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US 10,017,810 B2Granted
Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
AssigneeThe General Hospital Corporation
InventorsAnthony John Iafrate, Long Phi Le, Zongli Zheng
Life of this patent
Estimating the term…
Abstract
The technology described herein is directed to methods of determining oligonucleotide sequences, e.g. by enriching target sequences prior to sequencing the sequences.
Claims20
A method for preparing a nucleic acid for sequencing, the method comprising:
- (i) ligating a universal oligonucleotide tail adaptor that comprises a first ligatable duplex end and a second unpaired end to a nucleic acid comprising a known target nucleotide sequence to produce a ligation product, the universal oligonucleotide tail adaptor comprising an amplification strand and a blocking strand, wherein a 3′ duplex portion of the amplification strand and a 5′ duplex portion of the blocking strand are substantially complementary and form the first ligatable duplex end;
- (ii) amplifying the ligation product using a first target-specific primer that specifically anneals to the known target nucleotide sequence and a first adaptor primer having a nucleotide sequence identical to a first portion of the amplification strand;
- and
- (iii) amplifying an amplification product of (ii) using a second target-specific primer that specifically anneals to the amplification product of (ii) and a second adaptor primer having a nucleotide sequence identical to a second portion of the amplification strand, wherein the second target-specific primer is nested relative to the first target-specific primer.
1 more element
A method for preparing a nucleic acid for sequencing, the method comprising:
- (i) ligating a universal oligonucleotide tail adaptor that comprises a first ligatable duplex end and a second unpaired end to a nucleic acid comprising a known target nucleotide sequence to produce a ligation product, the universal oligonucleotide tail adaptor comprising an amplification strand and a blocking strand, wherein a 3′ duplex portion of the amplification strand and a 5′ duplex portion of the blocking strand are substantially complementary and form the first ligatable duplex end;
- (ii) amplifying the ligation product using a first target-specific primer that specifically anneals to the known target nucleotide sequence and a first adaptor primer having a nucleotide sequence identical to a first portion of the amplification strand;
- and
- (iii) amplifying an amplification product of (ii) using a second target-specific primer that specifically anneals to the amplification product of (ii) and a second adaptor primer having a nucleotide sequence identical to a second portion of the amplification strand, wherein ligating in step (i) comprises performing an overhang ligation reaction, and wherein the universal oligonucleotide tail adaptor further comprises a barcode portion.
1 more element
Dependencies are inferred from the claim text.