Non-Invasive Fetal Genetic Screening by Digital Analysis

• US 20100255492A1
• Filed: 06/15/2010
• Published: 10/07/2010
• Est. Priority Date: 02/02/2006
• Status: Abandoned Application

• 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
• Application Number:
  US12/815,674
• Publication Number:
  US 20100255492A1
• 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 differential detection of target sequences in a mixture of maternal and fetal genetic material, comprising the steps of:

(a) obtaining maternal tissue containing both maternal and fetal genetic material;

(b) distributing the genetic material into discrete samples, each sample containing on average not more than about one target sequence per sample;

(c) measuring the presence of different target sequences in the discrete samples; and

(d) analyzing a number of the discrete samples sufficient to obtain results distinguishing different target sequences.

2. The method of claim 1 wherein one of the different target sequences is diploid in maternal genetic material and aneuploid in fetal genetic material and another of the different target sequences is diploid in both maternal and fetal genetic material.

3. The method of claim 1 wherein the maternal tissue is maternal peripheral blood or blood plasma.

4. The method of claim 1 wherein the discrete samples are in reaction samples selected from the group consisting of:

wells in a microtiter plate, aqueous phases in an emulsion, and reaction chambers in a microfluidic device.

5. The method of claim 1 wherein the discrete samples are contacted with a plurality of PCR primers, including at least one primer directed specifically to a maternal sequence and at least one primer directed specifically to a fetal sequence, and further including the step of amplifying the maternal sequence and the fetal sequence.

6. The method of claim 5 wherein said measuring the presence of a target sequence further includes hybridizing it with a nucleic acid having a fluorescent label.

7. The method of claim 6 wherein said nucleic acid is in a sample for binding to a target sequence and generating a resulting change in fluorescence.

8. The method of claim 1 wherein the number of samples is at least about 10,000.

9. The method of claim 1 wherein said measuring the presence of different target sequences comprises the use of nucleic acids specific for human chromosome 21 and specific for another human chromosome.

10. The method of claim 1 wherein said measuring the presence of different target sequences comprises hybridization with nucleic acids which hybridize to a sequence selected from the group consisting of one or more of the genes:

CFTR, Factor VIII (F8 gene), beta globin, hemachromatosis, G6PD, neurofibromatosis, GAPDH, beta amyloid, and pyruvate kinase.

11. The method of claim 1 wherein said measuring the presence of different target sequences detects a sequence deleted in a human chromosome deletion, is moved in a translocation or inversion, or is duplicated in a chromosome duplication, wherein said sequence is characterized in a known genetic disorder in the fetal genetic material not present in the maternal genetic material.

12. The method of claim 1 wherein said measuring the presence of different target sequences detects a sequence of a mutated form of a human gene listed in the OMIM Morbid map, http:

//www.ncbi.nlm.nih.gov/Omim/getmorbid.cgi.

13. The method of claim 1 further comprising the step of enriching the mixture for fetal genetic material by size separation, whereby a preparation comprising only DNA fragments less than about 300 by are used for measuring in step (c).

14. The method of claim 1 in which the measuring comprises direct sequencing of the genetic material.

15. The method of claim 1 in which the measuring comprises sequencing of amplified derivatives of the target sequences clones or amplicons of the genetic material.

17. The method of claim 1 in which the genetic material is cDNA derived from RNA in the tissue.

16. The method of claim 16 where the amplified derivative is selected from the group consisting of an amplicon and a clone.

18. A method of differential detection of target sequences in a mixture of maternal and fetal genetic material, comprising the steps of:

(a) obtaining from maternal plasma a mixture containing maternal and fetal genetic material;

(b) distributing the mixture into at least five hundred separate reaction samples, each reaction sample containing less than about one target sequence;

(c) hybridizing DNA in each separate reaction sample with nucleic acids hybridizing to one of two different target sequences, one of which is used as a control sequence to detect targets equally present on both maternal and fetal DNA and the other is used to detect a fetal genetic abnormality; and

(d) analyzing results of said labeling to obtain results distinguishing a difference in target sequences in fetal DNA and maternal DNA.

19. The method of claim 18 wherein the distributing is into at least 10,000 separate reaction samples.

20. The method of claim 18 wherein said difference in target sequences is a fetal aneuploidy.

21. The method of claim 18 wherein said probes comprise a probe specific for human chromosome 21.

22. A kit for differential detection of target sequences in maternal and fetal DNA in a mixed DNA sample, comprising:

(a) primers specific for two chromosomes, one of which is possibly aneuploid and one of which is presumed diploid;

(b) a PCR reaction buffer for forming a PCR reaction sample with the primers; and

(c) a size separation medium for separating the DNA sample into a fraction having less than about 1000 bp.

23. The kit of claim 22 where the primers comprise a pair of primers specific to chromosome 21.

24. The kit of claim 22 further comprising a reaction chamber having at least 1,000 discrete reaction samples.