PHASING
First Claim
Patent Images
1. A method of phasing allelic variants of a first and a second genetic locus on a nucleic acid template comprising the first and the second genetic locus and a first spacing region between the first and the second genetic locus, the method comprising steps of:
- (a) providing a sample comprising one or more types of the nucleic acid template;
(b) partitioning the sample into aliquots so that a plurality of the aliquots contain no more than one type of the template;
(c) generating, from at least one aliquot containing one type of the template, a plurality of nucleic acid polymers based on the template, wherein at least one of the nucleic acid polymers comprises the first and the second genetic locus and a second spacing region that is different from the first spacing region; and
(d) phasing the first and the second genetic locus on the at least one nucleic acid polymer to phase the allelic variants of the first and second genetic loci on the nucleic acid template.
1 Assignment
0 Petitions
Accused Products
Abstract
Among other things, the present disclosure pertains to methods and compositions related to phasing of allelic variants of genetic loci. Phasing of allelic variants of genetic loci on an individual patient'"'"'s chromosomes is highly valuable for many purposes, including patient stratification for allele-specific therapeutics.
17 Citations
69 Claims
-
1. A method of phasing allelic variants of a first and a second genetic locus on a nucleic acid template comprising the first and the second genetic locus and a first spacing region between the first and the second genetic locus, the method comprising steps of:
-
(a) providing a sample comprising one or more types of the nucleic acid template; (b) partitioning the sample into aliquots so that a plurality of the aliquots contain no more than one type of the template; (c) generating, from at least one aliquot containing one type of the template, a plurality of nucleic acid polymers based on the template, wherein at least one of the nucleic acid polymers comprises the first and the second genetic locus and a second spacing region that is different from the first spacing region; and (d) phasing the first and the second genetic locus on the at least one nucleic acid polymer to phase the allelic variants of the first and second genetic loci on the nucleic acid template. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69)
-
-
2. A method of phasing allelic variants of multiple genetic loci on a nucleic acid template comprising the multiple genetic loci and multiple spacing regions between the multiple genetic loci, the method comprising steps of:
-
(a) providing a sample comprising one or more types of the nucleic acid template; (b) partitioning the sample into aliquots so that a plurality of the aliquots contain no more than one type of the template; (c) generating, from at least one aliquot containing one type of the template, a plurality of nucleic acid polymers based on the template, wherein at least one of the nucleic acid polymers comprises the multiple genetic loci and wherein at least one of the multiple spacing regions on the nucleic acid polymer is different from the corresponding spacing region on the nucleic acid template; and (d) phasing the multiple genetic loci on the at least one nucleic acid polymer to phase the allelic variants of the multiple genetic loci on the nucleic acid template.
-
-
3. A method of phasing allelic variants of a first and a second genetic locus on a nucleic acid template comprising the first and the second genetic locus and a first spacing region between the first and the second genetic locus, the method comprising a step of:
-
(a) phasing the allelic variants on a nucleic acid polymer in order to phase the allelic variants on the nucleic acid template, wherein the nucleic acid polymer comprises the first and the second genetic locus, and a second spacing region which is different from the first spacing region between the first and the second genetic locus, wherein the nucleic acid polymer is generated from an aliquot containing one type of the nucleic acid template, and wherein the aliquot is generated by partitioning a sample comprising one or more types of the nucleic acid template into aliquots so that a plurality of the aliquots contain no more than one type of the template.
-
-
4. A method of phasing allelic variants of multiple genetic loci on a nucleic acid template comprising the multiple genetic loci and multiple spacing regions between the multiple genetic loci, the method comprising a step of:
-
phasing the allelic variants on a nucleic acid polymer in order to phase the allelic variants on the nucleic acid template, wherein the nucleic acid polymer comprises the multiple genetic loci, and wherein one or more of the multiple spacing regions on the nucleic acid polymer is different than one or more of the multiple spacing regions on the nucleic acid template, wherein the nucleic acid polymer is generated from an aliquot containing one type of the nucleic acid template, and wherein the aliquot is generated by partitioning a sample comprising one or more types of the nucleic acid template into aliquots so that a plurality of the aliquots contain no more than one type of the template.
-
-
5. A method of generating nucleic acid polymers comprising a first and a second genetic locus, the method comprising steps of:
-
(a) providing a sample comprising one or more types of the nucleic acid template comprising the first and the second genetic locus and a first spacing region between the first and the second genetic locus; (b) partitioning the sample into aliquots so that a plurality of the aliquots contain no more than one type of the template; and (c) generating, from each aliquot containing one type of the template, a plurality of nucleic acid polymers based on the template, wherein at least one of the nucleic acid polymers comprises the first and the second genetic locus and a second spacing region that is different from the first spacing region. - View Dependent Claims (6)
-
-
7. A method of phasing allelic variants of a first and a second genetic locus on a nucleic acid template comprising the first and the second genetic locus and a first spacing region between the first and the second genetic locus, the method comprising:
-
generating from a first aliquot of the nucleic acid template a plurality of nucleic acid polymers based on the template, wherein at least one of the nucleic acid polymers comprises the first and the second genetic locus and a second spacer region that is different from the first linking region; and wherein at least one second aliquot of the nucleic acid template comprises a different type of the nucleic acid template than the first aliquot. - View Dependent Claims (8, 9)
-
-
10. A method of generating a plurality of nucleic acid polymers, wherein at least one of the polymers comprises a first and a second genetic locus of a nucleic acid template comprising the first and the second genetic locus and a first spacing region between the first and the second genetic locus, the method comprising a step of:
-
generating from a first aliquot of the nucleic acid template a plurality of nucleic acid polymers based on the template, wherein at least one of the nucleic acid polymers comprises the first and the second genetic locus and a second spacing region that is different from the first spacing region; and
wherein;the first aliquot contains no more than one type of the template; and at least one second aliquot of the nucleic acid template comprises a different type of the nucleic acid template than the first aliquot. - View Dependent Claims (11)
-
-
12. A method comprising steps of:
-
(a) providing a collection of discrete reaction aliquots, at least one of which contains; a single molecule of a nucleic acid template whose base sequence includes a first sequence element and a second sequence element, wherein the first and second sequence elements are separated from one another in the template nucleic acid by a spacing sequence element comprising a plurality of residues, and further wherein at least one of the first and/or second sequence elements is present in two or more different forms within the set of template nucleic acids present in the collection; and reagents for amplifying nucleic acids; (b) incubating the at least one reaction aliquot under conditions and for a time sufficient to generate, within the at least one reaction aliquot, a product nucleic acid in which the first and second sequences, or the complement sequences thereof, are linked to one another and some or all of the residues of the spacing element, or the complement thereof, are absent. - View Dependent Claims (13)
-
-
14. A method comprising:
-
incubating one or more discrete reaction aliquots of a collection, wherein each reaction aliquot of the collection contains; a single molecule of a template nucleic acid whose base sequence includes a first sequence element and a second sequence element, wherein the first and second sequence elements are separated from one another in the template nucleic acid by a spacing sequence element that comprises a plurality of residues, and further wherein at least one of the sequence elements is present in two or more different forms within the set of template nucleic acids present in the collection; and reagents for amplifying nucleic acids; under conditions and for a time sufficient to generate, within each discrete reaction aliquot, a product nucleic acid in which the first and second sequences, or the complement sequences thereof, are linked to one another and some or all of the residues of the spacing element, or the complement thereof, are absent. - View Dependent Claims (15)
-
-
16. A composition comprising:
-
a single molecule of a nucleic acid template whose base sequence includes a first sequence element and a second sequence element, wherein the first and second sequence elements are separated from one another in the template nucleic acid by a spacing sequence element that comprises a plurality of residues; a plurality of nucleic acid molecules in each of which the first and second sequence elements, or the complement sequences thereof, are linked to one another and some or all of the residues of the spacing sequence element, or the complement thereof, are absent; and optionally an reagent for amplifying the template nucleic acid, wherein no molecules other than the nucleic acid template and the plurality of nucleic acid molecules contain both the first and the second sequences.
-
-
17. A composition comprising a collection of discrete compositions, each of which independently contains:
-
a single molecule of a template nucleic acid whose base sequence includes a first sequence element and a second sequence element, wherein the first and second sequence elements are separated from one another in the template nucleic acid by a spacing sequence element that comprises a plurality of residues, and further wherein at least one of the first and/or second sequence elements is present in two or more different forms within the set of template nucleic acids present in the collection; and a plurality of nucleic acid molecules, in each of which the first and second sequences, or the complement sequences thereof, are linked to one another and some or all of the residues of the spacing sequence element, or the complement thereof, are absent; and optionally an reagent for amplifying the template nucleic acid.
-
-
18. A composition comprising a collection of discrete compositions, each of which independently contains:
-
a single molecule of a template nucleic acid whose base sequence includes a first sequence element and a second sequence element, wherein the first and second sequence elements are separated from one another in the template nucleic acid by a spacing sequence element that comprises a plurality of residues, and further wherein at least one of the sequence elements is present in two or more different forms within the set of template nucleic acids present in the collection; a plurality of nucleic acid molecules in each of which the first and second sequences, or the complement sequences thereof, are linked to one another and some or all of the residues of the spacing element, or the complement thereof, are absent; and optionally an reagent for amplifying the template nucleic acid; wherein in each composition, no molecules other than the template nucleic acid and the plurality of nucleic acid molecules contain both the first and the second sequences.
-
-
19. A method comprising:
incubating one or more discrete reaction systems of a collection, wherein each reaction system of the collection is discrete and contains; a single molecule of a template nucleic acid whose base sequence includes a first target sequence element and a second target sequence element, wherein the first and second target sequence elements are separated from one another in the template nucleic acid by a spacer sequence element that comprises a plurality of residues, and further wherein at least one of the target sequence elements is present in two or more different forms within the set of template nucleic acids present in the collection; and reagents for amplifying nucleic acids; under conditions and for a time sufficient that, within each discrete reaction system, a product nucleic acid in which the first and second target sequences, or the complement sequences thereof, are linked to one another and some or all of the residues of the spacer element, or the complement thereof, are absent.
-
20. A method of phasing allelic variants of a first and a second genetic locus on a chromosome or chromosomal fragment comprising the first and the second genetic locus, the method comprising the steps of:
-
(a) providing a sample comprising one or more haplotypes of the chromosome or chromosomal fragment; (b) diluting and partitioning the sample into aliquots so that a plurality of the aliquots contain no more than one haplotype of the chromosome or chromosomal fragment; (c) generating a plurality of amplicons from at least one chromosome or chromosomal fragment, wherein the amplicons are generated by polymerase chain reaction in the presence of a first and second pair of primers for the first and second genetic locus, respectively, each pair comprising a forward and a reverse primer, and wherein one of the first pair comprises a region of complementarity to one of the second pair, and wherein one or more of the amplicons comprises the first and the second genetic locus; and (d) determining the allelic variants of the first and second genetic loci on the one or more of the amplicons comprising the first and the second genetic locus to determine the phasing of the allelic variants of the first and second genetic locus on the chromosome or chromosomal fragment.
-
-
21. A method of phasing allelic variants of a plurality of genetic loci on a nucleic acid template comprising the plurality of loci, the method comprising the steps of:
-
(a) providing a sample comprising one or more types of the nucleic acid template; (b) partitioning the sample into aliquots so that a plurality of the aliquots contain no more than one type of the template; (c) generating, from each aliquot containing one type of the template, a plurality of nucleic acid polymers based on the template, wherein at least one of the nucleic acid polymers comprises each of the plurality of genetic loci; and (d) sequencing one or more of the nucleic acid polymers comprising each of the genetic loci to determine the phasing of the allelic variants of the plurality of genetic loci.
-
Specification