Non-invasive fetal genetic screening by digital analysis
First Claim
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.
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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.
96 Citations
9 Claims
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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:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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Specification