By a News Reporter-Staff News Editor at Life Science Weekly — From Washington, D.C., NewsRx journalists report that a patent application by the inventors Svoboda, Michal (Muttenz, CH); Svoboda, Xenia (Muttenz, CH), filed on December 20, 2013, was made available online on July 3, 2014 (see also Hutman Diagnostics AG).
The patent’s assignee is Hutman Diagnostics AG.
News editors obtained the following quote from the background information supplied by the inventors: “Microarrays are a useful tool for analysing the gene expression, genetic mutations, and detecting pathogens. Such microarrays are commonly prepared as square arrays of spots, with each spot containing nucleic acid probes or antibodies that are able to bind to a specific target. Binding of the target to a spot on the array is detected using a detectable label that is attached to the target either before or after contacting the target with the microarray.
“For example, nucleic acid microarrays are generally formed as a square array of spots, each containing a nucleic acid probe having a complementary sequence to a target of interest. Such arrays are contacted with a solution containing detectably labelled target nucleic acids. The target nucleic acid will become immobilized on a particular spot if it contains a probe with a complementary nucleic acid sequence. After washing away non-immobilized nucleic acids, a suitable reader can be used to detect the presence of the immobilized nucleic acids using the detectable label. For example, a fluorescence reader can be used to detect spots on which fluorescently labelled targets are immobilized. Alternatively, targets labelled with certain enzymes can be contacted with a chromogenic substrate and the resulting coloration change can be read as an absorbance value by a suitable device.
“Low density DNA, protein or mixed DNA/protein microarrays are useful for the simultaneous detection of multiple pathogens. The spots on such an array are usually between 50 and 150 micrometres in diameter, and therefore clearly visible with minimal magnification. In a typical application, the fluorescence, (chemi)luminescence or absorbance signals of the positive array spots are read by a suitable reader, and the resulting data is interpreted with a computer program. When a specific pathogen is present in the sample, usually only a few spots are fluorescing, and no spots may fluoresce with a negative sample. Minor defects in the array, like dried droplets of liquid, dust particles or haze, which would not prevent a human from determining whether a spot is positive or negative can render the array unintelligible for a machine. This aspect of computer-aided pathogen detection creates a requirement for high quality arrays, further demanding a great deal of care to be taken with array handling and reading.
“An additional risk associated with the use of microarrays is that the image of the microarray is inadvertently rotated and/or flipped, thereby producing erroneous results. With high-density genomic arrays, the array holder and dedicated instrumentation are often specially designed such that the array only fits into the instrument in a single orientation in order to safeguard against the array misreading. However, no such safeguard exists for low-density arrays read with generic laboratory equipment.
“Thus, there exists a great need for improved microarrays that reduce the risk of array misalignment and/or facilitate the visual interpretation of an array image by a human operator.”
As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors’ summary information for this patent application: “Provided herein are microarrays (protein and/or nucleic acid microarrays) containing an array of spots on a solid substrate, wherein the spots are arranged to reduce the risk of array misalignment and/or to facilitate the visual interpretation of an array image by a human operator. Also provided herein are methods of using such arrays and kits containing such arrays.
“In certain embodiments, described herein are nucleic acid and/or protein microarrays containing an array of spots on a solid substrate (e.g., a rectangular grid of spots such as a square grid of spots). In some embodiments the array of spots includes a plurality of pathogen-specific spots. Such pathogen-specific spot can contain a pathogen-specific nucleic acid probe or antibody immobilized on the solid substrate. In some embodiments the array of spots includes one or more always-detectable spots containing a detectable substance immobilized to the solid substrate (e.g., an always-fluorescing spot containing a fluorescent dye immobilized on the solid substrate). In some embodiments the array of spots includes one or more never-detectable spots. Such spots, for example, may be empty positions in the array or they may be spotted with spotting buffer that does not contain a detectable substance, a nucleic acid probe or an antibody (e.g., never-fluorescing spots containing neither a fluorescent dye nor a nucleic acid probe immobilized to the solid substrate). In some embodiments the array of spots also includes one or more positive-control spots containing, for example, a nucleic acid probe having a sequence complementary to a positive control nucleic acid (e.g., a conserved eubacterial 16S rRNA sequence).
“In certain embodiments, the one or more always-detectable spots and the one or more never-detectable spots are positioned such that the array of spots has neither rotational symmetry nor mirror symmetry. For example, in some embodiments the position of one or more always-detectable spots and one or more never-detectable spots are such that rotation of the microarray by 90 degrees, 180 degrees or 270 degrees results in at least one always-detectable spot being in a position occupied by a never-detectable spot in an un-rotated array. In another embodiment, the position of one or more always-detectable spots and one or more never-detectable spots are such that flipping the microarray on its horizontal or vertical axis results in at least one always-detectable spot being in a position occupied by a never-detectable spot in an un-rotated array. In some embodiments the position of one or more always-detectable spots and one or more never-detectable spots are such that rotation of the microarray by 90 degrees, 180 degrees or 270 degrees and flipping the microarray on its horizontal or vertical axis results in at least one always-detectable spot being in a position occupied by a never-detectable spot in an un-rotated array. In certain embodiments the position of one or more always-detectable spots and one or more never-detectable spots are such that rotation of the microarray by 90 degrees, 180 degrees or 270 degrees results in at least one never-detectable spot being in a position occupied by an always-detectable spot in an un-rotated array. In some embodiments the position of one or more always-detectable spots and one or more never-detectable spots are such that flipping the microarray on its horizontal or vertical axis results in at least one never-detectable spot being in a position occupied by an always-detectable spot in an un-rotated array. In certain embodiments the position of one or more always-detectable spots and one or more never-detectable spots are such that rotation of the microarray by 90 degrees, 180 degrees or 270 degrees and flipping the microarray on its horizontal or vertical axis results in at least one never-detectable spot being in a position occupied by an always-detectable spot in an un-rotated array.
“In some embodiments, the microarray is a rectangular grid of spots that is made up of multiple of sub-arrays of spots. In some embodiments the distance between adjacent sub-arrays is different than the distance between adjacent spots within the sub-arrays. For example, in some embodiments the distance between adjacent sub-arrays is greater than the distance between adjacent spots within the sub-arrays. In some embodiments the distance between adjacent sub-arrays is about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3 times, 4 times or 5 times the distance between spots within the sub-arrays. In some embodiments, the rectangular grid of spots contains at least 2, 3, 4, 5, 6, 7, 8 or 9 sub-arrays. In certain embodiments the rectangular grid of spots contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 sub-arrays. In some embodiments, the rectangular grid of spots contains 4, 9, 16 or 25 sub-arrays. In some embodiments each sub-array is a square grid of spots.
“In some embodiments, the microarray contains a rectangular grid of spots (e.g., a square grid of spots), and an always-fluorescing spot is positioned in at least one corner of the rectangular grid of spots. In some embodiments, always-fluorescing spots are positioned at 2 or 3 corners of the rectangular grid of spots and a never-fluorescing spot is positioned in the other corners of the rectangular grid of spots.
“In some embodiments, the microarray contains a plurality of pathogen-specific spots that are organized as one or more identification groups. For example, in some embodiments, in such an identification group, the pathogen-specific nucleic acid probe or antibody contained by each spot within an identification group is specific for a target nucleic acid or protein from a related group of pathogens. In some embodiments, the identification groups contain at least 2, 3, 4, 5, 6, 7 or 8 spots arranged in a square, rectangle and/or line. In some embodiments, the identification groups contain no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 spots arranged in a square, rectangle and/or line. In some embodiments the related group of pathogens contains no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 pathogens.
“In some embodiments, provided herein is a method of performing a nucleic acid microarray analysis using a microarray described herein. In some embodiments, the method includes the step of contacting a sample with the microarray. In some embodiments, the sample contains nucleic acids and/or proteins. In some embodiments, the nucleic acids or proteins are detectably labeled (e.g., fluorescently labeled). In some embodiments the method includes the step of incubating the microarray under conditions that would permit target proteins and/or target nucleic acids in the sample, if present, to become immobilized on spots of the microarray containing nucleic acid probes or antibodies specific for such target nucleic acids or proteins. In some embodiments the method includes the step of washing the microarray to remove non-immobilized nucleic acids and/or proteins. In some embodiments the method includes the step of detecting the presence of proteins or nucleic acids from the sample immobilized on at least one of the spots of the microarray. In some embodiments, the method includes the step of detecting fluorescence emitted by the spots of the microarray. In some embodiments the method includes the step of contacting the microarray with a chromogenic substrate and detecting a color change of the spots of the microarray. In some embodiments, the method includes performing an amplification reaction (e.g., a PCR reaction) on a nucleic acid of the sample before or after contacting it with the microarray.
“In some embodiments, the method includes the step of generating an image of the microarray during the detection step. In certain embodiments the method includes the step of visually interpreting the image of the microarray. In some embodiments the step of visually interpreting the image of the microarray is performed by the operator without the aid of image recognition software.
“In some embodiments, provided herein is a kit comprising a microarray described herein. In some embodiments the kit includes a microarray pattern identification aid, such as a rotary dial device and/or a printed pattern identification tree. In some embodiments the kit also includes instructions for using the microarray device.
BRIEF DESCRIPTION OF THE FIGURES
“FIG. 1 shows an array that includes four sub-arrays of three times three spots, the distance between the rows or columns within the sub-arrays (c, a) is different from that between the adjacent sub-arrays (d or b). The array also includes four always-fluorescing spots (always positive, 1a through 1d) and two never fluorescing spots (never positive, 2a and 2b) spots. Spots 3 to 32 are target selective probes.
“FIG. 2 shows that the array of FIG. 1 lacks rotational symmetry (a. correct position, b. 90 deg clockwise rotated image, c. 180 deg clockwise rotated image, d. 270 deg clockwise rotated image).
“FIG. 3 shows that the array of FIG. 1 lacks mirror-image symmetry (a. correct position, b. mirror image (flipped) of the array, c. 90 deg clockwise rotated flipped image, d. 180 deg clockwise rotated flipped image, d. 270 deg clockwise rotated flipped image).
“FIG. 4 shows that if the array of FIG. 1 is misaligned by one row (shifted grid) from the correct position, it results in pivotal elements 1b and 1d not being detected and thus the alignment can be rejected. For the sake of image clarity, the grid is also shifted by about half of a spot diameter.
“FIG. 5 shows a layout that includes 4 sectors of 6 times 6 spots with 8 always-detectable spots (1a through 1h), sixteen never-detectable spots (2a through 2p), and eight pathogen-specific spots (3a through 3d, 4a through 4d) that turn positive if the sample contains bacteria targeted by the specific probes or antibodies on the array (other spots, ‘x’).
“FIG. 6 shows dimensions of the array according to Example 1, elements 1(x) are always-fluorescing spots, elements 2(x) are never-fluorescing spots, and elements 3(x) and 4(x) are amplification-control spots (must be on for the result to be valid). All lengths in millimeters. The dimensions indicated are in mm, the respective dimensions are 0.012” and 0.018” in US units.
“FIG. 7 shows a print layout of the array according to Example 1, individual probes were printed only positions denoted with ‘x’, other positions were left empty
“FIG. 8 shows a scan of the array according to Example 2 hybridised with a positive control sample, location of representative position control elements is emphasised by arrow and circle, 1(x) through 4(x) have the same meaning as in FIGS. 5 and 6.
“FIG. 9 shows grid positioning over (9a) properly oriented scan, (9b) scan rotated 90 deg, (9c) scan rotated 180 deg, (9d) scan rotated 270 deg, and (9e) scan flipped horizontally. Representative mismatched positioning elements are emphasised by arrows.
“FIG. 10 shows a layout of a micro-array indicating the position of orientation (always-fluorescing spots–1, never-fluorescing spots–2, control spots–3, 4, and specific/multispecific probes (unlabelled positions). The array contains three identical sub-arrays A, B and C to provide robust reading through redundancy.
“FIG. 11 shows the probe layout on the Array of Example 3. Only one of the three identical sub-arrays is shown.
“FIG. 12 shows the fluorescence patterns for a subset of pathogens. Only one of the three identical sub-arrays is included, white spot indicates intense fluorescence, grey spot very weak to weak fluorescence, no spot indicates no fluorescence. 12A shows an example of the fluorescence patterns for a subset of pathogens selected from the enteric rods group, A–bacteraemia indicating spots (eubacterial universal probes), B–E. coli/Citrobacter spp. group indicator spots, C–E. coli/Citrobacter spp. identification square, D–enteric rods indicating spot (except for Citrobacter and E. coli), E–Klebsiella/Enterobacter identification square. 12B shows an example of the fluorescence patterns for a subset of pathogens–Streptococci, Enterococci and Staphylococcus, A–bacteraemia indicating spots (eubacterial universal probes), B–enterococcus group indicator, C–Enterococcus identification square, D–Streptococcus group indicator, E–Streptococcus identification square, F–Staphylococcus group indicator, G–Staphylococcus identification square. 12C shows an example of the fluorescence patterns for a subset of fungal pathogens–A bacteraemia indicating spots (eubacterial universal probes)–not fluorescing, B–Candida identification square.
“FIG. 13 shows the probe layout on the Array of Example 4. Only one of the three identical sub-arrays is shown.
“FIG. 14 shows the fluorescence patterns for a subset of pathogens, selected from the pathogen list in Table 5. Only one of the three identical sub-arrays is included, white spot indicates intense fluorescence, grey spot very weak to weak fluorescence, no spot indicates no fluorescence. 14A shows an example of the fluorescence patterns from a subset of pathogens selected from the enteric rods group, A–bacteraemia indicating spots (eubacterial universal probes), B–E. coli specific probes, C–E. coli/Citrobacter spp. group indicator spots, D–Klebsiella/Enterobacter identification field, E–enteric rod multispecific probe (except for Citrobacter and E. coli). 14B shows the fluorescence patterns for a subset of pathogens–Streptococci, Enterococci and Staphylococcus, A–bacteraemia indicating spots (eubacterial universal probes), B–Enterococcus identification area, C–enterococcus group probe, D–Streptococcus group probe, E–Streptococcus identification area, F–Staphylococcus group probes, G–Staphylococcus identification area. 14C shows the fluorescence patterns for a subset of fungal pathogens–A–bacteraemia indicating spots (eubacterial universal probes) not fluorescing, B Candida albicans probes, C Candida parapsilosis probes.”
For additional information on this patent application, see: Svoboda, Michal; Svoboda, Xenia. Microarrays. Filed December 20, 2013 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2484&p=50&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626
Keywords for this news article include: Antibodies, Immunology, Escherichia, Blood Proteins, Immunoglobulins, Molecular Probes, Enterobacteriaceae, Gammaproteobacteria, Nucleic Acid Probes, Hutman Diagnostics AG.
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