Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
Inventors
Iafrate, Anthony John • Le, Long Phi • Zheng, Zongli
Assignees
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Publication Number
US-9487828-B2
Publication Date
2016-11-08
Expiration Date
Abstract
The technology described herein is directed to methods of determining oligonucleotide sequences, e.g. by enriching target sequences prior to sequencing the sequences.
Core Innovation
The invention provides a method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides. The method ligates a universal oligonucleotide tail-adaptor to a nucleic acid comprising the known target nucleotide sequence to produce a ligation product. The adaptor is described as universally ligatable to the target-containing nucleic acid and as providing adaptor sequence regions for downstream amplification and sequencing.
After ligation, the ligation product is amplified by polymerase chain reaction using adaptor primers and target-specific primers. The method includes amplifying the ligation product using a first adaptor primer and a first target-specific primer, followed by amplifying the amplification product by polymerase chain reaction using a second adaptor primer and a second target-specific primer. 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, to generate a sequencing-ready amplification product.
The amplification product is sequenced using sequencing primers complementary to opposite strands of the amplification product. The adaptor is described in embodiments including a universal oligonucleotide tail-adaptor and a truncated Y-shaped universal oligonucleotide tail-adaptor. The workflow determines the contiguous sequence adjacent to a known target sequence using nested PCR together with sequencing primers complementary to opposite strands.
Claims Coverage
The provided content includes two independent claims. Both define the same core nested-PCR sequencing workflow for determining contiguous sequence adjacent to a known target sequence, with the adaptor form differing between a universal oligonucleotide tail-adaptor and a truncated Y-shaped universal oligonucleotide tail-adaptor.
Universal tail-adaptor nested-PCR and sequencing to determine contiguous target sequence
A method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides by ligating a universal oligonucleotide tail-adaptor to a nucleic acid comprising the known target nucleotide sequence, amplifying the ligation product by polymerase chain reaction using a first adaptor primer and a first target-specific primer, amplifying an amplification product by polymerase chain reaction using a second adaptor primer and a second target-specific primer that are nested relative to the first adaptor primer and the first target-specific primer, and sequencing the resulting amplification product using sequencing primers complementary to opposite strands of the amplification product.
Truncated Y-shaped universal adaptor nested-PCR and sequencing to determine contiguous target sequence
A method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides by ligating a truncated Y-shaped universal oligonucleotide tail-adaptor to a nucleic acid comprising the known target nucleotide sequence, amplifying the ligation product by polymerase chain reaction using a first adaptor primer annealing to the truncated Y-shaped universal oligonucleotide tail-adaptor and a first target-specific primer, amplifying an amplification product by polymerase chain reaction using a second adaptor primer and a second target-specific primer nested relative to the first adaptor primer and the first target-specific primer, and sequencing the resulting amplification product using sequencing primers complementary to opposite strands.
The inventive concept centers on ligating a universal adaptor to the target-containing nucleic acid, using nested PCR with nested adaptor and target-specific primers to generate an amplification product, and sequencing using primers complementary to opposite strands of that product. The independent claims differ in the adaptor form, including a truncated Y-shaped universal oligonucleotide tail-adaptor in one claim.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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