Direct molecular diagnosis of fetal aneuploidy
First Claim
Patent Images
1. A method for detecting an abnormal copy number of a target fetal chromosome, comprising the steps of:
- (a) obtaining a fetal sample containing genomic DNA including a target chromosome sequence and a reference chromosome sequence, said fetal sample being at least one of amniotic fluid, uncultured amniocytes and chorionic villus tissue;
(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 each of said plurality of reaction areas by;
(1) using primers to amplify a first ultraconserved element unique to said target chromosome sequence;
(2) binding a first detection probe to said amplified first ultraconserved element; and
(3) counting the number of reaction areas containing a bound first detection probe, to produce a target count;
(d) detecting whether said reference chromosome sequence is present in each of said plurality of reaction areas by;
(1) using primers to amplify a second ultraconserved element unique to said reference chromosome sequence;
(2) binding a second detection probe to said amplified second ultraconserved element; and
(3) counting the number of reaction areas containing a bound second detection probe, to produce a reference count; and
(e) obtaining sufficient numbers in said target count and said reference count to achieve statistical significance, wherein a statistically significant difference between said target count and said reference count indicates an abnormal number of the target fetal chromosome.
0 Assignments
0 Petitions
Accused Products
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.
-
Citations
17 Claims
-
1. A method for detecting an abnormal copy number of a target fetal chromosome, comprising the steps of:
-
(a) obtaining a fetal sample containing genomic DNA including a target chromosome sequence and a reference chromosome sequence, said fetal sample being at least one of amniotic fluid, uncultured amniocytes and chorionic villus tissue; (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 each of said plurality of reaction areas by;
(1) using primers to amplify a first ultraconserved element unique to said target chromosome sequence;
(2) binding a first detection probe to said amplified first ultraconserved element; and
(3) counting the number of reaction areas containing a bound first detection probe, to produce a target count;(d) detecting whether said reference chromosome sequence is present in each of said plurality of reaction areas by;
(1) using primers to amplify a second ultraconserved element unique to said reference chromosome sequence;
(2) binding a second detection probe to said amplified second ultraconserved element; and
(3) counting the number of reaction areas containing a bound second detection probe, to produce a reference count; and(e) obtaining sufficient numbers in said target count and said reference count to achieve statistical significance, wherein a statistically significant difference between said target count and said reference count indicates an abnormal number of the target fetal chromosome. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
-
-
11. A method for detecting an abnormal copy number of a target fetal chromosome, comprising the steps of:
-
(a) directly extracting genomic DNA from a sample, said DNA including target chromosome sequence and reference chromosome sequence, said sample being a fetal sample of at least one of amniotic fluid, uncultured amniocytes and chorionic villus tissue; (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, wherein a first amplification primer set amplifies an ultraconserved element on a reference chromosome and a second amplification primer set amplifies an ultraconserved element on the target chromosome, and carrying out a plurality of amplification reactions concurrently in the plurality of reaction areas; (d) adding a first probe having a first detectable label and allowing said first probe to bind to the ultraconserved element on said target chromosome sequence and a second probe having a second detectable label and allowing said second probe to bind to the ultraconserved element on said reference chromosome sequence; (e) counting a number of reaction areas in the plurality of reaction areas that contain said target chromosome by detecting the first detectable label to produce a target count, and counting a number of reaction areas in the plurality of reaction areas that contain said reference chromosome by measuring the second detectable label to produce a reference count; and (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; wherein a statistically significant difference between said target count and said reference count indicates an abnormal number of the target fetal chromosome. - View Dependent Claims (12, 13, 14, 15, 16, 17)
-
Specification