Direct Molecular Diagnosis of Fetal Aneuploidy

• US 20110151442A1
• Filed: 12/22/2009
• Published: 06/23/2011
• Est. Priority Date: 12/22/2009
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Fan, Hei-Mun Christina, Quake, Stephen R.
• Granted Patent: US 8,574,842 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

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.

Claims

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.

2. The method of claim 1 wherein said detecting step comprises amplification using one pair of primers and a detection probe for the target chromosome and another pair of primers and a detection probe for the reference chromosome.

3. The method of claim 2 wherein said primers hybridize to an ultraconserved sequence in the target chromosome and an ultraconserved sequence in the reference chromosome.

4. The method of claim 3 wherein said primers comprise sequences according to SEQ ID NO:

1 and SEQ ID NO;

2 and hybridize to reference chromosome 1.

5. The method of claim 3 wherein said primers comprise sequences according to SEQ ID NO:

3 and SEQ ID NO;

4 and hybridize to target chromosome 13.

6. The method of claim 3 wherein said primers comprise sequences according to SEQ ID NO:

19 and SEQ ID NO;

20 and hybridize to target chromosome 18.

7. The method of claim 3 wherein said primers comprise sequences according to SEQ ID NO:

7 and SEQ ID NO;

8 and hybridize to target chromosome 21.

8. The method of claim 7 wherein said primers comprise sequences according to SEQ ID NO:

1 and SEQ ID NO;

2 and hybridize to reference chromosome 1.

9. The method of claim 3 wherein said primers comprise sequences according to SEQ ID NO:

9 and SEQ ID NO;

10 and hybridize to target chromosome X.

10. The method of claim 3 wherein said primers comprise sequences according to SEQ ID NO:

11 and SEQ ID NO;

12 and hybridize to target chromosome Y.

11. The method of claim 1 wherein said detecting is detecting chromosomes 13, 18, and 21, and said reference chromosome is chromosome 1.

12. The method of claim 1 wherein the step of comparing said target count to said reference count further includes the step of determining whether or not each count is within a confidence interval of at least 99% in order to determine statistical significance of said abnormal difference.

13. A method for detecting a chromosomal aneuploidy of a target chromosome, comprising the steps of:

(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.

14. The method of claim 13 wherein said amplification reactions comprise heating and denaturing primers in the presence of a DNA polymerase.

15. The method of claim 13 wherein said amplification primers amplify regions of similar size in both the target chromosome sequence and reference chromosome sequence.

16. The method of claim 13 wherein the detecting comprises contacting an amplified sequence with a fluorescent probe.

17. The method of claim 16 wherein a fluorescent probe having one label is used for detecting amplified target sequence and a fluorescent probe having another label is used for detecting amplified reference sequence.

18. The method of claim 17 wherein said primers hybridize to invariant sequences in the target chromosome and the reference chromosome.

19. The method of claim 17 wherein said primers hybridize to ultraconserved elements.

20. The method of claim 17 wherein said primers comprise multiple primers directed to a single chromosome.

21. A kit comprising a primer set for detecting a specific human chromosome, wherein the primers hybridize to invariant sequences on that chromosome.

22. A primer set according to claim 21 for detecting human chromosome 1 comprising polynucleotides according to SEQ ID NO:

1 and SEQ ID NO;

2.

23. A primer set according to claim 21 for detecting human chromosome 13 comprising polynucleotides according to SEQ ID NO:

3 and SEQ ID NO;

4.

24. A primer set according to claim 21 for detecting human chromosome 18 comprising polynucleotides according to SEQ ID NO:

5 and SEQ ID NO;

6.

25. A primer set according to claim 21 for detecting human chromosome 21 comprising polynucleotides according to SEQ ID NO:

7 and SEQ ID NO;

8.

26. A primer set according to claim 21 for detecting human chromosome X comprising polynucleotides according to SEQ ID NO:

9 and SEQ ID NO;

10.

27. A primer set according to claim 21 for detecting human chromosome Y comprising polynucleotides according to SEQ ID NO:

11 and SEQ ID NO;

12.