Ligation-based detection of genetic variants
First Claim
Patent Images
1. A method for detecting a fetal aneuploidy and alleles at polymorphic loci in a maternal plasma or serum sample, comprising the steps of:
- providing a maternal plasma or serum sample;
introducing between 48 and 2000 first sets of first and second fixed sequence oligonucleotides to the maternal plasma or serum sample under conditions that allow the between 48 and 2000 first sets of first and second fixed sequence oligonucleotides to specifically hybridize to non-polymorphic regions on a first chromosome of interest, wherein each first set comprises a binding region that binds selectively to a first array feature and a region that binds to a first label, and wherein melting temperatures (Tms) of first fixed sequence oligonucleotides of each first set of first and second fixed sequence oligonucleotides vary in a range of two degrees centigrade;
introducing between 48 and 2000 second sets of first and second fixed sequence oligonucleotides to the maternal plasma or serum sample under conditions that allow the between 48 and 2000 second sets of first and second fixed sequence oligonucleotides to specifically hybridize to non-polymorphic regions on a second chromosome of interest, wherein each second set comprises a binding region that binds selectively to the first array feature and a region that binds to a second label that is different from the first label, and wherein Tms of first fixed sequence oligonucleotides of each second set of first and second fixed sequence oligonucleotides vary in a range of two degrees centigrade;
introducing between 48 and 2000 third sets of first, second and third fixed sequence oligonucleotides to the maternal plasma or serum sample under conditions that allow the between 48 and 2000 third sets of first, second and third fixed sequence oligonucleotides to specifically hybridize to polymorphic regions of interest, wherein each third set comprises a first fixed sequence oligonucleotide specific for a first allele and a third label, a second fixed sequence oligonucleotide specific for a second allele and a fourth label different from the third label, a binding region that binds selectively to an array feature, and wherein there is a different binding region and a different array feature for each polymorphic region;
hybridizing the first, second and third sets of fixed sequence oligonucleotides to nucleic acids in the maternal plasma or serum sample;
ligating the hybridized fixed sequence oligonucleotides to create contiguous ligation products complementary to the non-polymorphic and polymorphic regions of interest;
introducing the contiguous ligation products to an array comprising array features complementary to the binding regions in the contiguous ligation products;
detecting hybridization of the contiguous ligation products from the first and second sets of fixed sequence oligonucleotides to the array by detection of the first and second labels at the first array feature;
determining a frequency of the first and second chromosomes of interest based on the level of hybridization of the first and second sets of fixed sequence oligonucleotides;
detecting a fetal aneuploidy based on the frequency of the first and second chromosomes of interest;
detecting hybridization of the contiguous ligation products from the third sets of fixed sequence oligonucleotides to the array by detecting the third and fourth labels at the other array features; and
determining a frequency of the first and second alleles at each polymorphic locus based on level of hybridization of the third sets of fixed sequence oligonucleotides comprising the third and fourth labels at the other array features.
2 Assignments
0 Petitions
Accused Products
Abstract
The present invention provides assays systems and methods for detection of genetic variants in a sample, including copy number variation and single nucleotide polymorphisms. The invention preferably employs the technique of tandem ligation—, e.g., the ligation of two or more fixed sequence oligonucleotides and one or more bridging oligonucleotides complementary to a region between the fixed sequence oligonucleotides—combined with detection of levels of particular genomic regions using array hybridization.
-
Citations
11 Claims
-
1. A method for detecting a fetal aneuploidy and alleles at polymorphic loci in a maternal plasma or serum sample, comprising the steps of:
-
providing a maternal plasma or serum sample; introducing between 48 and 2000 first sets of first and second fixed sequence oligonucleotides to the maternal plasma or serum sample under conditions that allow the between 48 and 2000 first sets of first and second fixed sequence oligonucleotides to specifically hybridize to non-polymorphic regions on a first chromosome of interest, wherein each first set comprises a binding region that binds selectively to a first array feature and a region that binds to a first label, and wherein melting temperatures (Tms) of first fixed sequence oligonucleotides of each first set of first and second fixed sequence oligonucleotides vary in a range of two degrees centigrade; introducing between 48 and 2000 second sets of first and second fixed sequence oligonucleotides to the maternal plasma or serum sample under conditions that allow the between 48 and 2000 second sets of first and second fixed sequence oligonucleotides to specifically hybridize to non-polymorphic regions on a second chromosome of interest, wherein each second set comprises a binding region that binds selectively to the first array feature and a region that binds to a second label that is different from the first label, and wherein Tms of first fixed sequence oligonucleotides of each second set of first and second fixed sequence oligonucleotides vary in a range of two degrees centigrade; introducing between 48 and 2000 third sets of first, second and third fixed sequence oligonucleotides to the maternal plasma or serum sample under conditions that allow the between 48 and 2000 third sets of first, second and third fixed sequence oligonucleotides to specifically hybridize to polymorphic regions of interest, wherein each third set comprises a first fixed sequence oligonucleotide specific for a first allele and a third label, a second fixed sequence oligonucleotide specific for a second allele and a fourth label different from the third label, a binding region that binds selectively to an array feature, and wherein there is a different binding region and a different array feature for each polymorphic region; hybridizing the first, second and third sets of fixed sequence oligonucleotides to nucleic acids in the maternal plasma or serum sample; ligating the hybridized fixed sequence oligonucleotides to create contiguous ligation products complementary to the non-polymorphic and polymorphic regions of interest; introducing the contiguous ligation products to an array comprising array features complementary to the binding regions in the contiguous ligation products; detecting hybridization of the contiguous ligation products from the first and second sets of fixed sequence oligonucleotides to the array by detection of the first and second labels at the first array feature; determining a frequency of the first and second chromosomes of interest based on the level of hybridization of the first and second sets of fixed sequence oligonucleotides; detecting a fetal aneuploidy based on the frequency of the first and second chromosomes of interest; detecting hybridization of the contiguous ligation products from the third sets of fixed sequence oligonucleotides to the array by detecting the third and fourth labels at the other array features; and determining a frequency of the first and second alleles at each polymorphic locus based on level of hybridization of the third sets of fixed sequence oligonucleotides comprising the third and fourth labels at the other array features. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
-
Specification