation
`
`probes is directed to a different region of a first chromosome, and wherein said second ligation
`
`probe comprises a plurality of second ligation probes, wherein each probe of said plurality of
`
`second ligation probes is directed to a different region of a second chromosome.
`
`37.
`
`(Original) The method of claim 35, wherein said first target polynucleotide is a test
`
`chromosome and said second target polynucleotide is a reference chromosome.
`
`38.
`
`(Original) The method of claim 14, wherein said first and second ligation probes are
`
`conjugated to a signaling agent with the same signaling color.
`
`39.
`
`(Original) A method of detecting copy number of a target polynucleotide within a
`
`population of genetic material comprising:
`
`a. binding a first ligation probe to a first target polynucleotide;
`
`b. binding a second ligation probe to a second target polynucleotide;
`
`c.
`
`subjecting said first and second ligation probes to a ligation reaction in order to
`
`obtain one or more ligated products;
`
`d. partitioning said one or more ligated products into two or more aqueous
`
`droplets within a continuous oil phase;
`
`e. amplifying a sequence within said one or more ligated products to obtain
`
`amplified products;
`
`f. determining a number of said two or more aqueous droplets that contain said
`
`amplified products; and
`
`g. calculating a copy number of said first target polynucleotide.
`
`40-58. (Canceled).
`
`6
`
`Atty. Docket No. 38938-705201
`
`

`

`US. Application No. 12/954,634
`Response to Restriction Requirement mailed September 13, 2012
`Electronically filed November 5, 2012
`
`59.
`
`(Currently amended) A method of detecting a fetal genetic condition comprising:
`
`a. obtaining a mixture of maternal and fetal genetic material comprising target
`
`polynucleotides;
`
`b. combining said mixture with targeting oligonucleotides that bind said target
`
`polynucleotides;
`
`c. subdividing said targeting oligonucleotides into reaction volumes, wherein at
`
`least one of said reaction volumes comprises no said target polynucleotide and no said
`
`targeting oligonucleotide;
`
`d. performing an amplification reaction within said reaction volumes;
`
`e. detecting a presence of said target polynucleotide or said targeting
`
`oligonucleotide within said reaction volumes; and
`
`f. determining [[the]]a relative level of said target polynucleotide in said mixture
`
`in order to detect a fetal genetic condition.
`
`60-74. (Canceled).
`
`75.
`
`(Withdrawn) A microcapsule comprising a ligated probe wherein said microcapsule is
`
`obtained by:
`
`a. selectively binding a plurality of ligation probes to target polynucleotides within
`
`a genetic sample;
`
`b.
`
`ligating a 5’ end of at least one of said bound ligation probes to a 3’ end of the
`
`same or different bound ligation probe, thereby obtaining at least one ligation product;
`
`0.
`
`introducing an aqueous solution comprising said at least one ligation product
`
`into a device for generating droplets;
`
`(1. using said device to produce an aqueous droplet comprising said at least one
`
`ligation product, wherein said aqueous droplet is within an immiscible fluid; and
`
`e. converting said droplet into a microcapsule comprising a solid-phase exterior.
`
`76-88 (Canceled).
`
`7
`
`Atty. Docket No. 38938-705201
`
`

`

`US. Application No. 12/954,634
`Response to Restriction Requirement mailed September 13, 2012
`Electronically filed November 5, 2012
`
`89.
`
`(Withdrawn) A water-in-oil mixture comprising two or more aqueous droplets, wherein at
`
`least one of said two or more aqueous droplets comprises a first ligation probe directed to a first
`
`target polynucleotide and at least one of said two or more aqueous droplets comprises a second
`
`ligation probe directed to a second target polynucleotide.
`
`90-100 (Canceled).
`
`101.
`
`(New) The method of claim 1, fiirther 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.
`
`102.
`
`(New) The method of claim 59, wherein, for a particular target polynucleotide, an
`
`algorithm is used to calculate an average number of copies per reaction volume of said particular
`
`target polynucleotide.
`
`8
`
`Atty. Docket No. 38938-705201
`
`

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