Assay systems for genetic analysis
First Claim
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1. A method of detecting a presence or absence of copy number variation (CNV) in a mixed sample from a single subject, said mixed sample comprising nucleic acids from at least two sources comprising a major source and a minor source, the method comprising:
- a) hybridizing at least 24 first sets of two fixed sequence oligonucleotides to the nucleic acids in the mixed sample, wherein each first set of two fixed sequence oligonucleotides is complementary to a locus in a first region of interest, each first set of two fixed sequence oligonucleotides comprising universal primer regions, and wherein melting temperatures (Tms) of first fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade;
b) hybridizing at least 24 second sets of two fixed sequence oligonucleotides to the nucleic acids in the mixed sample, wherein each second set of two fixed sequence oligonucleotides is complementary to a locus in a second region of interest, each second set of two fixed sequence oligonucleotides comprising universal primer regions, and wherein Tms of first fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade;
c) extending one of said hybridized fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides with a polymerase between the hybridized oligonucleotides of each first set of two fixed sequence oligonucleotides to produce adjacently hybridized fixed sequence oligonucleotides and extending one of said hybridized fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides with a polymerase between the hybridized oligonucleotides of each second set of two fixed sequence oligonucleotides to produce adjacently hybridized fixed sequence oligonucleotides;
d) ligating the adjacently hybridized fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides to create contiguous ligation products and ligating the adjacently hybridized fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides to create contiguous ligation products;
e) amplifying the contiguous ligation products using the universal primer regions on each first and second sets of two fixed sequence oligonucleotides to produce amplification products;
f) detecting the amplification products; and
g) detecting a copy number variation of the first region of interest relative to the second region of interest.
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Abstract
The present invention provides assay systems and methods for detection of copy number variation at one or more loci and polymorphism detection at one or more loci in a mixed sample from an individual.
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Citations
19 Claims
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1. A method of detecting a presence or absence of copy number variation (CNV) in a mixed sample from a single subject, said mixed sample comprising nucleic acids from at least two sources comprising a major source and a minor source, the method comprising:
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a) hybridizing at least 24 first sets of two fixed sequence oligonucleotides to the nucleic acids in the mixed sample, wherein each first set of two fixed sequence oligonucleotides is complementary to a locus in a first region of interest, each first set of two fixed sequence oligonucleotides comprising universal primer regions, and wherein melting temperatures (Tms) of first fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade; b) hybridizing at least 24 second sets of two fixed sequence oligonucleotides to the nucleic acids in the mixed sample, wherein each second set of two fixed sequence oligonucleotides is complementary to a locus in a second region of interest, each second set of two fixed sequence oligonucleotides comprising universal primer regions, and wherein Tms of first fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade; c) extending one of said hybridized fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides with a polymerase between the hybridized oligonucleotides of each first set of two fixed sequence oligonucleotides to produce adjacently hybridized fixed sequence oligonucleotides and extending one of said hybridized fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides with a polymerase between the hybridized oligonucleotides of each second set of two fixed sequence oligonucleotides to produce adjacently hybridized fixed sequence oligonucleotides; d) ligating the adjacently hybridized fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides to create contiguous ligation products and ligating the adjacently hybridized fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides to create contiguous ligation products; e) amplifying the contiguous ligation products using the universal primer regions on each first and second sets of two fixed sequence oligonucleotides to produce amplification products; f) detecting the amplification products; and g) detecting a copy number variation of the first region of interest relative to the second region of interest. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method of detecting a presence or absence of genetic variation in a mixed sample from a single subject, said mixed sample comprising nucleic acids from at least two sources comprising a major source and a minor source, the method comprising:
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a) hybridizing at least 24 sets of two fixed sequence oligonucleotides to the nucleic acids in the mixed sample, wherein each set of two fixed sequence oligonucleotides is complementary to a polymorphic locus in a region of interest, each set of two fixed sequence oligonucleotides comprising universal primer regions, and each locus in the region of interest having different genotypes in the major source and the minor source, and wherein Tms of first fixed sequence oligonucleotides of each set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade; b) extending one of said hybridized two fixed sequence oligonucleotides of each set with a polymerase between the hybridized oligonucleotides of the set to produce adjacently hybridized fixed sequence oligonucleotides; c) ligating the adjacently hybridized oligonucleotides to create contiguous ligation products complementary to the locus; d) amplifying the contiguous ligation products using the universal primer regions on the two fixed sequence oligonucleotides of each set of fixed sequence oligonucleotides to produce amplification products; e) detecting the amplification products; and f) detecting a presence or absence of genetic variation in the locus of interest between the major and minor sources from the detected amplified products.
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Specification