PROCESSES AND COMPOSITIONS FOR METHYLATION-BASED ENRICHMENT OF FETAL NUCLEIC ACID FROM A MATERNAL SAMPLE USEFUL FOR NON INVASIVE PRENATAL DIAGNOSES
First Claim
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1. A method for preparing fetal nucleic acid, which comprises:
- a) providing a sample from a pregnant female;
b) separating fetal nucleic acid from maternal nucleic acid from the sample of the pregnant female according to a different methylation state between the fetal nucleic acid and the maternal nucleic acid counterpart, wherein the fetal nucleic acid comprises one or more CpG sites from one or more of the polynucleotide sequences of SEQ ID NOs;
90-261; and
c) preparing nucleic acid comprising fetal nucleic acid by a process in which fetal nucleic acid separated in part (b) is utilized as a template.
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Abstract
Provided are compositions and processes that utilize genomic regions that are differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample. The compositions and processes described herein are particularly useful for non-invasive prenatal diagnostics, including the detection of chromosomal aneuplodies.
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Citations
30 Claims
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1. A method for preparing fetal nucleic acid, which comprises:
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a) providing a sample from a pregnant female; b) separating fetal nucleic acid from maternal nucleic acid from the sample of the pregnant female according to a different methylation state between the fetal nucleic acid and the maternal nucleic acid counterpart, wherein the fetal nucleic acid comprises one or more CpG sites from one or more of the polynucleotide sequences of SEQ ID NOs;
90-261; andc) preparing nucleic acid comprising fetal nucleic acid by a process in which fetal nucleic acid separated in part (b) is utilized as a template. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for determining the absolute amount of fetal nucleic acid in a maternal sample, wherein the maternal sample comprises differentially methylated maternal and fetal nucleic acid, comprising:
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a) digesting the maternal nucleic acid in a maternal sample using one or more methylation sensitive restriction enzymes, thereby enriching the fetal nucleic acid, wherein the fetal nucleic acid comprises one or more CpG sites from one or more of the polynucleotide sequences of SEQ ID NOs;
90-261; andb) determining the absolute amount of fetal nucleic acid from step a) using a non-polymorphic-based and non-bisulfite-based quantitative method. - View Dependent Claims (14, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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15. A method for determining the concentration of fetal nucleic acid in a maternal sample, wherein the maternal sample comprises differentially methylated maternal and fetal nucleic acid, comprising:
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a) determining the total amount of nucleic acid present in the maternal sample; b) digesting the maternal nucleic acid in a maternal sample using a methylation sensitive restriction enzyme thereby enriching the fetal nucleic acid; c) determining the amount of fetal nucleic acid from step b) using a non-polymorphic-based and non-bisulfite-based quantitative method, wherein the fetal nucleic acid comprises one or more CpG sites from one or more of the polynucleotide sequences of SEQ ID NOs;
90-261; andd) comparing the amount of fetal nucleic acid from step c) to the total amount of nucleic acid from step a), thereby determining the concentration of fetal nucleic acid in the maternal sample. - View Dependent Claims (16)
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17. A method for determining the presence or absence of a fetal aneuploidy using fetal nucleic acid from a maternal sample, wherein the maternal sample comprises differentially methylated maternal and fetal nucleic acid, comprising:
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a) digesting the maternal nucleic acid in a maternal sample using a methylation sensitive restriction enzyme thereby enriching the fetal nucleic acid; b) determining the amount of fetal nucleic acid from a target chromosome using a non-polymorphic-based and non-bisulfite-based quantitative method, wherein the fetal nucleic acid comprises one or more CpG sites from one or more of the polynucleotide sequences of SEQ ID NOs;
164-261;c) determining the amount of fetal nucleic acid from a reference chromosome using a non-polymorphic-based and non-bisulfite-based quantitative method, wherein the fetal nucleic acid comprises one or more CpG sites from one or more of the polynucleotide sequences of SEQ ID NOs;
90-163;d) comparing the amount of fetal nucleic acid from step b) to step c), wherein a statistically significant difference between the amount of target and reference fetal nucleic acid is indicative of the presence of a fetal aneuploidy. - View Dependent Claims (18)
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Specification