Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
DC CAFCFirst Claim
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1. 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.
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Abstract
The technology described herein is directed to methods of determining oligonucleotide sequences, e.g. by enriching target sequences prior to sequencing the sequences.
47 Citations
20 Claims
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1. A method for preparing a nucleic acid for sequencing, the method comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for preparing a nucleic acid for sequencing, the method comprising:
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(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. - View Dependent Claims (17, 18, 19, 20)
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Specification