Hybridization immunoprecipitation sequencing (HIP-SEQ)
Inventors
McFarland, Kirsty A. • Magyar, Andrew P. • Pang, Ting • Springer, Michael
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Assignees
Charles Stark Draper Laboratory Inc • Harvard University
Member
DraperDraper is an independent nonprofit engineering innovation company with a legacy spanning over 90 years, dedicated to delivering transformative solutions for national security, prosperity, and global challenges. Renowned for its pioneering work in guidance, navigation, and control (GN&C) systems, Draper partners with government, industry, and academia to engineer advanced technologies in space, defense, biotechnology, and electronic systems. The company leverages multidisciplinary expertise, digital engineering, and a collaborative approach to provide field-ready prototypes, mission-critical systems, and innovative research. Draper’s mission is to ensure the nation's security and prosperity by delivering sustainable, cutting-edge solutions that address the toughest problems of today and tomorrow, while fostering an inclusive and diverse workforce. Draper also invests in the next generation of innovators through robust educational programs, including internships, co-ops, and the Draper Scholars Program, integrating academic research with real-world problem-solving.
Draper is an independent nonprofit engineering innovation company with a legacy spanning over 90 years, dedicated to delivering transformative solutions for national security, prosperity, and global challenges. Renowned for its pioneering work in guidance, navigation, and control (GN&C) systems, Draper partners with government, industry, and academia to engineer advanced technologies in space, defense, biotechnology, and electronic systems. The company leverages multidisciplinary expertise, digital engineering, and a collaborative approach to provide field-ready prototypes, mission-critical systems, and innovative research. Draper’s mission is to ensure the nation's security and prosperity by delivering sustainable, cutting-edge solutions that address the toughest problems of today and tomorrow, while fostering an inclusive and diverse workforce. Draper also invests in the next generation of innovators through robust educational programs, including internships, co-ops, and the Draper Scholars Program, integrating academic research with real-world problem-solving.
Publication Number
US-11802306-B2
Publication Date
2023-10-31
Expiration Date
Abstract
Methods of detecting unknown genetic modifications in a DNA sample from an organism are disclosed.
Core Innovation
Methods of detecting unknown genetic modifications in a DNA sample from an organism are disclosed. The present invention encompasses methods of identifying or detecting the presence of, or absence of, genetic modifications in a sample comprising nucleic acids, including insertions, deletions (indels) and single nucleotide polymorphisms (SNPs). The genetic modifications are not previously known and the sample can be obtained from prokaryotic or eukaryotic organisms or a virus.
The methods hybridize sample DNA with labelled capture probes generated from reference/cognate DNA to produce match duplex structures, mismatch duplex structures, and unhybridized ssDNA. One or more mismatch recognition proteins, including endonucleases, mismatch repair proteins, or DNA binding proteins, which can be genetically engineered and affinity-tagged, are used to specifically interact with mismatch duplex structures to form protein/mismatch duplex structure complexes that are recovered and from which mismatch DNA is released for sequencing. [procedural detail omitted for safety]
The invention addresses the problem that whole genome analysis by next generation sequencing can lose sequences associated with rare engineered organisms in a background of unmodified organisms and that selective capture and enrichment of sequences associated with unknown genetic modifications will dramatically reduce sequencing costs and enable identification of rare changes not currently detectable. The methods partition mismatched DNA from match duplexes and unhybridized DNA, and preserve unhybridized sample targets having no regions of identity with the reference for sequencing or further processing. These methods can identify unknown insertions, deletions and SNPs and have the potential to identify engineered genetic modification even from a complex mixture of organisms.
Claims Coverage
Overview: The coverage addresses inventive features extracted from two independent claims (1 and 21), totaling 14 inventive features.
Obtaining a DNA sample
obtaining a sample from the organism, wherein the sample comprises DNA
Hybridizing DNA with labelled nucleic acid probes
hybridizing the DNA in the sample with labelled nucleic acid probes complementary to cognate organism DNA under conditions suitable for the formation of a hybridization mixture comprising probe/sample DNA match duplex structures and/or mismatch duplex structures
Contacting with genetically-engineered non-catalytic endonuclease
contacting the mixture of hybridized probe/sample DNA comprising the match and mismatch duplex structures with one, or more mismatch recognition proteins comprising a genetically-engineered, non-catalytic endonuclease capable of specifically interacting with the mismatch duplex structures of the mixture under conditions suitable for the formation of a protein/mismatch duplex structure complex
Recovering protein/mismatch duplex complexes
recovering the protein/mismatch duplex structure complexes
Releasing mismatch duplex DNA from the protein
releasing the mismatch duplex structure DNA from the protein
Sequencing released DNA to determine modifications
sequencing the released DNA to determine if a genetic modification is present in the sample DNA that is not present in the cognate organism DNA
Enriching target DNA with genomic capture probes
contacting the sample with specific genomic capture probes under suitable conditions for the capture probes to hybridize with target DNA in the sample, thereby enriching the target DNA in the sample
Fragmenting enriched target DNA
fragmenting the enriched target DNA
Contacting fragmented target DNA with labelled cognate probes
contacting the fragmented target DNA with labelled, cognate probes generated from the reference genome of the organism of interest under conditions suitable for the formation of a hybridization mixture comprising target DNA/probe duplexes, wherein the duplexes comprise match duplex structures and/or mismatch duplex structures
Contacting with affinity-tagged mismatch recognition proteins
contacting the hybridization mixture with one, or more affinity-tagged mismatch recognition proteins comprising a genetically-engineered, non-catalytic endonuclease capable of specifically interacting with the mismatch duplex structures of the mixture under conditions suitable for the formation of a protein/mismatch duplex structure complex
Immunoprecipitating protein/mismatch duplex complexes with antibody
contacting the protein/mismatch duplex structure complexes with an antibody, or antibody fragment, that specifically binds to the affinity tag of the protein, thereby immunoprecipitating the protein/mismatch duplex complexes
Separating and denaturing labelled mismatch duplex structures
separating the mismatch duplex structure from the antibody-bound protein to obtain labelled mismatch duplex structures and denaturing the labelled mismatch duplex structures to obtain mismatch target DNA
Sequencing mismatch target DNA to detect genetic modifications
sequencing the mismatch target DNA to detect the presence or absence of a genetic modification
Partitioning supernatant to recover unhybridized DNA
recovering the supernatant from the immunoprecipitation step wherein the supernatant comprises labelled match duplex structures and unhybridized DNA, contacting the supernatant with streptavidin coated magnetic beads for labelled match duplex structures to bind to the beads, and recovering the unhybridized DNA for optional amplification and sequencing
The independent claims recite methods that hybridize sample DNA with labelled probes, use genetically-engineered non-catalytic endonucleases and other mismatch recognition proteins (optionally affinity-tagged) to form and recover protein/mismatch duplex structure complexes, and sequence released mismatch DNA to detect genetic modifications; claim 21 further recites enrichment of target DNA, fragmentation, immunoprecipitation with antibodies, separation/denaturation to obtain mismatch target DNA, and recovery of unhybridized DNA for sequencing.
Stated Advantages
Selectively capture and enrich sequences associated with unknown genetic modifications, thereby dramatically reducing the sequencing costs required to identify rare changes and enabling identification of genetic changes not currently detectable.
Identify unknown insertions, deletions and single nucleotide polymorphisms in a DNA sample from a known organism.
Potential to identify engineered genetic modification even from a complex mixture of organisms.
Partition mismatched DNA from matched duplexes and preserve unhybridized sample targets having no regions of identity with the reference, enabling sequencing or further processing of this DNA.
Permit successive testing on samples, allowing probing for additional genetic information.
Documented Applications
Identification of engineered organisms in the environment.
Identifying sequences associated with cancer or other diseases.
Identifying off-target effects of gene editing, including assessment of CRISPR-mediated genome changes and off-target CRISPR effects.
Identification of engineered genetic modification in a complex mixture of organisms and metagenomics enrichment of target genetic material (genomic fragment enrichment, GFE).
Analysis of DNA from cells obtained from humans or other organisms, including blood cells, epithelial cells, saliva, cerebral spinal fluid, sweat, feces or tears.
Interested in licensing this patent?