Detection of target nucleic acids using hybridization

Inventors

Oliphant, Arnold • Zahn, Jacob • Juneau, Kara • Bogard, Patrick • Huang, Stephanie

Assignees

Roche Molecular Systems Inc

Abstract

The present invention provides detection systems and methods for detection of loci and genomic regions in a sample, including mixed samples, using hybridization to an array.

Core Innovation

The patent describes an assay method that provides a statistical likelihood of a fetal copy number variation and/or a likelihood of a fetal aneuploidy using a maternal plasma or serum sample comprising maternal and fetal cell free DNA. The method interrogates at least 48 non-polymorphic loci from a first target genomic region and at least 48 non-polymorphic loci from a second target genomic region by hybridizing fixed sequence oligonucleotides that comprise capture regions and label binding regions, followed by ligating the hybridized fixed sequence oligonucleotides and amplifying the ligated products to create amplicons.

Restriction sites are used to cleave amplicons to create cleaved amplicons comprising a capture region and a label binding region. The cleaved amplicons are detected via hybridization of the capture regions to an array comprising capture probes complementary to the capture regions, where the cleaved amplicons from the first and second target genomic regions hybridize competitively to the capture probes. The method quantifies the capture regions of the cleaved amplicons to determine a relative frequency of the interrogated loci from the first and second target genomic regions by detecting the first and second label binding regions.

The method additionally interrogates at least 48 polymorphic loci from at least one target genomic region different from the first and second target genomic regions using a third set of fixed sequence oligonucleotides for each allele at each polymorphic locus, followed by ligation, amplification, restriction cleavage, competitive hybridization to array capture probes, and quantification by detecting allele-specific label binding regions to determine the fraction of fetal DNA. Finally, the patent describes calculating a statistical likelihood of a fetal copy number variation or calculating a likelihood of a fetal aneuploidy using the estimated relative frequency of the first and second target genomic regions together with the fraction of fetal DNA.

Claims Coverage

The partial content includes three independent claims that cover assay methods for statistical likelihood of fetal copy number variation or likelihood of fetal aneuploidy. The independent claims introduce three inventive features centered on fixed sequence oligonucleotides targeting loci with capture and label binding regions, competitive array detection with restriction-site cleavage, and integration with polymorphic loci to determine the fraction of fetal DNA.

Across the independent claims, the core claim coverage is the use of fixed sequence oligonucleotide sets with capture regions and label binding regions, restriction sites, and optional universal primer sites, followed by ligation, amplification, cleavage, and competitive array hybridization to measure relative frequencies for first and second non-polymorphic target genomic regions. The method also interrogates polymorphic loci to determine the fraction of fetal DNA and then calculates a fetal aneuploidy or copy-number statistical likelihood using the relative frequencies and the fetal DNA fraction.

Stated Advantages

Documented Applications