Methods of nucleic acid sample preparation
Inventors
Stahl, Joshua • Myers, Jason • Culver, Brady • Kudlow, Brian
Assignees
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Publication Number
US-10704082-B2
Publication Date
2020-07-07
Expiration Date
Abstract
Aspects of the technology disclosed herein relate to methods of 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 provides methods of preparing nucleic acids for analysis by adding one or more nucleotides to a 3′ end of a double-stranded nucleic acid comprising a target nucleotide sequence, wherein at least one nucleotide is a capture moiety modified nucleotide. An adapter nucleic acid is ligated to the double-stranded nucleic acid so that a sequence at a 3′ end of the adapter nucleic acid is complementary to the nucleotides added to the 3′ end, and the ligation product is captured by contacting the ligation product with a binding partner of the capture moiety modified nucleotide.
The ligation product is amplified by polymerase chain reaction using a first target-specific primer that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid. Additional amplification can include a second adapter primer and a second target-specific primer, where the second target-specific primer is nested relative to the first target-specific primer.
In additional embodiments, cDNA is prepared from RNA using a randomly-primed first strand synthesis reaction and a second strand synthesis reaction using the product of the randomly-primed first strand synthesis reaction as a template. The cDNA is end repaired to produce a blunt-ended, double-stranded nucleic acid comprising the target nucleotide sequence, then tailed at the 3′ end with one or more nucleotides including at least one capture moiety modified nucleotide before ligation of an adapter nucleic acid with a ligatable duplex portion and an overhang sequence complementary to the added nucleotides.
Claims Coverage
The independent claim set includes two independent claims and centers on capture-moiety modified 3′ ends, complementary adapter ligation, capture with a binding partner, and PCR amplification using target-specific and adapter primers, including nested target-specific primers.
Capture-moiety modified 3′ end ligation and capture
Adding one or more nucleotides to a 3′ end of a double-stranded nucleic acid comprising a target nucleotide sequence, wherein at least one nucleotide is a capture moiety modified nucleotide; ligating an adapter nucleic acid so a sequence at a 3′ end of the adapter nucleic acid is complementary to the added nucleotides; capturing the ligation product by contacting it with a binding partner of the capture moiety modified nucleotide.
PCR amplification using target-specific and adapter primers
Amplifying the ligation product by polymerase chain reaction using a first target-specific primer that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid.
Nested second amplification round
Further amplifying an amplification product using a second adapter primer and a second target-specific primer, where the second target-specific primer is nested relative to the first target-specific primer.
Randomly-primed cDNA generation with end repair
Preparing cDNA from RNA by a randomly-primed first strand synthesis reaction and a second strand synthesis reaction, then end repairing the cDNA to produce a blunt-ended, double-stranded nucleic acid comprising the target nucleotide sequence.
The inventive features combine formation of a capture-moiety modified 3′ end, ligation of a complementary adapter and capture of the ligation product, and PCR amplification with target-specific and adapter primers, including an embodiment with nested amplification.
Stated Advantages
The workflow is positioned to reduce unligated adapter carryover and related false positives associated with barcode inflation.
Documented Applications
Preparation of nucleic acids for analysis.
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