METHODS AND COMPOSITIONS FOR DETECTING GENETIC MATERIAL
First Claim
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1. 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;
d. 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.
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Abstract
This invention provides compositions and methods for detecting differences in copy number of a target polynucleotide. In some cases, the methods and compositions provided herein are useful for diagnosis of fetal genetic abnormalities, when the starting sample is maternal tissue (e.g., blood, plasma). The methods and materials described apply techniques for allowing detection of small, but statistically significant, differences in polynucleotide copy number.
243 Citations
101 Claims
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1. A method of detecting copy number of a target polynucleotide within a population of genetic material comprising:
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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; d. 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. - View Dependent Claims (2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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29. The method of 27, wherein said site cleavable by an enzyme comprises a restriction site.
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40. A method of detecting copy number of a target polynucleotide within a population of genetic material comprising:
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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. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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60. 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 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 relative level of said target polynucleotide in said mixture in order to detect a fetal genetic condition. - View Dependent Claims (61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75)
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76. A microcapsule comprising a ligated probe wherein said microcapsule is obtained by:
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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;c. introducing an aqueous solution comprising said at least one ligation product into a device for generating droplets; d. 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. - View Dependent Claims (77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89)
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- 90. 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.
Specification