US. Patent Application No. 12/954,634
`Final Office Action mailed on October 3, 2013
`Response filed on January 29, 2014
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`Listing of claims:
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`AMENDMENTS TO THE CLAIMS
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`1.
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`(Currently amended) A method of detecting copy number of a target polynucleotide within
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`a population of genetic material comprising:
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`a) binding a first ligation probe to a first target polynucleotide;
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`b) binding a second ligation probe to a second target polynucleotide;
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`c)
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`subjecting said first and second ligation probes to a ligation reaction to obtain
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`one or more ligated products, wherein said one or more ligated products are circular;
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`d) partitioning said one or more ligated products into two or more partitions,
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`wherein said two or more partitions are agueous droplets;
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`e) amplifying a region within said one or more ligated products to obtain amplified
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`products, wherein the amplifying occurs in said two or more partitions;
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`f) detecting said partitions to determinedetermining a number of said partitions
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`that contain said amplified products; and
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`g) calculating a copy number of said first target polynucleotide based on said
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`number of said partitions.
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`2.
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`(Previously presented) The method of claim 1, wherein said first and second target
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`polynucleotides are not partitioned into said two or more partitions.
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`3.
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`(Previously presented) The method of claim 1, wherein during said amplification process
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`said two or more partitions remain intact or stable.
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`4.
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`(Previously presented) The method of claim 1, wherein during said determining of step (i),
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`said two or more partitions remain intact or stable.
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`5.
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`(Previously presented) The method of claim 1, wherein said first and second ligation
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`probes are each designed to bind to said first target polynucleotide.
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`6.
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`(Previously presented) The method of claim 1, wherein said partitioning of step ((1) does
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`not comprise partitioning said first and second target polynucleotides.
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`Attorney Docket No. 44347-705201
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`US. Patent Application No. 12/954,634
`Final Office Action mailed on October 3, 2013
`Response filed on January 29, 2014
`
`7.
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`(Previously presented) The method of claim 1, wherein said two or more partitions
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`comprise an amplification reaction that is initiated from said one or more ligated products.
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`8.
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`(Original) The method of claim 1, wherein said first ligation probe is designed to bind to a
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`polynucleotide sequence that is conserved between individuals within a species.
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`9.
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`(Canceled)
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`10.
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`(Currently amended) The method of claim [[9]]1, wherein a continuous oil phase
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`comprises said aqueous droplets.
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`11.
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`(Original) The method of claim 1, fiirther comprising binding at least four ligation probes
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`to said first target polynucleotide.
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`12.
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`(Original) The method of claim 1, fiirther comprising binding at least four ligation probes
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`to said first target polynucleotide and at least four ligation probes to said second target
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`polynucleotide.
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`13.
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`(Original) The method of claim 1, wherein said first ligation probe is designed to bind to a
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`first region within said first target polynucleotide and said second ligation probe is designed to
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`bind to a second region within said first target polynucleotide, wherein said first and second
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`regions do not have identical sequences.
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`14.
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`(Previously presented) The method of claim 1, wherein said first target polynucleotide is
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`not identical to said second target polynucleotide.
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`15.
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`(Previously presented) The method of claim 14, wherein said first target polynucleotide is
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`a test chromosome and said second target polynucleotide is a reference chromosome.
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`16.
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`(Previously presented) The method of claim 15, wherein said test chromosome is selected
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`from the group consisting of: chromosome 21, chromosome 13, chromosome 18, and an X
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`chromosome.
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`US. Patent Application No. 12/954,634
`Final Office Action mailed on October 3, 2013
`Response filed on January 29, 2014
`
`17.
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`(Previously presented) The method of claim 1, wherein said first target polynucleotide is a
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`chromosome selected from the group consisting of chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12,
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`13,14,15,16,17,18,19, 20, 21, 22, X, and Y.
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`18.
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`(Original) The method of claim 1, wherein said first target polynucleotide is a segment of a
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`chromosome.
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`19.
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`(Previously presented) The method of claim 18, wherein said segment of a chromosome is
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`associated with fetal aneuploidy.
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`20.
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`(Previously presented) The method of claim 1, wherein said 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
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`a circular ligated product.
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`21.
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`(Previously presented) The method of claim 1, wherein said ligation reaction results in
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`ligation of a 5 ’ region of said first ligation probe to a 3 ’ region of said second ligation probe to
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`obtain a linear ligated product comprising at least a portion of said first and second ligation probes.
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`22.
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`(Previously presented) The method of claim 20, wherein said 5’ region and said 3’ region
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`of said first ligation probe are each designed to bind adjacent sequences within said first target
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`polynucleotide.
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`23.
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`(Previously presented) The method of claim 20, wherein said 5’ region and said 3’ region
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`of said first ligation probe are each designed to bind neighboring sequences within said first target
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`polynucleotide.
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`24.
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`(Previously presented) The method of claim 23, wherein said neighboring sequences are
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`separated by at least one nucleotide.
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`25.
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`(Previously presented) The method of claim 24, wherein said ligation reaction fiirther
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`comprises a template-driven gap fill reaction to incorporate nucleotides in a region between said 5’
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`region and said 3 ’ region of said first ligation probe.
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`US. Patent Application No. 12/954,634
`Final Office Action mailed on October 3, 2013
`Response filed on January 29, 2014
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`26.
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`(Original) The method of claim 1, wherein said first ligation probe comprises a site
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`cleavable by an enzyme.
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`27.
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`(Previously presented) The method of claim 26, wherein said site cleavable by an enzyme
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`comprises one or more uracils.
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`28.
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`(Previously presented) The method of claim 26, wherein said site cleavable by an enzyme
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`comprises a restriction site.
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`29.
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`(Original) The method of claim 1, wherein said first ligation probe is a padlock probe.
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`30.
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`(Original) The method of claim 1, wherein said first ligation probe is a molecular inversion
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`probe.
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`31.
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`(Original) The method of claim 1, fiarther comprising performing an enzymatic reaction to
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`remove linear polynucleotides.
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`32.
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`(Original) The method of claim 1, fiarther comprising performing an enzymatic reaction to
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`remove single-stranded polynucleotides.
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`33.
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`(Previously presented) The method of claim 1, 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
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`second signaling agent.
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`34.
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`(Previously presented) The method of claim 33, wherein said first signaling agent is a
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`fluorescent marker of a first color and said second signaling agent is a fluorescent marker of a
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`second color.
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`35.
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`(Previously presented) The method of claim 1, wherein said determining of step (f)
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`comprises detecting said first ligation probe with a first signaling agent and detecting said second
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`ligation probe with a second signaling agent.
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`36.
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`(Previously presented) The method of claim 1, wherein said first ligation probe comprises
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`a plurality of first ligation probes, wherein each probe of said plurality of first ligation probes is
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`US. Patent Application No. 12/954,634
`Final Office Action mailed on October 3, 2013
`Response filed on January 29, 2014
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`directed to a different region of a first chromosome, and wherein said second ligation probe
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`comprises a plurality of second ligation probes, wherein each probe of said plurality of second
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`ligation probes is directed to a different region of a second chromosome.
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`37.
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`(Original) The method of claim 35, wherein said first target polynucleotide is a test
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`chromosome and said second target polynucleotide is a reference chromosome.
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`38.
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`(Original) The method of claim 14, wherein said first and second ligation probes are
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`conjugated to a signaling agent with the same signaling color.
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`39.
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`(Currently amended) A method of detecting copy number of a target polynucleotide within
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`a population of genetic material comprising:
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`a) binding a first ligation probe to a first target polynucleotide;
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`b) binding a second ligation probe to a second target polynucleotide;
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`c)
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`subjecting said first and second ligation probes to a ligation reaction in order to
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`obtain one or more ligated products, wherein said one or more ligated products are circular;
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`d) partitioning said one or more ligated products into two or more aqueous
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`droplets within a continuous oil phase;
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`e) amplifying a sequence within said one or more ligated products to obtain
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`amplified products, wherein the amplifying occurs in said two or more aqueous droplets;
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`f) detecting said two or more aqueous droplets to determinedetermining a number
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`of said two or more aqueous droplets that contain said amplified products; and
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`g) calculating a copy number of said first target polynucleotide.
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`40-58. (Canceled).
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`59.
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`(Currently amended) A method of detecting a fetal genetic condition comprising:
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`a) obtaining a mixture of maternal and fetal genetic material comprising target
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`polynucleotides;
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`b) combining said mixture with targeting oligonucleotides that bind said target
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`polynucleotides, wherein said targeting oligonucleotides are circular;
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`US. Patent Application No. 12/954,634
`Final Office Action mailed on October 3, 2013
`Response filed on January 29, 2014
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`c) subdividing said targeting oligonucleotides into reaction volumes, which
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`reaction volumes are aqueous droplets, wherein at least one of said reaction volumes
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`comprises no said target polynucleotide and no said targeting oligonucleotide;
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`d) performing an amplification reaction within said reaction volumes;
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`e) detecting said reactions volumes to detecting a presence of said target
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`polynucleotide or said targeting oligonucleotide within said reaction volumes; and
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`f) determining a relative level of said target polynucleotide in said mixture in order
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`to detect a fetal genetic condition.
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`60-74. (Canceled).
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`75.
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`(Withdrawn) A microcapsule comprising a ligated probe wherein said microcapsule is
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`obtained by:
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`a) selectively binding a plurality of ligation probes to target polynucleotides within
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`a genetic sample;
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`b) ligating a 5’ end of at least one of said bound ligation probes to a 3’ end of the
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`same or different bound ligation probe, thereby obtaining at least one ligation product;
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`0) introducing an aqueous solution comprising said at least one ligation product
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`into a device for generating droplets;
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`(1) using said device to produce an aqueous droplet comprising said at least one
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`ligation product, wherein said aqueous droplet is within an immiscible fluid; and
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`e) converting said droplet into a microcapsule comprising a solid-phase exterior.
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`76-88 (Canceled).
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`89.
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`(Withdrawn) A water-in-oil mixture comprising two or more aqueous droplets, wherein at
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`least one of said two or more aqueous droplets comprises a first ligation probe directed to a first
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`target polynucleotide and at least one of said two or more aqueous droplets comprises a second
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`ligation probe directed to a second target polynucleotide.
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`90-100 (Canceled).
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`Attorney Docket No. 44347-705201
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`US. Patent Application No. 12/954,634
`Final Office Action mailed on October 3, 2013
`Response filed on January 29, 2014
`
`101.
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`(Previously Presented) The method of claim 1, further comprising performing an
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`enzymatic reaction to remove said first and second target polynucleotides, wherein said first and
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`second target polynucleotides are linear polynucleotides or single-stranded polynucleotides and
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`wherein said enzymatic reaction is performed prior to the amplification step.
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`102.
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`(Previously Presented) The method of claim 59, wherein, for a particular target
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`polynucleotide, an algorithm is used to calculate an average number of copies per reaction volume
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`of said particular target polynucleotide.
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`103.
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`(Canceled)
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`104.
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`(Canceled)
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`105.
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`(Previously presented) The method of claim 59, wherein an algorithm is used to calculate
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`an average number of copies/droplet of a particular genetic target
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