Methods of preparing nucleic acids for sequencing
Inventors
Iafrate, Anthony John • Le, Long Phi • Zheng, Zongli • Myers, Jason • Stahl, Joshua
Assignees
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Publication Number
US-12371732-B2
Publication Date
2025-07-29
Expiration Date
Abstract
Aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.
Core Innovation
The invention relates to a method of determining a nucleotide sequence contiguous to a known target nucleotide sequence. A target nucleic acid molecule comprising the known target nucleotide sequence and the nucleotide sequence contiguous to it is contacted with a first target primer under hybridization conditions, followed by a template-dependent extension reaction primed by the first target primer.
The extension product is contacted with a population of tailed random primers under hybridization conditions. Each tailed random primer comprises a common sequence together with 6 to 12 random nucleotides in a 5′ to 3′ nucleic acid sequence, and the extension product is further extended by at least one tailed random primer that hybridizes to a portion of the extension product. The resulting extension product is amplified using a second target primer and a first tail primer comprising a 3′ sequence that specifically anneals to a complement of the common sequence.
The nucleotide sequence contiguous to the known target nucleotide sequence is determined by sequencing an amplicon using a first and/or second sequencing primer. The document additionally describes nested relationships between target primers, removal of unhybridized primers, constraints on the tailed random primers and common sequence relative to sequencing primers, and implementations including barcode sequences within the tailed random primers and applications involving gene rearrangements and fusion mRNA, including oncogene fusion contexts.
Claims Coverage
The independent claim recites a method with six main inventive features: target-primed extension into unknown contiguous sequence, priming with a population of tailed random primers containing a common sequence and random nucleotides, second extension, amplification using a second target primer plus a tail primer that anneals to a complement of the common sequence, and sequencing of the resulting amplicon using first and/or second sequencing primers.
Determining nucleotide sequence contiguous to a known target
A method of determining a nucleotide sequence contiguous to a known target nucleotide sequence by contacting a target nucleic acid molecule with a first target primer under hybridization conditions, then progressing through extension, amplification, and sequencing of the resulting amplicon.
First target-primed extension from known target
A template-dependent extension reaction primed by a first target primer that hybridizes to the known target nucleotide sequence, producing an extension product for further priming.
Population of tailed random primers with common sequence and 6 to 12 random nucleotides
Contacting the extension product with a population of tailed random primers under hybridization conditions, wherein each tailed random primer comprises a common sequence and a 5′ to 3′ nucleic acid sequence of 6 to 12 random nucleotides.
Tailed-random-primed extension of the extension product
Performing a template-dependent extension reaction primed by at least one of the tailed random primers that hybridizes to a portion of the extension product of the first extension step.
Amplification using second target primer and first tail primer complementary to the common sequence
Amplifying the extension product using a second target primer and a first tail primer comprising a 3′ sequence that specifically anneals to a complement of the common sequence.
Sequencing the amplicon with first and/or second sequencing primers
Determining the nucleotide sequence contiguous to the known target nucleotide sequence by sequencing an amplicon using a first and/or second sequencing primer.
Nested target primer relationship
The second target primer is nested with respect to the first target primer.
Removal of unhybridized primers after extension/amplification
Unhybridized primers are removed from a reaction after an extension or amplification reaction.
Gene rearrangement target nucleic acid context
The target nucleic acid molecule comprises a gene rearrangement.
Unique barcode per tailed random primer in the population
Each tailed random primer in the population comprises a unique barcode sequence.
Tailed random primer common sequence identical to a sequencing primer
The common sequence is identical to the first or second sequencing primer.
Single-stranded oligonucleotide tailed random primers
The tailed random primers comprise single-stranded oligonucleotide molecules.
Across the independent claim and its refinements, coverage centers on determining unknown nucleotide sequence contiguous to a known target by sequentially extending from a known-target-specific primer, re-priming with a population of tailed random primers containing a common sequence and 6 to 12 random nucleotides, amplifying using a second target primer together with a tail primer complementary to the common sequence, and determining the contiguous sequence by sequencing the resulting amplicon with first and/or second sequencing primers. Dependent claims further constrain relationships between primers, add optional removal of unhybridized primers, specify target context as gene rearrangement, and include barcoded tailed random primers and common-sequence identity to sequencing primer sequences.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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