Detection of target nucleic acids using hybridization

• US 10,533,223 B2
• Filed: 12/20/2016
• Issued: 01/14/2020
• Est. Priority Date: 08/06/2010
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Ariosa Diagnostics Incorporated (Roche Holding AG)
• Inventors: Oliphant, Arnold, Zahn, Jacob, Juneau, Kara, Bogard, Patrick, Huang, Stephanie
• Primary Examiner(s): Dauner, Joseph G.
• Application Number:
  US15/385,785
• Publication Number:
  US 20170145510A1
• Time in Patent Office: 1,120 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding

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.

Claims

1. An assay method for providing a statistical likelihood of a fetal copy number variation comprising:

providing a maternal plasma or serum sample comprising maternal and fetal cell free DNA;

interrogating at least 48 non-polymorphic loci from a first target genomic region by hybridizing at least 48 first sets of at least two fixed sequence oligonucleotides to the maternal and fetal cell free DNA, wherein each of the at least two fixed sequence oligonucleotides in each first set comprises a region complementary to a locus in the first target genomic region, wherein one of the fixed sequence oligonucleotides of each first set comprises a capture region, a first label binding region, and two restriction sites;

interrogating at least 48 non-polymorphic loci from a second target genomic region by hybridizing at least 48 second sets of at least two fixed sequence oligonucleotides to the maternal and fetal cell free DNA, wherein each of the at least two fixed sequence oligonucleotides in each second set comprises a region complementary to a locus in the second target genomic region, wherein one of the fixed sequence oligonucleotides of each second set comprises a capture region, a second label binding region, and two restriction sites, wherein the capture region in each second set is the same as a capture region in at least one first set;

ligating the hybridized fixed sequence oligonucleotides;

amplifying the ligated fixed sequence oligonucleotides to create amplicons;

cleaving the amplicons at the restriction sites to create cleaved amplicons, wherein each cleaved amplicon comprises a capture region and the first or second label binding region;

detecting the cleaved amplicons from the first and second target genomic regions via hybridization of the capture regions of the cleaved amplicons to an array comprising capture probes complementary to the capture regions, wherein the cleaved amplicons from the first and second target genomic regions hybridize competitively to the capture probes complementary to the capture regions;

quantifying the capture regions of the cleaved amplicons to determine a relative frequency of the interrogated non-polymorphic loci from the first and second target genomic regions by detecting the first and second label binding regions;

estimating the relative frequency of the first and second target genomic regions based on the determined relative frequency of the first and second label binding regions;

interrogating at least 48 polymorphic loci from at least one target genomic region different from the first and second target genomic regions by hybridizing a third set of at least three fixed sequence oligonucleotides for each allele at each polymorphic locus, wherein one of the at least three fixed sequence oligonucleotides of each third 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;

ligating the hybridized fixed sequence oligonucleotides;

amplifying the ligated fixed sequence oligonucleotides to create allele-specific amplicons;

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;

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 the array;

quantifying the alleles of the polymorphic loci by detecting the allele-specific label binding regions for each allele on the cleaved allele-specific amplicons to determine the fraction of fetal DNA in the sample;

determining the fraction of fetal DNA; and

calculating a statistical likelihood of a fetal copy number variation in the maternal sample using the estimated relative frequency of the first and second target genomic regions in the sample and the fraction of fetal DNA.

2. The assay method of claim 1, wherein three or more fixed sequence oligonucleotides for each first and second set are used to interrogate each non-polymorphic locus in the first and second target genomic regions.

3. The assay method of claim 1, wherein each amplifying step uses universal amplification through a polymerase chain reaction.

4. The assay method of claim 1, further comprising:

identifying low frequency alleles from the quantified alleles of the polymorphic loci where the maternal DNA is homozygous and the fetal DNA is heterozygous; and

using the low frequency alleles from the polymorphic loci to determine the fraction of fetal DNA.

5. The assay method of claim 1, wherein the first target genomic region is a chromosome.

6. The assay method of claim 1, wherein the second target genomic region is a chromosome.

7. The assay method claim 1, wherein the first target genomic region is a sub-chromosomal region.

8. The assay method claim 1, wherein the second target genomic region is a sub-chromosomal region.

9. The assay method of claim 1, wherein the label binding regions of the fixed sequence oligonucleotides each comprise a label, wherein the first label binding regions each comprise a first label, and the second label binding regions each comprise a second label.

10. The assay method of claim 1, wherein the label binding regions of the fixed sequence oligonucleotides each comprise a sequence complementary to an oligonucleotide comprising a label, wherein the first label binding regions each comprise a sequence complementary to a first oligonucleotide comprising a first label, and the second label binding regions each comprise a sequence complementary to a second oligonucleotide comprising a second label.

11. An assay method for determining a likelihood of a fetal aneuploidy comprising the steps of:

providing a maternal plasma or serum sample comprising maternal and fetal cell free DNA;

introducing at least fifty first sets of two or more fixed sequence oligonucleotides to the maternal and fetal cell free DNA, wherein each of the two or more fixed sequence oligonucleotides in each first set is complementary to a non-polymorphic locus in a first target genomic region, under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the non-polymorphic locus, wherein at least one of the fixed sequence oligonucleotides of each first set comprises a universal primer site, a capture region, a first label binding region, and two restriction sites;

introducing at least fifty second sets of two or more fixed sequence oligonucleotides to the maternal and fetal cell free DNA, wherein each of the two or more fixed sequence oligonucleotides in each second set is complementary to a non-polymorphic locus in a second target genomic region, under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the non-polymorphic locus, wherein at least one of the fixed sequence oligonucleotides of each second set comprises a universal primer site, a capture region, a second label binding region, and two restriction sites, wherein the capture region in each second set is the same as the capture region in at least one first set;

introducing a third set of three or more fixed sequence oligonucleotides for each allele of a set of at least fifty polymorphic loci to the maternal and fetal cell free DNA under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to a polymorphic locus, wherein one of the three fixed sequence oligonucleotides of each third set comprises a universal primer site, a sequence complementary to one allele at a polymorphic locus, an allele-specific label binding region, two restriction sites, and a polymorphic locus-specific capture region, wherein the capture region for each polymorphic locus is different from the capture region for every other polymorphic locus and different from the capture regions of the first and second sets;

hybridizing the first, second and third sets of fixed sequence oligonucleotides to the first and second target genomic regions and the polymorphic loci;

extending at least one of the hybridized fixed sequence oligonucleotides of the first, second and third sets to form adjacently hybridized fixed sequence oligonucleotides;

ligating the hybridized fixed sequence oligonucleotides of the first, second and third sets to create ligation products;

amplifying the ligation products using the universal primer sites to create amplicons corresponding to the polymorphic loci;

cleaving the amplicons at the restriction sites to create cleaved amplicons, wherein each cleaved amplicon comprises one capture region and one label binding region;

applying the cleaved amplicons to an array, wherein the array comprises capture probes complementary to the capture regions on the cleaved amplicons from the first and second target genomic regions, and wherein the array comprises capture probes complementary to the capture regions on the cleaved amplicons from each polymorphic locus;

hybridizing the capture regions of the cleaved amplicons from the first and second target genomic regions to capture probes on the array;

hybridizing the capture regions of the cleaved amplicons from the polymorphic loci to capture probes on the array;

detecting the hybridized cleaved amplicons;

quantifying a relative frequency of the cleaved amplicons corresponding to loci from the first target genomic region and a relative frequency of the cleaved amplicons corresponding to loci from the second target genomic region by detecting the first and second label binding regions;

quantifying a relative frequency of each allele from the polymorphic loci by detecting the allele-specific label binding regions for each allele on the cleaved amplicons to determine a percent fetal cell free DNA; and

computing a likelihood of fetal aneuploidy using the relative frequency of the cleaved amplicons corresponding to loci from the first and second target genomic regions to determine the likelihood of a fetal aneuploidy and the determined percent fetal cell free DNA.

12. The assay method of claim 11, wherein the relative frequencies of the cleaved amplicons corresponding to the loci from the first target genomic region are summed and the relative frequencies of the cleaved amplicons corresponding to the loci from the second target genomic region are summed, and the sum from the cleaved amplicons corresponding to the loci from the first target genomic region are compared to the sum from the cleaved amplicons corresponding to the loci from the second target genomic region to calculate a target genomic region ratio.

13. The assay method of claim 11, wherein a single hybridized fixed sequence oligonucleotide of each first, second and third set is extended to form adjacently hybridized fixed sequence oligonucleotides.

14. The assay method of claim 11, wherein the label binding regions of the fixed sequence oligonucleotides each comprise a label, wherein the first label binding regions each comprise a first label, and the second label binding regions each comprise a second label.

15. The assay method of claim 11, wherein the label binding regions of the fixed sequence oligonucleotides each comprise a sequence complementary to an oligonucleotide comprising a label, wherein the first label binding regions each comprise a sequence complementary to a first oligonucleotide comprising a first label, and the second label binding regions each comprise a sequence complementary to a second oligonucleotide comprising a second label.

16. An assay method for determining a likelihood of a fetal aneuploidy comprising the steps of:

providing a maternal plasma or serum sample comprising maternal and fetal cell free DNA;

introducing at least fifty first sets of two or more fixed sequence oligonucleotides to the maternal and fetal cell free DNA, wherein each of the two or more fixed sequence oligonucleotides in each first set is complementary to a set of non-polymorphic loci in a first target genomic region, under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the non-polymorphic loci, wherein at least one of the fixed sequence oligonucleotides of each first set comprises a universal primer site, a capture region, a first label binding region, and two restriction sites;

introducing at least fifty second sets of two or more fixed sequence oligonucleotides to the maternal and fetal cell free DNA, wherein each of the two or more fixed sequence oligonucleotides in each second set is complementary to a set of non-polymorphic loci in a second target genomic region, under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the non-polymorphic loci, wherein at least one of the fixed sequence oligonucleotides of each second set comprises a universal primer site, a capture region, a second label binding region, and two restriction sites, wherein the capture region in each second set is the same as the capture region in at least one first set;

introducing a third set of three or more fixed sequence oligonucleotides for each allele of a set of two or more polymorphic loci to the maternal and fetal cell free DNA under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to a polymorphic locus, wherein one of the three or more fixed sequence oligonucleotides of each third set comprises a universal primer site, a sequence complementary to one allele at a polymorphic locus, an allele-specific label binding region, two restriction sites, and a polymorphic locus-specific capture region, wherein the capture region for each polymorphic locus is different from the capture region for every other polymorphic locus and different from the capture regions of the first and second sets;

hybridizing the first, second and third sets of fixed sequence oligonucleotides to the first and second target genomic regions and the polymorphic loci;

extending at least one of the hybridized fixed sequence oligonucleotides of the first, second and third sets to form adjacently hybridized fixed sequence oligonucleotides for each set;

ligating the adjacently hybridized fixed sequence oligonucleotides from the first, second and third sets to create ligation products;

amplifying the ligation products using the universal primer sites to create amplicons;

cleaving the amplicons at the restriction sites to create cleaved amplicons, wherein each cleaved amplicon comprises one capture region and one label binding region;

applying the cleaved amplicons to an array, wherein the array comprises capture probes complementary to the capture regions on the cleaved amplicons from the first and second target genomic regions, and wherein the array comprises capture probes complementary to the capture regions on the cleaved amplicons from each polymorphic locus;

hybridizing the capture regions of the cleaved amplicons from the first and second target genomic regions to capture probes on the array;

hybridizing the capture regions of the cleaved amplicons from the polymorphic loci to capture probes on the array;

detecting the hybridized cleaved amplicons;

quantifying a relative frequency of each allele from the polymorphic loci by detecting the allele-specific label binding regions for each allele on the cleaved amplicons to determine a percent fetal cell free DNA;

determining the percent of fetal cell free DNA by identifying low frequency alleles from the quantified alleles where a maternal locus is homozygous and a corresponding fetal locus is heterozygous;

quantifying a relative frequency of cleaved amplicons corresponding to loci from the first target genomic region and a relative frequency of cleaved amplicons corresponding to loci from the second target genomic region by detecting the first and second label binding regions; and

computing a likelihood of a fetal aneuploidy using the relative frequency of cleaved amplicons corresponding to loci from the first and second target genomic regions and the percent fetal cell-free DNA.

17. The assay method of claim 16, further comprising comparing the relative frequencies of the cleaved amplicons corresponding to loci from the first and second target genomic regions and adjusting the likelihood of a fetal aneuploidy based on the percent fetal cell-free DNA.

18. The assay method of claim 16, wherein the relative frequencies of the cleaved amplicons corresponding to the loci from the first target genomic region are summed and the relative frequencies of the cleaved amplicons corresponding to the loci from the second target genomic region are summed, and the sum from the cleaved amplicons corresponding to the loci from the first target genomic region are compared to the sum from the cleaved amplicons corresponding to the loci from the second target genomic region to calculate a target genomic region ratio.

19. The assay method of claim 16, wherein the polymorphic loci are autosomal loci.

20. The assay method of claim 16, wherein one or more corresponding fetal polymorphic loci comprise more than one genetic variation as compared to a maternal locus.

21. The assay method of claim 16, wherein the cleaved amplicons from loci of the first and second target genomic regions and the cleaved amplicons from the polymorphic loci are created in a single vessel.

22. The assay method of claim 16, wherein the label binding regions of the fixed sequence oligonucleotides each comprise a label, wherein the first label binding regions each comprise a first label, and the second label binding regions each comprise a second label.

23. The assay method of claim 16, wherein the label binding regions of the fixed sequence oligonucleotides each comprise a sequence complementary to an oligonucleotide comprising a label, wherein the first label binding regions each comprise a sequence complementary to a first oligonucleotide comprising a first label, and the second label binding regions each comprise a sequence complementary to a second oligonucleotide comprising a second label.