Non-Invasive Fetal Genetic Screening by Digital Analysis
First Claim
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.
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Accused Products
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.
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Citations
24 Claims
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1. A method of differential detection of 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) 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17)
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16. The method of claim 16 where the amplified derivative is selected from the group consisting of an amplicon and a clone.
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18. A method of differential detection of target sequences in a mixture of maternal and fetal genetic material, comprising the steps of:
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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. - View Dependent Claims (19, 20, 21)
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22. A kit for differential detection of target sequences in maternal and fetal DNA in a mixed DNA sample, comprising:
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(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. - View Dependent Claims (23, 24)
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Specification