Assay systems for genetic analysis
Inventors
Sparks, Andrew • Struble, Craig • Wang, Eric • Oliphant, Arnold
Assignees
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Publication Number
US-12344887-B2
Publication Date
2025-07-01
Expiration Date
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.
Core Innovation
The invention provides an assay method for calculation of source contribution and detection of the presence or absence of copy number variations (CNVs) in one or more genomic regions within a mixed sample. The method interrogates selected loci by introducing a first set of fixed sequence oligonucleotides and a second set of fixed sequence oligonucleotides, each under conditions that allow specific hybridization to complementary regions on target loci. The first set is used to identify loci interrogated for polymorphisms of interest corresponding to a major source or a minor source, while the second set is used to interrogate at least one polymorphic locus.
The assay further introduces a pool of one or more bridging oligonucleotides that hybridize to complementary regions in the two or more selected loci. The bridging oligonucleotides are complementary to a region between and adjacent to the regions complementary to the fixed sequence oligonucleotides of each set, and the bridging oligonucleotides are designed to have a melting temperature in a range of ±5°C. After hybridization, the hybridized oligonucleotides are ligated to create contiguous ligation products complementary to the loci, and the contiguous ligation products are amplified to create amplification products.
The method analyzes individual amplification products through sequence determination or hybridization techniques, including interrogating one or more selected loci for copy number and interrogating one or more selected loci for polymorphisms of interest. Source contribution is calculated by determining loci content from amplification products derived from fixed sequence oligonucleotides that identify selected loci interrogated for polymorphisms corresponding to major and minor sources. CNVs are detected by determining loci frequency from amplification products derived from fixed sequence oligonucleotides that identify selected loci interrogated for copy number.
Claims Coverage
The document includes one independent claim. It contains a single integrated assay method with multiplexed locus interrogation, bridging oligonucleotide hybridization, ligation, amplification, individual amplification product analysis, and linked determinations of source contribution and CNV presence or absence.
Multiplex locus interrogation with first and second fixed sequence oligonucleotides
Interrogating selected loci in the mixed sample by introducing a first set of fixed sequence oligonucleotides and a second set of fixed sequence oligonucleotides, under conditions allowing specific hybridization to complementary regions on target loci and at least one polymorphic locus.
Bridging oligonucleotide pool enabling contiguous ligation products across selected loci
Introducing a pool of one or more bridging oligonucleotides that specifically hybridize to complementary regions in the two or more selected loci, where the bridging oligonucleotides are complementary to a region between and adjacent to the regions complementary to the fixed sequence oligonucleotides of each set and have a melting temperature in a range of ±5°C.
Ligation and amplification of contiguous ligation products
Ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the loci, and amplifying the contiguous ligation products to create amplification products.
Individual amplification product analysis by sequence determination or hybridization
Analyzing the individual amplification products through sequence determination or hybridization techniques, interrogating one or more selected loci for copy number and one or more selected loci for polymorphisms of interest.
Source contribution calculation from polymorphism-interrogating fixed oligonucleotides
Calculating source contribution by determining loci content from amplification products derived from fixed sequence oligonucleotides that identify selected loci interrogated for polymorphisms of interest corresponding to a major source or a minor source.
CNV detection from copy-number-interrogating loci frequency
Detecting CNVs by determining loci frequency from amplification products derived from fixed sequence oligonucleotides that identify selected loci interrogated for copy number.
The inventive approach combines dual fixed-sequence interrogation, bridging oligonucleotides with defined melting temperature, ligation and amplification, individual amplification product analysis, and output determinations for source contribution and CNV presence or absence.
Stated Advantages
Enables calculation of source contribution from polymorphisms corresponding to major versus minor sources in a mixed sample.
Enables detection of the presence or absence of CNVs in one or more genomic regions.
Supports interrogating individual amplification products through sequence determination or hybridization techniques.
Documented Applications
Assay method for genetic analysis in a mixed sample, including calculation of source contribution and detection of CNVs in one or more genomic regions.
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