Determination of fetal dna fraction in a sample

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.

1 - 20 . (canceled) 
     
     
         21 . An assay method for determining the fraction of fetal DNA in a sample, comprising the steps of:
 (a) providing a maternal plasma or serum sample comprising maternal and fetal cell free DNA;   (b) interrogating at least 48 polymorphic loci from at least one target genomic region in the maternal plasma or serum sample by hybridizing a set of at least two fixed sequence oligonucleotides for each allele at each polymorphic locus to the maternal and fetal cell free DNA, wherein one of the at least two fixed sequence oligonucleotides of each set comprises a sequence complementary to one allele at a polymorphic locus, a capture region specific for each polymorphic locus, an allele-specific label binding region, and two restriction sites;   (c) ligating each set of hybridized fixed sequence oligonucleotides;   (d) amplifying the ligated fixed sequence oligonucleotides to create allele-specific amplicons;   (e) cleaving the allele-specific amplicons at the restriction sites to create cleaved allele-specific amplicons, wherein each cleaved allele-specific amplicon comprises a polymorphic locus-specific capture region and an allele-specific label binding region;   (f) detecting the cleaved allele-specific amplicons from the polymorphic loci via competitive hybridization of the polymorphic locus-specific capture regions of the cleaved allele-specific amplicons to capture probes on an array; and   (g) quantifying the alleles of the polymorphic loci by detecting the allele-specific label binding regions for each allele on the cleaved allele-specific amplicons on the array,   (g) thereby determining the fraction of fetal DNA in the sample.   
     
     
         22 . The assay method of  claim 21 , wherein three or more fixed sequence oligonucleotides for set are used to interrogate each polymorphic locus. 
     
     
         23 . The assay method of  claim 21 , wherein the amplifying step comprises universal amplification through a polymerase chain reaction. 
     
     
         24 . The assay method of  claim 21 , wherein the at least one target genomic region comprises at least one chromosome. 
     
     
         25 . The assay method of  claim 21 , wherein the at least one target genomic region comprises two or more chromosomes. 
     
     
         26 . The assay method of  claim 24 , wherein the at least one chromosome is not chromosome 13, 18, or 21. 
     
     
         27 . The assay method of  claim 21 , wherein the allele-specific label binding regions each comprise a label specific for one allele at a polymorphic locus. 
     
     
         28 . The assay method of  claim 21 , wherein the allele-specific label binding regions each comprise a sequence complementary to an allele-specific oligonucleotide comprising an allele-specific label. 
     
     
         29 . An assay method for determining the fraction of fetal DNA in a sample, comprising the steps of:
 (a) providing a maternal plasma or serum sample comprising maternal and fetal cell free DNA;   (b) interrogating at least 48 polymorphic loci from at least one target genomic region in the maternal plasma or serum sample by hybridizing a set of at least two fixed sequence oligonucleotides for each allele at each polymorphic locus to the maternal and fetal cell free DNA, wherein at least two of the fixed sequence oligonucleotides of each set comprise a universal primer site, and wherein at least one of the fixed sequence oligonucleotides of each set comprises a sequence complementary to one allele at a polymorphic locus, a capture region specific for each polymorphic locus, an allele-specific label binding region, and two restriction sites;   (c) extending at least one of the hybridized fixed sequence oligonucleotides of each set to form adjacently hybridized fixed sequence oligonucleotides.   (c) ligating each set of hybridized fixed sequence oligonucleotides;   (d) amplifying the ligated fixed sequence oligonucleotides using the universal primer sites to create allele-specific amplicons;   (e) cleaving the allele-specific amplicons at the restriction sites to create cleaved allele-specific amplicons, wherein each cleaved allele-specific amplicon comprises a polymorphic locus-specific capture region and an allele-specific label binding region;   (f) applying the cleaved amplicons to an array, wherein the array comprises allele-specific capture probes complementary to the allele-specific capture regions of the cleaved amplicons;   (g) hybridizing the allele-specific capture regions of the cleaved amplicons to the allele-specific capture probes on the array;   (h) detecting the hybridized cleaved amplicons;   (i) quantifying the relative frequency of cleaved amplicons corresponding to each allele of each polymorphic locus by detecting each allele-specific label binding region; and   (j) calculating the fraction of fetal DNA in the sample using the relative frequency of cleaved amplicons corresponding to each allele of each polymorphic locus.   
     
     
         30 . The assay method of  claim 29 , wherein a single hybridized fixed sequence oligonucleotide of each set is extended to form adjacently hybridized fixed sequence oligonucleotides. 
     
     
         31 . The assay method of  claim 29 , wherein three or more fixed sequence oligonucleotides for each set are used to interrogate each polymorphic locus. 
     
     
         32 . The assay method of  claim 29 , wherein the amplifying step comprises universal amplification through a polymerase chain reaction. 
     
     
         33 . The assay method of  claim 29 , wherein the at least one target genomic region comprises at least one chromosome. 
     
     
         34 . The assay method of  claim 29 , wherein the at least one target genomic region comprises two or more chromosomes. 
     
     
         35 . The assay method of  claim 33 , wherein the at least one chromosome is not chromosome 13, 18, or 21. 
     
     
         36 . The assay method of  claim 29 , wherein the allele-specific label binding regions each comprise a label specific for one allele at a polymorphic locus. 
     
     
         37 . The assay method of  claim 21 , wherein the allele-specific label binding regions each comprise a sequence complementary to an allele-specific oligonucleotide comprising an allele-specific label. 
     
     
         38 . An assay method for determining the fraction of fetal DNA in a sample, comprising the steps of:
 (a) providing a maternal plasma or serum sample comprising maternal and fetal cell free DNA;   (b) interrogating at least 48 polymorphic loci from at least one target genomic region in the maternal plasma or serum sample by hybridizing a set of at least three fixed sequence oligonucleotides for each allele at each polymorphic locus to the maternal and fetal cell free DNA, wherein at least two of the fixed sequence oligonucleotides of each set comprises a universal primer site, and wherein at least one of the fixed sequence oligonucleotides of each set comprises a sequence complementary to one allele at a polymorphic locus, a capture region specific for each polymorphic locus, an allele-specific label binding region, and two restriction sites;   (c) ligating each set of hybridized fixed sequence oligonucleotides;   (d) amplifying the ligated fixed sequence oligonucleotides to create allele-specific amplicons;   (e) cleaving the allele-specific amplicons at the restriction sites to create cleaved allele-specific amplicons, wherein each cleaved allele-specific amplicon comprises a polymorphic locus-specific capture region and an allele-specific label binding region;   (f) applying the cleaved amplicons to an array, wherein the array comprises allele-specific capture probes complementary to the allele-specific capture regions of the cleaved amplicons;   (g) hybridizing the allele-specific capture regions of the cleaved amplicons to the allele-specific capture probes on the array;   (h) detecting the hybridized cleaved amplicons;   (i) quantifying the relative frequency of cleaved amplicons corresponding to each allele of each polymorphic locus by detecting each allele-specific label binding region; and   (j) calculating the fraction of fetal DNA in the sample using the relative frequency of cleaved amplicons corresponding to each allele of each polymorphic locus.   
     
     
         39 . The assay method of  claim 38 , wherein the amplifying step comprises universal amplification through a polymerase chain reaction. 
     
     
         40 . The assay method of  claim 38 , wherein the at least one target genomic region comprises at least one chromosome. 
     
     
         41 . The assay method of  claim 38 , wherein the at least one target genomic region comprises two or more chromosomes. 
     
     
         42 . The assay method of  claim 40 , wherein the at least one chromosome is not chromosome 13, 18, or 21. 
     
     
         43 . The assay method of  claim 38 , wherein the allele-specific label binding regions each comprise a label specific for one allele at a polymorphic locus. 
     
     
         44 . The assay method of  claim 38 , wherein the allele-specific label binding regions each comprise a sequence complementary to an allele-specific oligonucleotide comprising an allele-specific label.