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US 2024/0076727 A1Published application
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
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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.
Claims17
A method for preparing a nucleic acid for sequencing, the method comprising:
- (a) amplifying a single strand of a nucleic acid template comprising a known target nucleotide sequence, a first nucleotide sequence contiguous to the known target nucleotide sequence, and a 5′ portion comprising an amplification strand of a ligated universal oligonucleotide tail-adaptor, using (i) a first primer that anneals to a 3′ portion of the nucleic acid template comprising the known target nucleotide sequence and (ii) a second primer that comprises a nucleotide sequence identical to a portion of the amplification strand of the ligated universal oligonucleotide tail-adaptor, thereby generating a first amplification product comprising the first nucleotide sequence and the known target nucleotide sequence;
- (b) amplifying the first amplification product of (a) using
- (i) a third primer that comprises a nucleotide sequence identical to a portion of the first primer and a nucleotide sequence that is identical to a portion of a first sequencing primer, and
- (ii) a fourth primer that comprises a 3′ nucleotide sequence identical to a portion of the second primer and a 5′ portion comprising a nucleic acid sequence that is identical to a portion of a second sequencing primer,
- thereby generating a second amplification product.
2 more elements
A method for preparing a nucleic acid for sequencing, the method comprising:
- (a) amplifying a single strand of a nucleic acid template comprising a known target nucleotide sequence, a first nucleotide sequence contiguous to the known target nucleotide sequence, and a 5′ portion comprising an amplification strand of a ligated universal oligonucleotide tail-adaptor, using (i) a target-specific primer that anneals to the 3′ portion of the nucleic acid template comprising the known target nucleotide sequence and (ii) a first adaptor primer that comprises a nucleotide sequence identical to a portion of the amplification strand of the ligated universal oligonucleotide tail-adaptor, thereby generating a first amplification product comprising the first nucleotide sequence and the known target nucleotide sequence;
- (b) amplifying the first amplification product of (a) using
- (i) a second target-specific primer that comprises a nucleotide sequence identical to a portion of the first primer and anneals to the first amplification product of (a), and
- (ii) a second adaptor primer that comprises a 3′ nucleotide sequence identical to a portion of the first adaptor primer,
- thereby generating a second amplification product.
2 more elements
Dependencies are inferred from the claim text.