Non-invasive fetal genetic screening by digital analysis

Patent metadata

Field	Value
Publication number	US-9777328-B2
Application number	US-68951710-A
Country	US
Kind code	B2
Filing date	Jan 19, 2010
Priority date	Feb 2, 2006
Publication date	Oct 3, 2017
Grant date	Oct 3, 2017

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 for identifying a fetal aneuploidy from a biological sample of a female subject pregnant with at least one fetus, wherein the biological sample includes a mixture of nucleic acid molecules from the female subject and from the at least one fetus, the method comprising:
(a) distributing nucleic acids derived from the nucleic acid mixture into a plurality of reaction samples to randomly provide individual reaction samples that contain a nucleic acid representative of a clinically relevant nucleic acid sequence for which the sequence imbalance is to be tested, and individual reaction samples that do not contain a nucleic acid representative of a clinically relevant nucleic acid sequence for the fetal aneuploidy, the plurality of reaction samples comprising at least 10,000 reaction samples;
(b) simultaneously performing reactions on the at least 10,000 reaction samples to obtain data, wherein the data include:
(1) a first set of quantitative data derived from a number of reaction samples containing subsequences from the clinically relevant nucleic acid sequence; and
(2) a second set of quantitative data derived from a number of reaction samples containing subsequences from a background sequence, wherein said background sequence comprises at least one nucleic acid sequence different from the clinically relevant nucleic acid sequence; and

(c) calculating a difference between the first set of quantitative data and the second set of quantitative data,

wherein a statistically significant difference between the first set of quantitative data and the second set of quantitative data indicates that a fetal aneuploidy exists.

2. The method of claim 1 wherein the first set of data are obtained from one or more first markers that each detect a presence of a part of the clinically relevant nucleic acid sequence in a reaction, and wherein the second set of data are obtained from one or more second markers that each detect a presence of a part of the background nucleic acid sequence in a reaction.

3. The method of claim 1 wherein the clinically relevant nucleic acid sequence is an allele of a genetic polymorphism, and the background nucleic acid sequence is another allele of the genetic polymorphism.

4. The method of claim 1 wherein the biological sample is plasma or serum from a pregnant woman.

5. The method of claim 1 wherein a reaction is an amplification reaction.

6. The method of claim 5 wherein a reaction is a part of a digital PCR process.

7. The method of claim 1 wherein a reaction is a sequencing reaction.

8. The method of claim 1 wherein first portions of the clinically relevant nucleic acid sequence and the background nucleic acid sequence are from a first individual and second portions of the clinically relevant nucleic acid sequence and the background nucleic acid sequence are from a second individual.

9. The method of claim 1 wherein the classifications include disease state, non-disease state, and non-classifiable.

10. The method of claim 1 wherein the classifications include homozygous, heterozygous, and non-classifiable.

11. The method of claim 1 , wherein the statistical significance is calculated using a t-test.

12. The method of claim 1 , wherein
the clinically relevant sequence is chromosome 21 ,
the first quantitative data is based on a plurality of preselected subsequences of chromosome 21 ,
the second quantitative data is based on a plurality of preselected subsequences of at least one chromosome other than chromosome 21 .

13. The method of claim 12 , wherein the second quantitative data is based on preselected subsequences from at least two chromosomes.

14. The method of claim 1 , wherein the data from the reactions comprises characterizing individual reaction samples as either having or not having a nucleic acid from the clinically relevant sequence.

15. The method of claim 1 , wherein distributing is performed without selecting nucleic acids derived from the nucleic acid mixture.

Assignees

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

Inventors

• Quake Stephen
• Fan Hei-Mun Christina

Classifications

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title

• Y10T436/143333

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

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title