Methods of nucleic acid sample preparation for immune repertoire sequencing
Inventors
Myers, Jason • Stahl, Joshua • Culver, Brady • Kudlow, Brian • Eberlein, Jens
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10947582-B2
Publication Date
2021-03-16
Expiration Date
Abstract
Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids, e.g., nucleic acids encoding immune receptors and immunoglobulins. 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 preparing nucleic acids for analysis by a capture-enrichment workflow for target nucleotide sequences using a capture moiety modified primer. A nucleic acid molecule comprising a target nucleotide sequence is contacted with a capture moiety modified primer that specifically anneals to the target nucleotide sequence under hybridization conditions, and a first strand synthesis reaction is conducted that is primed by the hybridized capture moiety modified primer and uses the nucleic acid molecule as a template.
A second strand synthesis reaction is conducted using a product of the first strand synthesis reaction as a template to generate a double-stranded nucleic acid comprising a capture moiety. An adapter nucleic acid is ligated to the double-stranded nucleic acid to produce a ligation product comprising the capture moiety, and the ligation product is captured by contacting it with a binding partner of the capture moiety.
The captured ligation product is amplified by polymerase chain reaction using a target-specific primer and adapter primer sequences. The target-specific primer comprises a 3′ portion 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, and the target-specific primer further comprises a 5′ tail portion that does not specifically anneal to the target nucleotide sequence.
Claims Coverage
The patent includes one independent claim that defines a full ligation-based capture/enrichment method followed by PCR amplification with a target-specific primer having a non-annealing 5′ tail, for analysis of nucleic acids.
Capture-moiety primed hybridization, strand synthesis, and capture of ligation product
Contacting a nucleic acid molecule comprising a target nucleotide sequence with a capture moiety modified primer that specifically anneals under hybridization conditions; performing first strand synthesis primed by the hybridized capture moiety modified primer to generate a product; conducting second strand synthesis to generate a double-stranded nucleic acid comprising a capture moiety; ligating an adapter nucleic acid to produce a ligation product comprising the capture moiety; and capturing the ligation product by contacting it with a binding partner of the capture moiety.
PCR amplification using target-specific primer with non-annealing 5′ tail and adapter primer
Amplifying the captured ligation product by polymerase chain reaction using a target-specific primer that comprises a 3′ portion specifically annealing to the target nucleotide sequence and a first adapter primer specifically annealing to a complementary sequence of the adapter nucleic acid, wherein the target-specific primer comprises a 5′ tail portion that does not specifically anneal to the target nucleotide sequence.
Claim coverage centers on capturing an adapter-ligated double-stranded nucleic acid via a capture moiety modified primer and binding partner scheme, followed by PCR amplification using a target-specific primer that includes a non-annealing 5′ tail in addition to adapter-compatible annealing elements.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Immune repertoire sequencing contexts involving T cell receptor (TCR) and B cell receptor (BCR) analysis are discussed.
Gene rearrangement-related cancer therapy implications are described in the provided partial content.
Interested in licensing this patent?