Non-invasive fetal genetic screening by digital analysis

Patent metadata

Field	Value
Publication number	US-9441273-B2
Application number	US-81604310-A
Country	US
Kind code	B2
Filing date	Jun 15, 2010
Priority date	Feb 2, 2006
Publication date	Sep 13, 2016
Grant date	Sep 13, 2016

Abstract

The present methods are exemplified by a process in which maternal blood containing fetal DNA is diluted to a nominal value of approximately 0.5 genome equivalent of DNA per reaction sample. Digital PCR is then be used to detect aneuploidy, such as the trisomy that causes Down Syndrome. Since aneuploidies do not present a mutational change in sequence, and are merely a change in the number of chromosomes, it has not been possible to detect them in a fetus without resorting to invasive techniques such as amniocentesis or chorionic villi sampling. Digital amplification allows the detection of aneuploidy using massively parallel amplification and detection methods, examining, e.g., 10,000 genome equivalents.

First claim

What is claimed is:

1. A method of identifying an abnormal fetal chromosome copy number by analysis of a blood sample comprising a mixture of fetal and maternal genomic DNA, the method comprising:
a) obtaining a blood sample comprising a mixture of fetal and maternal genomic DNA;
b) isolating the mixture of fetal and maternal genomic DNA from the blood sample;
c) distributing the isolated mixture of fetal and maternal DNA obtained in step b) into a plurality of at least 500 discrete reaction samples at discrete locations, to randomly provide individual reaction samples that contain a target sequence from a target chromosome and individual reaction samples that do not contain a target sequence from a target chromosome;
d) at each discrete location, amplifying genomic DNA with multiple primers to multiple target sequences; and
e) counting the number of amplified target sequences representing a fetal chromosome which may be of an abnormal copy number and the number of amplified target sequences representing a fetal chromosome of presumably normal copy number to detect a significantly different number of amplified target sequences representing a fetal chromosome which may be of abnormal copy number compared to the number of amplified target sequences representing a fetal chromosome of presumably normal, thereby identifying an abnormal fetal chromosome copy number.

2. The method of claim 1 , wherein the abnormal fetal chromosome copy number is a trisomy.

3. The method of claim 2 , wherein the trisomy is trisomy 21 .

4. The method of claim 1 , wherein the blood sample is a maternal blood sample.

5. The method of claim 1 , wherein the isolated mixture of fetal and maternal DNA obtained in step b) is distributed into at least 1000 discrete reaction samples at discrete locations.

6. The method of claim 1 , wherein the discrete locations are recordable locations on an array.

7. The method of claim 1 , wherein at each discrete location, a portion of the mixture of fetal and maternal genomic DNA of step d) is amplified by a polymerase chain reaction (PCR).

8. A method of identifying an abnormal fetal chromosome copy number by analysis of a blood sample comprising a mixture of fetal and maternal genomic DNA, the method comprising:
a) obtaining a blood sample comprising a mixture of fetal and maternal genomic DNA;
b) isolating the mixture of fetal and maternal genomic DNA from the blood sample;
c) distributing the isolated mixture of fetal and maternal DNA obtained in step b) into a plurality of discrete reaction samples at discrete locations, wherein the discrete locations are recordable locations on an array, to randomly provide individual reaction samples that contain a target sequence from a target chromosome and individual reaction samples that do not contain a target sequence from a target chromosome;
d) at each discrete location, amplifying genomic DNA with multiple primers to multiple target sequences; and
e) counting the number of amplified target sequences representing a fetal chromosome which may be of an abnormal copy number and the number of amplified target sequences representing a fetal chromosome of presumably normal copy number to detect a significantly different number of amplified target sequences representing a fetal chromosome which may be of abnormal copy number compared to the number of amplified target sequences representing a fetal chromosome of presumably normal, thereby identifying an abnormal fetal chromosome copy number.

9. The method of claim 8 , wherein the abnormal fetal chromosome copy number is a trisomy.

10. The method of claim 9 , wherein the trisomy is trisomy 21 .

11. The method of claim 8 , wherein the blood sample is a maternal blood sample.

12. The method of claim 8 , wherein the isolated mixture of fetal and maternal DNA obtained in step b) is distributed into at least 1000 discrete reaction samples at discrete locations.

13. The method of claim 8 , wherein at each discrete location, the mixture of fetal and maternal genomic DNA of step d) is amplified by polymerase chain reaction (PCR).

14. A method of identifying an abnormal fetal chromosome copy number by analysis of a blood sample comprising a mixture of fetal and maternal genomic DNA, the method comprising:
a) obtaining a blood sample comprising a mixture of fetal and maternal genomic DNA;
b) isolating the mixture of fetal and maternal genomic DNA from the blood sample;
c) distributing the isolated mixture of fetal and maternal DNA obtained in step b) into a plurality of at least 500 discrete reaction samples at discrete locations, to randomly provide individual reaction samples that contain a target sequence from a target chromosome and individual reaction samples that do not contain a target sequence from a target chromosome;
d) at each discrete location, contacting the genomic DNA with labeled oligonucleotide probes to detect target DNA sequences within the genomic DNA; and
e) counting the number of target DNA sequences representing a fetal chromosome which may be of an abnormal copy number and the number of target DNA sequences representing a fetal chromosome of presumably normal copy number to detect a significantly different number of target DNA sequences representing a fetal chromosome which may be of abnormal copy number compared to the number of target DNA sequences representing a fetal chromosome of presumably normal, thereby identifying an abnormal fetal chromosome copy number.

15. The method of claim 14 , wherein the abnormal fetal chromosome copy number is a trisomy.

16. The method of claim 15 , wherein the trisomy is trisomy 21 .

17. The method of claim 14 , wherein the blood sample is a maternal blood sample.

18. The method of claim 14 , wherein the sample is distributed into at least 1000 discrete reaction samples at discrete locations.

19. The method of claim 14 , wherein the discrete locations are recordable locations on an array.

20. The method of claim 14 , wherein the genomic DNA of step d) is amplified by a polymerase chain reaction (PCR).

21. A method of identifying an abnormal fetal chromosome copy number by analysis of a blood sample comprising a mixture of fetal and maternal genomic DNA, the method comprising:
a) obtaining a blood sample comprising a mixture of fetal and maternal genomic DNA;
b) isolating the mixture of fetal and maternal genomic DNA from the blood sample;
c) distributing the isolated mixture of fetal and maternal DNA obtained in step b) into a plurality of at least 500 discrete reaction samples at discrete locations, wherein the discrete locations are recordable locations on an array, to randomly provide individual reaction samples that contain a target sequence from a target chromosome and individual reaction samples that do not contain a target sequence from a target chromosome;
d) detecting target DNA sequences in the genomic DNA by contacting the genomic DNA at each discrete location with oligonucleotide probes that become fluorescent upon binding to target DNA sequences in the genomic DNA; and
e) counting the number of target DNA sequences representing a fetal chromosome which may be of an abnormal copy number and the number of target DNA sequences representing a fetal chromosome of presumably normal copy number to detect a significantly different number of target DNA sequences representing a fetal chromosome which may be of abnormal copy number compared to the number of target DNA sequences representing a fetal chromosome of presumably normal, thereb

Assignees

• Quake Stephen
• Fan Hei-Mun Christina
• Univ Leland Stanford Junior

Inventors

• Quake Stephen
• Fan Hei-Mun Christina

Classifications

• C12Q1/6881

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title

• Y10T436/143333

  Saccharide [e.g., DNA, etc.] · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title