Non-invasive fetal genetic screening by digital analysis

• US 9,777,329 B2
• Filed: 01/19/2010
• Issued: 10/03/2017
• 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:
  US12/689,548
• Publication Number:
  US 20100124752A1
• Time in Patent Office: 2,814 Days
• 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 for identifying an abnormal fetal chromosome copy number by measuring target sequences in a mixture of maternal and fetal DNA, comprising the steps of:

a) obtaining a maternal blood sample comprising a mixture of fragments of maternal and fetal DNA;

b) distributing the mixture of maternal and fetal DNA into reaction samples containing on average one target sequence per reaction sample;

c) in each reaction sample, identifying target sequences that are specific to a human chromosome suspected of being of an abnormal fetal copy number and target sequences on one or more human chromosomes that are presumably present in a normal copy number, wherein the identifying comprises (1) sequencing target chromosomal sequences on a chromosome suspected of being of an abnormal fetal copy number and target chromosomal sequences on one or more chromosomes of presumably normal copy number;

(2) amplifying target chromosomal sequences on a chromosome suspected of being of an abnormal fetal copy number and target chromosomal sequences on one or more chromosomes of presumably normal copy number by polymerase chain reaction (PCR);

or (3) hybridizing target chromosomal sequences on a chromosome suspected of being of an abnormal fetal copy number with a sequence-specific probe and hybridizing target chromosomal sequences on one or more chromosomes of presumably normal copy number with a sequence-specific probe;

d) digitally counting the number of target sequences identified as belonging to a specific chromosome suspected of being of abnormal fetal copy number and the number of target sequences on one or more chromosomes of presumably normal copy number in the reaction samples; and

e) detecting a significantly different number of target sequences on chromosomes suspected of being of abnormal fetal copy number compared to the number of target sequences on the one or more chromosomes of presumably normal copy number in the reaction samples, thereby identifying an abnormal fetal chromosome copy number.

2. The method of claim 1 wherein the target sequences on the human chromosome suspected of being of abnormal fetal copy number comprise chromosome 21 sequences and wherein the abnormal number of chromosomes is trisomy for chromosome 21.

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

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

4. The method of claim 1 wherein the identifying in step (c) comprises amplifying target chromosomal sequences suspected of being present in an abnormal fetal copy number and target chromosomal sequences of presumably normal fetal copy number by PCR.

5. The method of claim 1 wherein the mixture of maternal and fetal DNA is distributed into at least about 10,000 reaction samples.

6. The method of claim 1 wherein the target sequences on the human chromosome suspected of being of abnormal fetal copy number comprise chromosome 21 sequences and wherein the target sequences on the one or more chromosomes of presumably normal fetal copy number are specific for another human chromosome.

7. The method of claim 1 further comprising, prior to step (b), the step of enriching the mixture for fetal DNA by size separation, whereby a preparation comprising only DNA fragments less than about 300 bp is used for distributing in step (b).

8. The method of claim 1 in which the identifying in step (c) comprises sequencing target chromosomal sequences suspected of being present in an abnormal fetal copy number and target chromosomal sequences of presumably normal fetal copy number.

9. The method of claim 4, wherein FOR is performed by contacting DNA in the reaction samples with a plurality of FOR primers.