Non-invasive fetal genetic screening by digital analysis

• US 9,441,273 B2
• Filed: 06/15/2010
• Issued: 09/13/2016
• Est. Priority Date: 02/02/2006
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Quake, Stephen, Fan, Hei-Mun Christina
• Primary Examiner(s): MYERS, CARLA J
• Application Number:
  US12/816,043
• Publication Number:
  US 20100255493A1
• Time in Patent Office: 2,282 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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.

Claims

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, thereby identifying an abnormal fetal chromosome copy number.

22. The method of claim 21, wherein the abnormal fetal chromosome copy number is a trisomy.

23. The method of claim 22, wherein the trisomy is trisomy 21.

24. The method of claim 21, wherein the blood sample is a maternal blood sample.

25. The method of claim 21, wherein the isolated mixture of fetal and maternal DNA obtained in step b) is distributed into at least 500 discrete reaction samples at discrete locations in step c).

26. The method of claim 25, 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 in step c).

27. The method of claim 21, wherein the target DNA sequences of step d) are amplified by a polymerase chain reaction (PCR).

28. 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) isolating a mixture of fetal and maternal genomic DNA from a blood sample from a pregnant subject;

b) distributing the isolated mixture of fetal and maternal genomic DNA obtained in step a) into a plurality of at least 500 discrete reaction samples at discrete locations of a surface which supports multiple parallel nucleic acid amplifications and detections;

c) at each discrete location;

i) amplifying genomic DNA in the reaction samples with primers to;

(1) target sequences present on fetal chromosome of presumably normal number; and

(2) target sequences on a fetal chromosome which may possibly be of abnormal copy number; and

ii) detecting the presence or absence of the target sequences in steps (i1) and (i2);

d) counting the number of discrete locations at which the target sequences of steps (i1) and (i2) are present; and

e) calculating a statistically significant difference between the number of target sequences detected from steps (i1) and (i2), to thereby detect an abnormal fetal chromosome copy number.