Non-Invasive Fetal Genetic Screening by Digital Analysis

• US 20100255493A1
• Filed: 06/15/2010
• Published: 10/07/2010
• 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
• Granted Patent: US 9,441,273 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 distinguishing the presence or absence of a fetal aneuploidy 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 into a plurality of at least 500 discrete reaction samples at discrete locations;

d) at each discrete location, tagging any genomic DNA by amplification;

e) analyzing the tagged genomic DNA of step d to detect a presence of any amplified fetal or maternal genomic DNA at the discrete locations;

f) generating data corresponding to the presence or absence of amplified fetal and maternal genomic DNA at each discrete location; and

g) distinguishing the presence or absence of a fetal aneuploidy from the data of step f).

2. The method of claim 1, wherein the fetal aneuploidy 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 sample 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, the portion of the mixture of fetal and maternal genomic DNA of step d is amplified and tagged by a polymerase chain reaction (PCR).

8. The method of claim 1, wherein the analysis of step d) comprises the use of a computer program.

9. The method of claim 8, wherein the suspected aneuploidy region is an entire chromosome.

10. A method of distinguishing the presence or absence of a fetal aneuploidy by analysis of a blood sample comprising a mixture of fetal and maternal genomic DNA 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 into a plurality of discrete reaction samples at discrete locations, wherein the discrete locations are recordable locations on an array;

d) at each discrete location, tagging any genomic DNA by amplification;

e) analyzing the tagged genomic DNA of step d to detect a presence of any amplified fetal or maternal genomic DNA at the discrete locations;

f) generating data corresponding to the presence or absence of amplified fetal or maternal genomic DNA at each discrete location; and

g) distinguishing the presence or absence of a fetal aneuploidy from the data of step f).

11. The method of claim 10, wherein the fetal aneuploidy is a trisomy.

12. The method of claim 11, wherein the trisomy is trisomy 21.

13. The method of claim 10, wherein the blood sample is a maternal blood sample.

14. The method of claim 10, wherein the sample is distributed into at least 500 discrete reaction samples at discrete locations.

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

16. The method of claim 10, wherein at each discrete location, the mixture of fetal and maternal genomic DNA of step d is amplified and tagged by polymerase chain reaction (PCR).

17. The method of claim 10, wherein the analysis of step e) comprises the use of a computer program to determine the ratios of suspected aneuploidy chromosome regions.

18. The method of claim 17, wherein the suspected aneuploidy region is an entire chromosome.

19. A method of distinguishing the presence or absence of a fetal aneuploidy 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 maternal and fetal genomic DNA;

b) isolating the mixture of fetal and maternal genomic DNA from the blood;

c) distributing the sample into at least 500 discrete reaction samples and directing each discrete reaction sample to a discrete location;

d) tagging genomic DNA regions of the genomic DNA in the discrete locations of step

c) by amplification;

e) generating data by quantitating the number of tagged genomic regions of step d) by counting the number of discrete locations containing amplified genomic DNA; and

f) distinguishing the presence or absence of a fetal aneuploidy from the data of step e).

20. The method of claim 19, wherein the fetal aneuploidy is a trisomy.

21. The method of claim 20, wherein the trisomy is trisomy 21.

22. The method of claim 19, wherein the blood sample is a maternal blood sample.

23. The method of claim 19, wherein the sample is distributed into at least 1000 discrete reaction samples at discrete locations.

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

25. The method of claim 19, wherein the tagged genomic DNA of step d) is amplified by a polymerase chain reaction (PCR).

26. The method of claim 19, wherein the quantitating of step e) and the determining of step f) each comprises the use of a computer program.

27. The method of claim 26, wherein the suspected aneuploidy region is an entire chromosome.

28. A method of distinguishing the presence or absence of a fetal aneuploidy 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 maternal and fetal genomic DNA;

b) isolating the mixture of maternal and fetal genomic DNA from the blood sample;

c) distributing the isolated mixture of maternal and fetal genomic DNA into a plurality of discrete reaction samples at discrete locations, wherein the discrete locations are recordable locations on an array;

d) tagging genomic DNA regions of the genomic DNA in the discrete locations of step

c) by amplification;

e) generating data by quantitating the number of amplified genomic regions of step d) by counting the number of discrete locations containing amplified genomic DNA; and

f) distinguishing the presence or absence of a fetal aneuploidy condition from the data of step e).

29. The method of claim 28, wherein the fetal aneuploidy is a trisomy.

30. The method of claim 29, wherein the trisomy is trisomy 21.

31. The method of claim 28, wherein the blood sample is a maternal blood sample.

32. The method of claim 28, wherein the sample is distributed into at least 500 discrete reaction samples at discrete locations in step c).

33. The method of claim 32, wherein the sample is distributed into at least 1000 discrete reaction samples at discrete locations in step c).

34. The method of claim 28, wherein the genomic DNA regions of step c) are amplified by a polymerase chain reaction (PCR).

35. The method of claim 28, wherein the quantitating of step e) and the determining of step f) comprises the use of a computer program.

36. The method of claim 28, wherein the suspected aneuploidy region is an entire chromosome.

37. A method of determining a presence or absence of a fetal aneuploidy condition 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) splitting the isolated mixture of fetal and maternal DNA into a plurality of at least 100 discrete portions and directing each discrete portion to a discrete location;

d) at each discrete location, tagging any genomic DNA by amplification;

e) analyzing the tagged genomic DNA of step d to detect a presence of any amplified fetal or maternal genomic DNA at the discrete locations;

f) generating data corresponding to the presence or absence of amplified fetal and maternal genomic DNA at each discrete location; and

g) determining the presence or absence of a fetal aneuploidy condition from the data of step f).

38. The method of claim 37, wherein the fetal aneuploidy is a trisomy.

39. The method of claim 38, wherein the trisomy is trisomy 21.

40. The method of claim 37, wherein the blood sample is a maternal blood sample.

41. The method of claim 37, wherein the sample is split into at least 1000 different sites.

42. The method of claim 37, wherein the discrete locations are addressable locations on a matrix.

43. The method of claim 37, wherein at each discrete location, the portion of the mixture of fetal and maternal genomic DNA of step d is amplified and tagged by polymerase chain reaction (PCR).

44. The method of claim 37, wherein the analysis of step e) comprises the use of computer executable logic.

45. The method of claim 44, wherein the suspected aneuploidy region is an entire chromosome.

46. A method of determining the presence or absence of a fetal aneuploidy condition 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) splitting the isolated mixture of fetal and maternal DNA sample into a plurality of discrete locations, wherein the discrete locations are addressable locations on a matrix;

d) at each discrete location, tagging any genomic DNA by amplification;

e) analyzing the tagged genomic DNA of step d to detect a presence of any amplified fetal or maternal genomic DNA at the discrete locations;

f) generating data corresponding to the presence or absence of amplified fetal or maternal genomic DNA at each discrete location; and

g) determining the presence or absence of a fetal aneuploidy from the data of step f).

47. The method of claim 46, wherein the fetal aneuploidy is a trisomy.

48. The method of claim 47, wherein the trisomy is trisomy 21.

49. The method of claim 46, wherein the blood sample is a maternal blood sample.

50. The method of claim 46, wherein the sample is split into at least 100 different sites.

51. The method of claim 50, wherein the sample is split into at least 1000 different sites.

52. The method of claim 46, wherein at each discrete location, the mixture of fetal and maternal genomic DNA of step d is amplified and tagged by polymerase chain reaction (PCR).

53. The method of claim 46, wherein the analysis of step e) comprises the use of computer executable logic to determine the ratios of suspected aneuploidy chromosome regions.

54. The method of claim 53, wherein the suspected aneuploidy region is an entire chromosome.

55. A method of detecting the presence or absence of a fetal aneuploidy condition 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;

c) splitting the sample into at least 100 discrete portions and directing each discrete portion to a discrete location;

d) tagging genomic DNA regions of the genomic DNA in the discrete locations of step c) by amplification;

e) generating data by quantitating the number of tagged genomic regions of step d) by counting the number of discrete locations containing amplified genomic DNA; and

f) determining the presence or absence of a fetal aneuploidy condition from the data of step e).

56. The method of claim 55, wherein the fetal aneuploidy is a trisomy.

57. The method of claim 56, wherein the trisomy is trisomy 21.

58. The method of claim 55, wherein the blood sample is a maternal blood sample.

59. The method of claim 55, wherein the sample is split into at least 1000 discrete portions.

60. The method of claim 55, wherein the discrete locations are addressable locations on a matrix.

61. The method of claim 55, wherein the tagged genomic DNA of step d) is amplified by a polymerase chain reaction (PCR).

62. The method of claim 55, wherein the quantitating of step e) and the determining of step f) each comprises the use of a computer executable logic.

63. The method of claim 62, wherein the suspected aneuploidy region is an entire chromosome.

64. A method of detecting the presence or absence of a fetal aneuploidy condition 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 maternal and fetal genomic DNA;

b) isolating the mixture of maternal and fetal genomic DNA from the blood sample;

c) splitting the isolated mixture of maternal and fetal genomic DNA into a plurality of discrete locations, wherein the discrete locations are addressable locations on a matrix;

d) tagging genomic DNA regions of the genomic DNA in the discrete locations of step c) by amplification;

e) generating data by quantitating the number of amplified genomic regions of step d) by counting the number of discrete locations containing amplified genomic DNA; and

f) determining the presence or absence of a fetal aneuploidy condition from the data of step e).

65. The method of claim 64, wherein the fetal aneuploidy is a trisomy.

66. The method of claim 65, wherein the trisomy is trisomy 21.

67. The method of claim 64, wherein the blood sample is a maternal blood sample.

68. The method of claim 64, wherein the sample is split into at least 100 discrete portions in step c).

69. The method of claim 68, wherein the sample is split into at least 1000 discrete locations in step c).

70. The method of claim 64, wherein the genomic DNA regions of step c) are amplified by a polymerase chain reaction (PCR).

71. The method of claim 64, wherein the quantitating of step e) and the determining of step f) comprises the use of a computer executable logic.

72. The method of claim 64, wherein the suspected aneuploidy region is an entire chromosome.