Assay systems for genetic analysis

An assay for detecting a presence or absence of a fetal copy number variation (CNV) in a genomic region within a maternal sample comprising fetal and maternal cell-free DNA, the assay comprising the steps of:

• (a) hybridizing a first set of fixed sequence oligonucleotides with the maternal and fetal cell-free DNA in the maternal sample, wherein:
• (i) the first set of fixed sequence oligonucleotides comprises first and second fixed sequence oligonucleotides complementary to regions of 24 or more loci in a first genomic region,
• (ii) the first and second fixed sequence oligonucleotides hybridize directly adjacent to each other at each locus or are separated by a gap of one or more nucleotides, and
• (iii) the melting temperatures (Tms) of the first fixed sequence oligonucleotides of the first set of fixed sequence oligonucleotides vary in a range of two degrees centigrade;
• (b) hybridizing a second set of fixed sequence oligonucleotides with the maternal and fetal cell-free DNA in the maternal sample, wherein:
• (i) the second set of fixed sequence oligonucleotides comprises first and second fixed sequence oligonucleotides complementary to regions of 24 or more loci in a second genomic region,
• (ii) the first and second fixed sequence oligonucleotides hybridize directly adjacent to each other at each locus or are separated by a gap of one or more nucleotides, and
• (iii) the Tms of the first fixed sequence oligonucleotides of the second set of fixed sequence oligonucleotides vary in a range of two degrees centigrade;
• (c) when the first and second fixed sequence oligonucleotides of each set are separated by a gap of one or more nucleotides,
• (i) hybridizing bridging oligonucleotides with the maternal and fetal cell-free DNA in the maternal sample, wherein the bridging oligonucleotides are complementary to regions in the loci between the regions complementary to the first and second fixed sequence oligonucleotides of the first and second sets, to create adjacently hybridized oligonucleotides;
• or
• (ii) extending the first and second fixed sequence oligonucleotides of each set with a polymerase to create adjacently hybridized oligonucleotides;
• (d) ligating the first set of fixed sequence oligonucleotides, the first set of fixed sequence oligonucleotides and the bridging oligonucleotides, or the first set of extended fixed sequence oligonucleotides to create contiguous ligation products complementary to the loci in the first genomic region, and ligating the second set of fixed sequence oligonucleotides, the second set of fixed sequence oligonucleotides and bridging oligonucleotides, or the second set of extended fixed sequence oligonucleotides to create contiguous ligation products complementary to the loci in the second genomic region;
• (e) amplifying each of the contiguous ligation products complementary to the loci in the first genomic region and each of the contiguous ligation products complementary to the loci in the second genomic region to create amplification products;
• (f) detecting the amplification products;
• (g) determining a relative frequency of the loci measured from the first and second genomic regions;
• and
• (h) detecting the presence or absence of a fetal CNV when the relative frequency of the loci measured from the first genomic region that is different from the relative frequency of the loci measured from the second genomic region.