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-11542498-B2
Publication Date
2023-01-03
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 method for fabricating the microarray system by first attaching the bifunctional polymer linkers to the solid support, followed by photochemical coupling of the nucleic probes to the microarray. 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 relates to a microarray system that forms a 3-dimensional lattice on a substantially flat glass support. The front surface of the glass support includes activated surface moieties selected from an epoxysilane group, an N-hydroxy-succinimide group, 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 moieties.
The system is configured so that there are a greater number of activated surface moieties attached to the front surface than the total 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. Each oligodeoxythymidine linker consists of 20 to 60 thymidine bases with its 5′terminus covalently linked to a fluorescent label.
Nucleic acid probes are crosslinked to adjacent oligodeoxythymidine linkers to create the 3-dimensional lattice. Each probe includes a pathogenic fungal nucleotide sequence selected from SEQ ID NOS: 86-125 and/or a Cannabis plant nucleotide sequence selected from SEQ ID NOS: 126-128, with each nucleotide sequence sandwiched between three to seven consecutive thymidine nucleotides attached at both the 3′terminus and the 5′terminus. 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, and each crosslinked probe is separated by both a vertical space and the lattice width to form the 3-dimensional lattice.
Claims Coverage
The partial content provides one independent claim (clm-00001). Its coverage includes a microarray system architecture based on an activated glass surface with covalently attached oligodeoxythymidine linkers carrying fluorescent labels, and nucleic acid probes that are crosslinked to adjacent linkers to form a 3-dimensional lattice with both vertical space and lattice width spacing. The independent claim includes three inventive features.
Activated glass front surface with spacer lattice width created by uncoupled moieties
A substantially flat glass support with a front surface comprising activated surface moieties selected from an epoxysilane group, an N-hydroxy-succinimide group, and an activated carboxylic acid ester attached to the front surface, wherein the number of activated surface moieties is greater than the total number of covalently coupled oligodeoxythymidine linkers and the uncoupled activated surface moieties create a lattice width spacing.
Covalently coupled 3′-terminated oligodeoxythymidine linkers carrying fluorescent labels
A plurality of oligodeoxythymidine linkers each covalently coupled at its 3′terminus via an amide bond to one of the activated surface moieties, where each linker consists of 20 to 60 thymidine bases and has its 5′terminus covalently linked to a fluorescent label.
Crosslinking nucleic acid probes between thymidine ends of adjacent linkers to form a 3-dimensional lattice
A plurality of nucleic acid probes selected from pathogenic fungal nucleotide probes selected from SEQ ID NOS: 86-125, Cannabis plant nucleotide probes selected from SEQ ID NOS: 126-128, or a combination thereof; wherein each probe comprises a pathogenic fungal nucleotide sequence or a Cannabis plant nucleotide sequence sandwiched between three to seven consecutive thymidine nucleotides attached at both the 3′terminus and the 5′terminus, and a thymidine nucleotide at each of the 3′ and 5′ termini of the probe is crosslinked to two adjacent oligodeoxythymidine linkers such that each crosslinked probe is separated by both a vertical space and the lattice width to form a 3-dimensional lattice.
Across clm-00001, the main coverage is directed to a microarray system that uses activated moieties on a substantially flat glass support to covalently anchor oligodeoxythymidine linkers, uses uncoupled activated moieties to create lattice width spacing, and crosslinks thymidine-ended nucleic acid probes between adjacent linkers so that probes are separated by vertical space and lattice width to form a 3-dimensional lattice.
Stated Advantages
Documented Applications
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