Methods for preserving methylation status during clustering
AssigneeIllumina, Inc.
InventorsColin Brown, Sarah E. Shutlzaberger
Life of this patent
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Abstract
The present disclosure is concerned with compositions, articles, kits, and methods for producing an array that includes clonal clusters. In one embodiment, methods include producing clonal clusters on an array that preserves methylation status, such as the methylation state of methylated CpG dinucleotides of the seed DNA molecules.
Claims22
An array comprising a plurality of amplification sites,
- wherein at least one amplification site comprises one double-stranded target nucleic acid immobilized thereto,
- wherein a first strand of the double-stranded target nucleic acid is attached to the amplification site at its 5′ end,
- wherein a second strand of the double-stranded target nucleic acid is complementary to and annealed to a region of the first strand comprising the 3′ end of the first strand, and the second strand is not attached to the amplification site, and
- wherein the first strand of the double-stranded target nucleic acid comprises at least one first strand CpG dinucleotide and the second strand comprises at least one second strand CpG dinucleotide complementary to the at least one first strand CpG dinucleotide, wherein each cytosine of the first strand CpG dinucleotide and the second strand CpG is methylated.
6-7. (canceled)
An array comprising a plurality of amplification sites comprising a plurality of single-stranded nucleic acids attached thereto by the 5′ end,
- wherein the plurality of single-stranded nucleic acids at each amplification site comprises two populations, a first population comprising a template strand and a second population comprising the complement of the template strand,
- wherein one of the complements of the template strand at the amplification sites comprises at least one methylated CpG dinucleotide and the other complements of the template strand at the amplification sites do not comprise the at least one methylated CpG dinucleotide.
10-17. (canceled)
A method for producing an array comprising clonal clusters, the method comprising:
- providing an array comprising a plurality of amplification sites, each amplification site comprising one double-stranded nucleic acid immobilized thereto,
- wherein a first strand of the double-stranded nucleic acid is attached to the amplification site at its 5′ end,
- wherein a second strand of the double-stranded nucleic acid is a member of a sequencing library, the second strand is complementary to and annealed to a region of the first strand comprising the 3′ end of the first strand, and the second strand is not attached to the amplification site, and
- wherein the second strand of the double-stranded nucleic acid attached to at least one of the plurality of amplification sites comprises at least one methylated CpG dinucleotide and the first strand comprises a first unmethylated cytosine complementary to the guanine of the methylated CpG dinucleotide;
- and
- exposing the array to a template-dependent DNA methyltransferase enzyme to result in conversion of the first unmethylated cytosine to a second methylated cytosine, thereby converting the first complementary unmethylated CpG dinucleotide of the first strand to a second methylated CpG dinucleotide.
30-42. (canceled)
Dependencies are inferred from the claim text.