Microarray based multiplex pathogen analysis and uses thereof
Inventors
Hogan, Michael Edward • May, Melissa Rose • Eggers, Frederick Henry
Assignees
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Publication Number
US-11512307-B2
Publication Date
2022-11-29
Expiration Date
Abstract
Provided herein is a 3-dimensional lattice microarray system for DNA sequence detection and analysis. The system has a plurality of bifunctional polymer linkers, on one end of which are attached nucleic acid probes where each have a sequence complementary to signature nucleotide sequences in pathogens, plants or animals. The other end of the bifunctional polymer linker is attached to a solid support by non-covalent or covalent means. Each of the nucleic acid probes have terminal thymidine bases at the 5′ and 3′ ends that permit attachment of the probes to the bifunctional polymer linkers. Also provided is a customizable microarray kit is provided that contains the solid support, linkers, probes, solvent mixture and instructions to use the kit.
Core Innovation
The invention describes a microarray system for nucleic acid analysis based on a substantially flat borosilicate glass slide having a front surface functionalized with activated surface moieties. The front surface includes activated surface moieties selected from epoxysilane, N-hydroxysuccinimide, and an activated carboxylic acid ester attached to the front surface. A plurality of oligodeoxythymidine linkers are covalently coupled at their 3′ terminus via an amide bond to the activated surface moiety groups.
The system is configured such that there are more activated surface moieties on the slide front surface than the number of covalently coupled oligodeoxythymidine linkers. The activated surface moieties that are not covalently coupled create a lattice width spacing between the covalently coupled oligodeoxythymidine linkers. The oligodeoxythymidine linkers each consist of 20 to 60 thymidine bases and include a 5′ terminus covalently linked to a fluorescent label.
Nucleic acid probes are crosslinked to the oligodeoxythymidine linkers to form a three-dimensional lattice on the substantially flat borosilicate glass slide. Each probe includes pathogenic bacterial nucleotide probes selected from SEQ ID NOS: 37-85, pathogenic fungal nucleotide probes selected from SEQ ID NOS: 86-125 and 86-126, or a combination thereof. A thymidine nucleotide at both the 3′ and 5′ termini of each nucleic acid probe is crosslinked to two adjacent oligodeoxythymidine linkers, and the probe termini are separated by both a vertical space and the lattice width to create the 3-dimensional lattice.
Claims Coverage
Independent claim clm-00001 defines a microarray system with covalently coupled oligodeoxythymidine linkers on a functionalized borosilicate glass slide, leaving uncoupled activated moieties to set lattice width spacing, and enabling crosslinking of thymidine-flanked pathogenic bacterial and/or fungal nucleotide probes at both 3′ and 5′ termini to form a 3-dimensional lattice.
Functionalized borosilicate glass front surface with activated moieties
A substantially flat borosilicate glass slide whose front surface includes activated surface moieties selected from an epoxysilane group, an N-hydroxysuccinimide group, and an activated carboxylic acid ester attached to the front surface.
Covalently coupled oligodeoxythymidine linkers via 3′ amide bond
A plurality of oligodeoxythymidine linkers each covalently coupled at its 3′ terminus via an amide bond to one of the activated surface moiety groups, with a greater number of activated surface moieties attached than the total number of covalently coupled oligodeoxythymidine linkers.
Lattice width spacing defined by uncoupled activated surface moieties
Activated surface moieties that are not covalently coupled create a lattice width spacing between the covalently coupled oligodeoxythymidine linkers.
Oligodeoxythymidine linker length and fluorescent label at 5′ terminus
Each oligodeoxythymidine linker consists of 20 to 60 thymidine bases, with its 5′ terminus covalently linked to a fluorescent label.
Pathogenic bacterial and fungal nucleotide probes with thymidine-sandwich flanks
Nucleic acid probes selected from pathogenic bacterial nucleotide probes selected from SEQ ID NOS: 37-85, pathogenic fungal nucleotide probes selected from SEQ ID NOS: 86-125 and 86-126, or a combination thereof.
3′ and 5′ termini crosslinking to adjacent oligodeoxythymidine linkers
A thymidine nucleotide at the 3′ terminus and a thymidine nucleotide at the 5′ terminus of each nucleic acid probe is crosslinked to two adjacent oligodeoxythymidine linkers attached to the substantially flat borosilicate glass slide.
Three-dimensional lattice formation using vertical space and lattice width
Each nucleic acid probe crosslinked at the 3′ terminus and the 5′ terminus to two adjacent oligodeoxythymidine linkers are separated by both a vertical space and the lattice width, such that they form a 3-dimensional lattice on the substantially flat borosilicate glass slide.
The claim coverage centers on a 3D lattice microarray system where activated moieties on a borosilicate glass slide covalently attach oligodeoxythymidine linkers, leaving uncoupled moieties to define lattice width spacing, and where thymidine-flanked pathogenic bacterial and/or fungal nucleotide probes are crosslinked at both termini to adjacent linkers to form a 3-dimensional lattice.
Stated Advantages
Documented Applications
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