Direct Molecular Diagnosis of Fetal Aneuploidy
First Claim
1. A method for detecting a fetal chromosomal aneuploidy of a target chromosome, comprising the steps of:
- (a) obtaining a fetal sample containing genomic DNA including a target chromosome sequence and a reference chromosome sequence;
(b) distributing said fetal sample into a plurality of reaction areas, each reaction area containing on average not more than one target chromosome sequence and not more than one reference chromosome sequence;
(c) detecting whether said target chromosome sequence is present in said plurality of reaction areas, said detecting comprising the step of detecting an invariant sequence, to produce a target count;
(d) detecting whether said reference chromosome sequence is present in said plurality of reaction areas, said detecting comprising the step of detecting an invariant sequence, to produce a reference count;
(e) obtaining sufficient numbers in said target count and said reference count to achieve statistical significance; and
(f) comparing said target count to said reference count, whereby an abnormal difference between said target count and said reference count indicates fetal chromosomal aneuploidy in the target chromosome.
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Abstract
Methods and materials for detection of aneuploidy and other chromosomal abnormalities using fetal tissue are disclosed. Results can be obtained rapidly, without cell culture. The method uses digital PCR for amplification and detection of single target sequences, allowing an accurate count of a specific chromosome or chromosomal region. Specific polynucleic acid primers and probes are disclosed for chromosomes 1, 13, 18, 21, X and Y. These polynucleic acid sequences are chosen to be essentially invariant between individuals, so the test is not dependent on sequence differences between fetus and mother.
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Citations
27 Claims
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1. A method for detecting a fetal chromosomal aneuploidy of a target chromosome, comprising the steps of:
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(a) obtaining a fetal sample containing genomic DNA including a target chromosome sequence and a reference chromosome sequence; (b) distributing said fetal sample into a plurality of reaction areas, each reaction area containing on average not more than one target chromosome sequence and not more than one reference chromosome sequence; (c) detecting whether said target chromosome sequence is present in said plurality of reaction areas, said detecting comprising the step of detecting an invariant sequence, to produce a target count; (d) detecting whether said reference chromosome sequence is present in said plurality of reaction areas, said detecting comprising the step of detecting an invariant sequence, to produce a reference count; (e) obtaining sufficient numbers in said target count and said reference count to achieve statistical significance; and (f) comparing said target count to said reference count, whereby an abnormal difference between said target count and said reference count indicates fetal chromosomal aneuploidy in the target chromosome. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for detecting a chromosomal aneuploidy of a target chromosome, comprising the steps of:
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(a) directly extracting genomic DNA from a sample, said DNA including target chromosome sequence and reference chromosome sequence; (b) distributing said fetal sample from step (a), concurrently into a plurality of reaction areas, each reaction area comprised in a microfluidic device and containing on average not more than one target chromosome sequence and not more than one reference chromosome sequence; (c) adding amplification primers, where one hybridization primer set hybridizes to a reference chromosome and one primer set hybridizes to the target chromosome, and carrying out a plurality of amplification reactions concurrently in the plurality of reaction areas; (d) adding a label for detecting presence and absence of said target chromosome sequence and said reference chromosome sequence to produce a target count; (e) detecting presence and absence of said reference chromosome sequence in said plurality of reaction areas to produce a reference count; (f) obtaining sufficient numbers in said target count and said reference count to achieve a predetermined statistical significance in any difference between said target count and said reference count; and (g) comparing said target count to said reference count, whereby an abnormal difference between said target count and said reference count indicates fetal chromosomal aneuploidy in the target chromosome. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20)
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- 21. A kit comprising a primer set for detecting a specific human chromosome, wherein the primers hybridize to invariant sequences on that chromosome.
Specification