Assay systems for genetic analysis

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.

1 . An assay system for detection of the presence or absence of copy number variation (CNV) of a genomic region and presence or absence of one or more polymorphisms in a mixed sample using a single assay, the assay comprising the steps of:
 introducing a first set of fixed sequence oligonucleotides to a mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci in or associated with a genomic region;   introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci with a putative polymorphism;   ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the loci corresponding to a genomic region and/or the loci with a putative polymorphism;   amplifying the contiguous ligation products using the universal primer regions to create amplification products; and   detecting the amplification products;   wherein detection of the amplification products correlates to the copy number of one or more genomic regions and the presence or absence of a polymorphism in one or more loci in the mixed sample.   
     
     
         2 . The assay system of  claim 1 , wherein the fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         3 . The assay system of  claim 1 , wherein the universal primer regions are used in sequence determination of the amplification products. 
     
     
         4 . The assay system of  claim 1 , wherein the fixed sequence oligonucleotides of each set hybridize to immediately adjacent regions in the loci. 
     
     
         5 . The assay system of  claim 1 , wherein one or both sets of fixed sequence oligonucleotides comprise separate fixed sequence oligonucleotides complementary to regions on the loci. 
     
     
         6 . The assay system of  claim 1 , wherein one or both sets of fixed sequence oligonucleotides comprise a precircle probe. 
     
     
         7 . The assay system of  claim 1 , wherein the amplification products are isolated prior to detection. 
     
     
         8 . The assay system of  claim 6 , wherein the amplification products are isolated as individual molecules prior to detection. 
     
     
         9 . The assay system of  claim 7 , wherein the individual isolated amplification products are further amplified to create identical copies of all or a portion of the individual amplification products prior to detection. 
     
     
         10 . The assay system of  claim 7 , wherein the individual isolated amplification products are further amplified to create identical copies of molecules complementary to all or a portion of the individual amplification products prior to detection. 
     
     
         11 . The assay system of  claim 1 , wherein at least one locus interrogated for copy number is different from all loci interrogated for polymorphisms. 
     
     
         12 . The assay system of  claim 10 , wherein several loci interrogated for copy number are different from the loci interrogated for polymorphisms. 
     
     
         13 . The assay system of  claim 1 , wherein the genomic region is a chromosome. 
     
     
         14 . The assay system of  claim 1 , wherein the genomic region is a sub-chromosomal region. 
     
     
         15 . The assay system of  claim 1 , wherein the genomic region is a single locus. 
     
     
         16 . An assay system for detection of the presence or absence of copy number variation (CNV) of a genomic region and presence or absence of one or more polymorphisms in a mixed sample using a single assay, the assay comprising the steps of:
 introducing a first set of fixed sequence oligonucleotides to a mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci in or associated with a genomic region;   introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci with a putative polymorphism;   extending the region between the first and second hybridized oligonucleotide of at least one fixed sequence oligonucleotide set with a polymerase and dNTPs to create two adjacently hybridized fixed sequence oligonucleotides from that set;   ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the loci corresponding to a genomic region and/or the loci with a putative polymorphism;   amplifying the contiguous ligation products using the universal primer regions to create amplification products; and   detecting the amplification products;   wherein detection of the amplification products correlates to the copy number of one or more genomic regions and the presence or absence of a polymorphism in one or more loci in the mixed sample.   
     
     
         17 . The assay system of  claim 16 , wherein the fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         18 . The assay system of  claim 16 , wherein the universal primer regions are used in sequence determination of the amplification products. 
     
     
         19 . The assay system of  claim 16 , wherein one or both sets of fixed sequence oligonucleotides comprise separate fixed sequence oligonucleotides complementary to regions on the loci. 
     
     
         20 . The assay system of  claim 16 , wherein one or both sets of fixed sequence oligonucleotides comprise a precircle probe. 
     
     
         21 . The assay system of  claim 16 , wherein the amplification products are isolated prior to detection. 
     
     
         22 . The assay system of  claim 21 , wherein the amplification products are isolated as individual molecules prior to detection. 
     
     
         23 . The assay system of  claim 22 , wherein the individual isolated amplification products are further amplified to create identical copies of all or a portion of the individual amplification products prior to detection. 
     
     
         24 . The assay system of  claim 22 , wherein the individual isolated amplification products are further amplified to create identical copies of molecules complementary to all or a portion of the individual amplification products prior to detection. 
     
     
         25 . The assay system of  claim 16 , wherein at least one locus interrogated for copy number is different from all loci interrogated for polymorphisms. 
     
     
         26 . The assay system of  claim 25 , wherein several loci interrogated for copy number are different from the loci interrogated for polymorphisms. 
     
     
         27 . The assay system of  claim 16 , wherein the genomic region is a chromosome. 
     
     
         28 . The assay system of  claim 16 , wherein the genomic region is a sub-chromosomal region. 
     
     
         29 . The assay system of  claim 16 , wherein the genomic region is a single locus. 
     
     
         30 . An assay system for detection of the presence or absence of copy number variation (CNV) of a genomic region and presence or absence of one or more polymorphisms in a mixed sample using a single assay, the assay comprising the steps of:
 introducing a first set of fixed sequence oligonucleotides to a mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci in or associated with a genomic region;   introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci with a putative polymorphism;   introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to regions in the loci between the regions complementary to the fixed sequence oligonucleotides of the sets;   ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the loci corresponding to a genomic region and/or the loci with a putative polymorphism;   amplifying the contiguous ligation products using the universal primer regions to create amplification products; and   detecting the amplification products;   wherein detection of the amplification products correlates to the copy number of one or more genomic regions and the presence or absence of a polymorphism in one or more loci in the mixed sample.   
     
     
         31 . The assay system of  claim 30 , wherein the fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         32 . The assay system of  claim 30 , wherein the universal primer regions are used in sequence determination of the amplification products. 
     
     
         33 . The assay system of  claim 30 , wherein the bridging oligonucleotides hybridize between and adjacent to both fixed sequence oligonucleotides of the sets. 
     
     
         34 . The assay system of  claim 30 , wherein one or both sets of fixed sequence oligonucleotides comprise separate fixed sequence oligonucleotides complementary to regions on the loci. 
     
     
         35 . The assay system of  claim 30 , wherein one or both sets of fixed sequence oligonucleotides comprise a precircle probe. 
     
     
         36 . The assay system of  claim 30 , wherein the bridging oligonucleotides are introduced simultaneously with the ligation mixture. 
     
     
         37 . The assay system of  claim 30 , wherein the amplification products are isolated prior to detection. 
     
     
         38 . The assay system of  claim 37 , wherein the amplification products are isolated as individual molecules prior to detection. 
     
     
         39 . The assay system of  claim 38 , wherein the individual isolated amplification products are further amplified to create identical copies of all or a portion of the individual amplification products prior to detection. 
     
     
         40 . The assay system of  claim 38 , wherein the individual isolated amplification products are further amplified to create identical copies of molecules complementary to all or a portion of the individual amplification products prior to detection. 
     
     
         41 . The assay system of  claim 30 , wherein at least one locus interrogated for copy number is different from all loci interrogated for polymorphisms. 
     
     
         42 . The assay system of  claim 41 , wherein several loci interrogated for copy number are different from the loci interrogated for polymorphisms. 
     
     
         43 . The assay system of  claim 30 , wherein the bridging oligonucleotide are between 2-45 nucleotides in length. 
     
     
         44 . The assay system of  claim 30 , wherein the bridging oligonucleotide are between 3-9 nucleotides in length. 
     
     
         45 . The assay system of  claim 30 , wherein the bridging oligonucleotides are between 10-30 nucleotides in length. 
     
     
         46 . An assay system for detection of the presence or absence of copy number variation (CNV) of a genomic region and presence or absence of one or more polymorphisms in a mixed sample using a single assay, the assay comprising the steps of:
 introducing a first set of fixed sequence oligonucleotides to a mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci in or associated with a genomic region;   introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci with a putative polymorphism;   introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to regions in the loci between the regions complementary to the fixed sequence oligonucleotides of the set;   extending the region between at least one fixed sequence oligonucleotide and a bridging oligonucleotide with a polymerase and dNTPs to create adjacently hybridized fixed sequence oligonucleotides and bridging oligonucleotides;   ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the loci corresponding to a genomic region and/or the loci with a putative polymorphism;   amplifying the contiguous ligation products using the universal primer regions to create amplification products; and   detecting the amplification products;   wherein detection of the amplification products correlates to the copy number of one or more genomic regions and the presence or absence of a polymorphism in one or more loci in the mixed sample.   
     
     
         47 . The assay system of  claim 46 , wherein the fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         48 . The assay system of  claim 46 , wherein the universal primer regions are used in sequence determination of the amplification products. 
     
     
         49 . The assay system of  claim 46 , wherein one or both sets of fixed sequence oligonucleotides comprise separate fixed sequence oligonucleotides complementary to regions on the loci. 
     
     
         50 . The assay system of  claim 46 , wherein one or both sets of fixed sequence oligonucleotides comprise a precircle probe. 
     
     
         51 . The assay system of  claim 46 , wherein the bridging oligonucleotides are introduced simultaneously with the ligation mixture. 
     
     
         52 . The assay system of  claim 46 , wherein the amplification products are isolated prior to detection. 
     
     
         53 . The assay system of  claim 52 , wherein the amplification products are isolated as individual molecules prior to detection. 
     
     
         54 . The assay system of  claim 53 , wherein the individual isolated amplification products are further amplified to create identical copies of all or a portion of the individual amplification products prior to detection. 
     
     
         55 . The assay system of  claim 53 , wherein the individual isolated amplification products are further amplified to create identical copies of molecules complementary to all or a portion of the individual amplification products prior to detection. 
     
     
         56 . The assay system of  claim 45 , wherein at least one locus interrogated for copy number is different from all loci interrogated for polymorphisms. 
     
     
         57 . The assay system of  claim 54 , wherein several loci interrogated for copy number are different from the loci interrogated for polymorphisms. 
     
     
         58 . The assay system of  claim 46 , wherein the bridging oligonucleotide are between 2-45 nucleotides in length. 
     
     
         59 . The assay system of  claim 46 , wherein the bridging oligonucleotide are between 3-9 nucleotides in length. 
     
     
         60 . The assay system of  claim 46 , wherein the bridging oligonucleotides are between 10-30 nucleotides in length. 
     
     
         61 . An assay system for detection of the presence or absence of a chromosomal abnormality and the presence or absence of one or more polymorphisms in a mixed sample using a single assay, the assay comprising the steps of:
 introducing a first set of fixed sequence oligonucleotides to a mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci with a putative polymorphism;   introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on two or more loci from a first chromosome;   introducing a third set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on two or more loci from a second chromosome;   introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to regions in the loci between the regions complementary to the fixed sequence oligonucleotides of the set;   ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the nucleic acids;   amplifying the contiguous ligation products to create amplification products; and   detecting the amplification products;   wherein detection of the amplification products correlates to the presence or absence of a chromosomal abnormality and the presence or absence of a polymorphism in one or more loci in the mixed sample.   
     
     
         62 . The assay system of  claim 61 , wherein the fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         63 . The assay system of  claim 62 , wherein the universal primer regions are used in sequence determination of the amplification products. 
     
     
         64 . The assay system of  claim 61 , wherein the bridging oligonucleotides hybridize between and adjacent to both fixed sequence oligonucleotides of the sets. 
     
     
         65 . The assay system of  claim 61 , wherein one or more sets of fixed sequence oligonucleotides comprise separate fixed sequence oligonucleotides complementary to regions on the loci. 
     
     
         66 . The assay system of  claim 61 , wherein one or more sets of fixed sequence oligonucleotides comprise a precircle probe. 
     
     
         67 . The assay system of  claim 61 , wherein the bridging oligonucleotides are introduced simultaneously with the ligation mixture. 
     
     
         68 . The assay system of  claim 61 , wherein the amplification products are isolated prior to detection. 
     
     
         69 . The assay system of  claim 68 , wherein the amplification products are isolated as individual molecules prior to detection. 
     
     
         70 . The assay system of  claim 69 , wherein the individual isolated amplification products are further amplified to create identical copies of all or a portion of the individual amplification products prior to detection. 
     
     
         71 . The assay system of  claim 69 , wherein the individual isolated amplification products are further amplified to create identical copies of molecules complementary to all or a portion of the individual amplification products prior to detection. 
     
     
         72 . The assay system of  claim 61 , wherein the bridging oligonucleotide are between 2-45 nucleotides in length. 
     
     
         73 . The assay system of  claim 61 , wherein the bridging oligonucleotide are between 3-9 nucleotides in length. 
     
     
         74 . The assay system of  claim 61 , wherein the bridging oligonucleotides are between 10-30 nucleotides in length. 
     
     
         75 . An assay system for detection of the presence or absence of a chromosomal abnormality and presence or absence of one or more polymorphisms in a mixed sample using a single assay, the assay comprising the steps of:
 introducing a first set of fixed sequence oligonucleotides to a mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci with a putative polymorphism;   introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on two or more loci from a first chromosome;   introducing a third set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on two or more loci from a second chromosome;   introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to regions in the loci between the regions complementary to the fixed sequence oligonucleotides of the set;   extending the region between the first and second hybridized oligonucleotide of at least one fixed sequence oligonucleotide set with a polymerase and dNTPs to create two adjacently hybridized fixed sequence oligonucleotides from that set;   ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the nucleic acids;   amplifying the contiguous ligation products to create amplification products; and   detecting the amplification products;   wherein detection of the amplification products correlates to the presence or absence of a chromosomal abnormality and the presence or absence of a polymorphism in one or more loci in the mixed sample.   
     
     
         76 . The assay system of  claim 75 , wherein the fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         77 . The assay system of  claim 75 , wherein the universal primer regions are used in sequence determination of the amplification products. 
     
     
         78 . The assay system of  claim 75 , wherein the same universal primer regions are used in all sets of fixed sequence oligonucleotides. 
     
     
         79 . The assay system of  claim 75 , wherein the fixed sequence oligonucleotides of each set hybridize to immediately adjacent regions in the loci. 
     
     
         80 . The assay system of  claim 75 , wherein the unhybridized fixed sequence oligonucleotides are removed prior to amplification of the contiguous ligation product. 
     
     
         81 . The assay system of  claim 75 , wherein the sets of fixed sequence oligonucleotides are introduced simultaneously. 
     
     
         82 . The assay system of  claim 75 , wherein the bridging oligonucleotides are introduced simultaneously with the ligation mixture. 
     
     
         83 . The assay system of  claim 75 , wherein the amplification products are isolated prior to detection. 
     
     
         84 . The assay system of  claim 83 , wherein the amplification products are isolated as individual molecules prior to detection. 
     
     
         85 . The assay system of  claim 83 , wherein the individual isolated amplification products are further amplified to create identical copies of all or a portion of the individual amplification products prior to detection. 
     
     
         86 . The assay system of  claim 85 , wherein the individual isolated amplification products are further amplified to create identical copies of molecules complementary to all or a portion of the individual amplification products prior to detection. 
     
     
         87 . The assay system of  claim 85 , wherein at least one locus interrogated for copy number is different from all loci interrogated for polymorphisms. 
     
     
         88 . The assay system of  claim 75 , wherein several loci interrogated for copy number are different from the loci interrogated for polymorphisms. 
     
     
         89 . The assay system of  claim 75 , wherein the bridging oligonucleotide are between 2-45 nucleotides in length. 
     
     
         90 . The assay system of  claim 75 , wherein the bridging oligonucleotide are between 3-9 nucleotides in length. 
     
     
         91 . The assay system of  claim 75 , wherein the bridging oligonucleotides are between 10-30 nucleotides in length. 
     
     
         92 . A single assay system with the ability to determine the presence or absence of one or more chromosomal abnormalities and the presence or absence of one or more polymorphisms in a minor source of DNA in a mixed sample from an individual. 
     
     
         93 . An assay system for detection of the presence or absence of genetic copy number variation (CNV) of a genomic region and the presence or absence of an infectious agent in a mixed sample using a single assay, the assay comprising the steps of:
 introducing a first set of fixed sequence oligonucleotides to a mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci in or associated with a genomic region;   introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions in nucleic acids indicative of an infectious agent;   introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acids, wherein one or more bridging oligonucleotides are complementary to a region of the nucleic acids between the regions complementary to the fixed sequence oligonucleotides of each set;   ligating the hybridized oligonucleotides to create a contiguous ligation product complementary to the nucleic acids;   amplifying the contiguous ligation product to create amplification products; and   detecting the amplification products;   wherein detection of the amplification products correlates to copy number of the genomic region and the presence or absence of an infectious agent in the mixed sample.   
     
     
         94 . The assay system of  claim 93 , wherein the assay system further comprises introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acids, wherein one or more bridging oligonucleotides are complementary to a region of the nucleic acids between the regions complementary to the fixed sequence oligonucleotides of each set. 
     
     
         95 . An assay system for detection of the presence or absence of copy number variation (CNV) of a genomic region, the presence or absence of one or more polymorphisms, and the presence or absence of an infectious agent in a mixed sample from an individual using a single assay, the assay comprising the steps of:
 introducing a first set of fixed sequence oligonucleotides to a mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci in or associated with a genomic region;   introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci with a putative polymorphism;   introducing a third set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions in nucleic acids indicative of an infectious agent;   introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acids, wherein one or more bridging oligonucleotides are complementary to a region of the nucleic acids between the regions complementary to the fixed sequence oligonucleotides of the set;   ligating the hybridized oligonucleotides to create a contiguous ligation product complementary to the nucleic acids;   amplifying the contiguous ligation product to create amplification products; and   detecting the amplification products;   wherein detection of the amplification products correlates to copy number of the genomic region, the presence or absence of a polymorphism at one or more loci, and the presence or absence of an infectious agent in the mixed sample.   
     
     
         96 . The assay system of  claim 95 , wherein the assay system further comprises introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acids, wherein one or more bridging oligonucleotides are complementary to a region of the nucleic acids between the regions complementary to the fixed sequence oligonucleotides of each set.