Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
First Claim
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1. A method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides, the method comprising:
- (i) ligating a universal oligonucleotide tail-adaptor to a nucleic acid comprising the known target nucleotide sequence to produce a ligation product;
(ii) amplifying the ligation product by polymerase chain reaction using a first adaptor primer that specifically anneals to the universal oligonucleotide tail-adaptor, and a first target-specific primer that specifically anneals to the known target nucleotide sequence;
(iii) amplifying an amplification product of (ii) by polymerase chain reaction using a second adaptor primer and a second target-specific primer, wherein the second adaptor primer and the second target-specific primer are nested relative to the first adaptor primer and the first target-specific primer, respectively; and
(iv) sequencing an amplification product of (iii) using a first sequencing primer and a second sequencing primer, wherein the first sequencing primer and the second sequencing primer are complementary to opposite strands of the amplification product of (iii).
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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.
24 Citations
47 Claims
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1. A method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides, the method comprising:
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(i) ligating a universal oligonucleotide tail-adaptor to a nucleic acid comprising the known target nucleotide sequence to produce a ligation product; (ii) amplifying the ligation product by polymerase chain reaction using a first adaptor primer that specifically anneals to the universal oligonucleotide tail-adaptor, and a first target-specific primer that specifically anneals to the known target nucleotide sequence; (iii) amplifying an amplification product of (ii) by polymerase chain reaction using a second adaptor primer and a second target-specific primer, wherein the second adaptor primer and the second target-specific primer are nested relative to the first adaptor primer and the first target-specific primer, respectively; and (iv) sequencing an amplification product of (iii) using a first sequencing primer and a second sequencing primer, wherein the first sequencing primer and the second sequencing primer are complementary to opposite strands of the amplification product of (iii). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides, the method comprising:
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(i) ligating a truncated Y-shaped universal oligonucleotide tail-adaptor to a nucleic acid comprising the known target nucleotide sequence to produce a ligation product; (ii) amplifying the ligation product by polymerase chain reaction using a first adaptor primer that specifically anneals to the truncated Y-shaped universal oligonucleotide tail-adaptor, and a first target-specific primer that specifically anneals to the known target nucleotide sequence; (iii) amplifying an amplification product of (ii) by polymerase chain reaction using a second adaptor primer and a second target-specific primer, wherein the second adaptor primer and the second target-specific primer are nested relative to the first adaptor primer and the first target-specific primer, respectively; and (iv) sequencing an amplification product of (iii) using a first sequencing primer and a second sequencing primer, wherein the first sequencing primer and the second sequencing primer are complementary to opposite strands of the amplification product of (iii). - View Dependent Claims (41, 42, 43, 44, 45, 46, 47)
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Specification