TARGETED SEQUENCING LIBRARY PREPARATION BY GENOMIC DNA CIRCULARIZATION
First Claim
1. A method of sequencing comprising:
- a) digesting a sample comprising genomic DNA using a restriction enzyme to produce a digested sample;
b) producing a circular nucleic acid comprising i. a splint oligonucleotide, ii. a vector oligonucleotide comprises a binding site for a first sequencing primer iii. a target genomic fragment, and iv. a duplex region in which the 5′
end of said vector oligonucleotide is ligatably adjacent to the 3′
end of the target genomic fragment, and the 3′
end of said vector oligonucleotide is ligatably adjacent to the 5′
end of said target genomic fragment by;
contacting, under hybridization conditions, said digested sample with;
i. said vector oligonucleotide; and
ii. said splint oligonucleotide, wherein said splint oligonucleotide comprises;
a central region that hybridizes to the entirety of said vector oligonucleotide;
a 5′
region that hybridizes to a first region in a target genomic fragment in said digested sample, anda 3′
region that hybridizes to a second region in said target genomic fragment;
and, optionally enzymatic treatment remove any 5′
overhang from said target genomic fragment to make the 3′
end of said vector oligonucleotide ligatably adjacent to the 5′
end of said target genomic fragment;
c) contacting said circular nucleic acid with a ligase, thereby ligating the 5′
end of said vector oligonucleotide to the 3′
end of the target genomic fragment and ligating the 3′
end of said vector oligonucleotide to the 5′
end of the target genomic fragment to produce a circular DNA molecule;
d) separating said circular DNA molecule from said splint oligonucleotide; and
e) sequencing the target genomic fragment of said circular DNA molecule using said first sequencing primer.
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Accused Products
Abstract
Certain embodiments provide a method of sequencing that comprises: a) contacting, under hybridization conditions, a target genomic fragment with: i. a vector oligonucleotide comprising a binding site for a sequencing primer; and ii. a splint oligonucleotide that hybridizes to the vector oligonucleotide and to the nucleotide sequences at the ends of a target genomic fragment, to produce a circular nucleic acid; b) contacting the circular nucleic acid with a ligase, thereby ligating the ends of the vector oligonucleotide to the ends of the target genomic fragment to produce a circular DNA molecule; c) separating the circular DNA molecule from the splint oligonucleotide; and d) sequencing the target genomic fragment of the circular DNA molecule using the first sequencing primer.
93 Citations
20 Claims
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1. A method of sequencing comprising:
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a) digesting a sample comprising genomic DNA using a restriction enzyme to produce a digested sample; b) producing a circular nucleic acid comprising i. a splint oligonucleotide, ii. a vector oligonucleotide comprises a binding site for a first sequencing primer iii. a target genomic fragment, and iv. a duplex region in which the 5′
end of said vector oligonucleotide is ligatably adjacent to the 3′
end of the target genomic fragment, and the 3′
end of said vector oligonucleotide is ligatably adjacent to the 5′
end of said target genomic fragment by;contacting, under hybridization conditions, said digested sample with; i. said vector oligonucleotide; and ii. said splint oligonucleotide, wherein said splint oligonucleotide comprises; a central region that hybridizes to the entirety of said vector oligonucleotide; a 5′
region that hybridizes to a first region in a target genomic fragment in said digested sample, anda 3′
region that hybridizes to a second region in said target genomic fragment;and, optionally enzymatic treatment remove any 5′
overhang from said target genomic fragment to make the 3′
end of said vector oligonucleotide ligatably adjacent to the 5′
end of said target genomic fragment;c) contacting said circular nucleic acid with a ligase, thereby ligating the 5′
end of said vector oligonucleotide to the 3′
end of the target genomic fragment and ligating the 3′
end of said vector oligonucleotide to the 5′
end of the target genomic fragment to produce a circular DNA molecule;d) separating said circular DNA molecule from said splint oligonucleotide; and e) sequencing the target genomic fragment of said circular DNA molecule using said first sequencing primer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A kit comprising:
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i. a vector oligonucleotide comprising a first binding site for a sequencing primer and a second binding site for a second sequencing primer; and ii. a splint oligonucleotide that hybridizes to said the vector oligonucleotide and to the nucleotide sequences at the ends of a plurality of restriction fragments in a mammalian genome, wherein said vector and splint oligonucleotides are characterized in that, when hybridized with said restriction fragment, they produce a circular nucleic acid comprising a duplex region in which at least the 5′
end of said vector oligonucleotide is ligatably adjacent to the 3′
end of the genomic fragment. - View Dependent Claims (19, 20)
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Specification