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US 11,781,179 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
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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.
Claims14
A method for determining a nucleotide sequence, the method comprising:
- (a) amplifying a nucleic acid template comprising a 3′-end comprising a known target nucleotide sequence, a first nucleotide sequence contiguous to the known target nucleotide sequence, and a 5-end comprising an amplification strand of a ligated universal oligonucleotide tail-adaptor, using (i) a first primer that comprises a nucleotide sequence identical to a 5′ portion of a first sequencing primer and (ii) a second primer that anneals to the 3-end of the nucleic acid template comprising the known target nucleotide sequence, 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 sequencing primer and is nested relative to the first primer and (ii) a fourth primer that comprises a 5′ portion comprising a nucleic acid sequence that is identical to a second sequencing primer, anneals to a 3′ portion of the first amplification product comprising the known target nucleotide sequence, and is nested relative to the second primer, thereby generating a second amplification product;
- and
- (c) determining the sequence of the first nucleotide sequence in the second amplification product.
1 more element
A method for determining a nucleotide sequence, the method comprising:
- (a) ligating an amplification strand of a universal oligonucleotide tail-adaptor to a 5′-end of a nucleic acid template comprising (i) a 3-end comprising a known target nucleotide sequence and (ii) a first nucleotide sequence contiguous to the known target nucleotide sequence;
- (b) amplifying the nucleic acid template using (i) a first primer that comprises a nucleotide sequence identical to a 5′ portion of a first sequencing primer and (ii) a second primer that anneals to the 3′-end of the nucleic acid template comprising the known target nucleotide sequence, thereby generating a first amplification product comprising the first nucleotide sequence and the known target nucleotide sequence;
- (c) amplifying the first amplification product of (b) using (i) a third primer that comprises a nucleotide sequence identical to a portion of the first sequencing primer and that is nested relative to the first primer and (ii) a fourth primer that comprises a 5′ portion comprising a nucleic acid sequence that is identical to a second sequencing primer, anneals to a 3′ portion of the first amplification product comprising the known target nucleotide sequence and is nested relative to the second primer, thereby generating a second amplification product comprising the first nucleotide sequence;
- and
- (d) determining the sequence of the first nucleotide sequence in the second amplification product by a process comprising next-generation sequencing.
2 more elements
Dependencies are inferred from the claim text.