Non-invasive fetal genetic screening by digital analysis
DC CAFCFirst Claim
1. A method of determining a fetal aneuploidy by determining the number of copies of maternal and fetal target chromosomes, having 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) obtaining a mixture of maternal and fetal genetic material from said maternal tissue;
c) distributing the genetic material obtained in step b) into a plurality of reaction samples, 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) analyzing the target sequences of genetic material present or absent in said individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples,e) determining the presence or absence of a fetal aneuploidy by comparing said first number of step (d) with said second number of step (d), wherein a differential between the first number and the second number indicates the presence of a fetal aneuploidy.
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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.
310 Citations
28 Claims
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1. A method of determining a fetal aneuploidy by determining the number of copies of maternal and fetal target chromosomes, having target sequences in a mixture of maternal and fetal genetic material, comprising the steps of:
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a) obtaining maternal tissue containing both maternal and fetal genetic material; b) obtaining a mixture of maternal and fetal genetic material from said maternal tissue; c) distributing the genetic material obtained in step b) into a plurality of reaction samples, 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) analyzing the target sequences of genetic material present or absent in said individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples, e) determining the presence or absence of a fetal aneuploidy by comparing said first number of step (d) with said second number of step (d), wherein a differential between the first number and the second number indicates the presence of a fetal aneuploidy. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 23, 24, 25, 26, 27, 28)
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17. A method for determination of the presence or absence of a fetal aneuploidy in a maternal tissue sample comprising fetal and maternal genomic DNA, wherein the method comprises:
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a) obtaining a mixture of fetal and maternal genomic DNA from said maternal tissue sample; b) distributing random fragments from the mixture of fetal and maternal genomic DNA of step a to provide reaction samples containing a single genomic DNA molecule or amplification products of a single genomic DNA molecule; c) conducting massively parallel DNA sequencing of the random fragments of genomic DNA in the reaction samples of step b) to determine the sequence of said random fragments; d) identifying the chromosomes to which the sequences obtained in step c) belong; e) analyzing the data of step d) to determine i) the number of copies of at least one first target chromosome in said mixture of fetal and maternal genomic DNA, wherein said at least one first target chromosome is presumed to be diploid in both the mother and the fetus, and ii) the number of copies of a second target chromosome in said mixture of fetal and maternal genomic DNA, wherein said second chromosome is suspected to be aneuploid in the fetus; f) conducting a statistical analysis that compares the number of copies of said at least one first target chromosome to the number of copies of said second target chromosome; and g) determining the presence or absence of a fetal aneuploidy from the results of the statistical analysis of step f). - View Dependent Claims (18, 19, 20, 21, 22)
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Specification