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WO2008140494A2 - Criblage haut débit à l'aide de micro-réseaux - Google Patents

Criblage haut débit à l'aide de micro-réseaux Download PDF

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Publication number
WO2008140494A2
WO2008140494A2 PCT/US2007/024291 US2007024291W WO2008140494A2 WO 2008140494 A2 WO2008140494 A2 WO 2008140494A2 US 2007024291 W US2007024291 W US 2007024291W WO 2008140494 A2 WO2008140494 A2 WO 2008140494A2
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WIPO (PCT)
Prior art keywords
probes
nucleic acid
target
dna
sequences
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PCT/US2007/024291
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English (en)
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WO2008140494A9 (fr
WO2008140494A3 (fr
Inventor
Syed Anwar Hashsham
James M. Teidje
Erdogan Gulari
Dieter Tourlousse
Robert Stedtfeld
Farhan Ahmad
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Michigan State University MSU
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Michigan State University MSU
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Publication of WO2008140494A2 publication Critical patent/WO2008140494A2/fr
Publication of WO2008140494A3 publication Critical patent/WO2008140494A3/fr
Publication of WO2008140494A9 publication Critical patent/WO2008140494A9/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the design and manufacture of microarrays for use in detecting multiple organisms in a sample in a high throughput manner.
  • the present invention relates to the detection of waterborne pathogens in a sample of water.
  • Waterborne pathogens are estimated to cause 2.2 and 5 million cases deaths per year. Waterborne illnesses continue to be a problem even in developed countries where outbreaks of unknown etiology are routine and chlorine resistant pathogens are emerging as a threat to public health. Mackenzie, W.R., et al., A Massive Outbreak in Milwaukee of Cryptosporidium Infection Transmitted through the Public Water-Supply. New England Journal of Medicine, 1994. 331(3): p. 161-167, herein incorporated by reference. Historically, people have been exposed to waterborne pathogens through accidental or inadvertent contamination of their water supply. Additionally, measures are needed for monitoring and guarding against deliberate contamination of the water supply.
  • Wilson et al. High-density microarray of small-subunit ribosomal DNA probes. Applied and Environmental Microbiology, 2002. 68(5): p. 2535-2541, herein incorporated by reference
  • Wilson et al. describes development of a microarray targeting the small-subunit ribosomal DNA of 18 pathogenic bacteria (not all are human pathogens).
  • Wilson et al. Sequence-specific identification of 18 pathogenic microorganisms using microarray technology. Molecular and Cellular Probes, 2002. 16(2): p. 119-127, herein incorporated by reference
  • the present invention relates to the design and manufacture of microarrays for use in detecting multiple organisms in a sample in a high throughput manner.
  • the present invention relates to the detection of waterborne pathogens in a sample of water.
  • the present invention provides methods for identifying regions of DNA that will specifically identify groups of pathogenic organisms.
  • the present invention provides methods for identifying regions of DNA that will specifically identify a single pathogenic organism.
  • the present invention provides methods for providing DNA probes for identifying regions of pathogen DNA.
  • the present invention provides methods for providing DNA probes for identifying subregions of pathogen DNA.
  • the present invention provides methods for providing nonoverlapping DNA probes.
  • the "maximum number of nonoverlapping probes” depends on the size of the gene sequence and the predetermined length of the probes. For example, where the gene sequence is 1000 bases and the predetermined length of the probes is 100 bases, the maximum number of nonoverlapping probes is 10. Where the gene sequence is 1000 bases and the predetermined length of the probes is 50 bases, the maximum number of nonoverlapping probes is 20 (and so on).
  • the probes can be "generated” electronically or chemically (synthetically) or even enzymatically.
  • a "subregion” is smaller in size than the complete gene sequence. Typical subregions are in the size range of 50-500 nucleotides.
  • the predetermined number of data base microorganisms depends on the degree of specificity desired.
  • the total database comprises 100 organisms and the predetermined number is 5. In one embodiment, the total database comprises 100 organisms and the predetermined number is 4. In one embodiment, the total database comprises 100 organisms and the predetermined number is 3. In one embodiment, the total database comprises 100 organisms and the predetermined number is 2. In one embodiment, the total database comprises 100 organisms and the predetermined number is l.
  • the present invention provides methods of providing target probes for detecting a target pathogen comprising, a) generating a maximum number of nonoverlapping probes of a predetermined length from a gene sequence derived from a target microorganism, b) comparing the sequence of each nonoverlapping probe to sequences from a plurality of database microorganisms, c) identifying at least one subregion in the gene sequence derived from a target microorganism, wherein the at least one subregion contains exact matches to less than a predetermined number of the database microorganisms; and d) generating a set of overlapping probes of a predetermined length for the at least one subregion.
  • the present invention is not limited to any particular length of nonoverlapping probes.
  • the predetermined length of the nonoverlapping probes is from 10 to 30 bases. In some embodiments, the predetermined length of the nonoverlapping probes is from 30 to 60 bases.
  • the present invention is not limited to subregions containing exact matches to any particular number of database microorganisms. In some embodiments, the subregion contains exact matches to less than from 5 up to 100 database microorganisms. The present invention is not limited to subregions of any particular length. In some embodiments, the subregion is from about 50 to about 500 bases in length. In some embodiments, multiple subregions containing exact matches to less than a predetermined number of database sequences are identified. In some preferred embodiments, overlapping probes of a predetermined length are generated for the multiple subregions.
  • the set of overlapping probes of a predetermined length are from 15 to 30 bases in length. In other preferred embodiments, the set of overlapping probes of a predetermined length are from 40 to 60 bases in length.
  • the methods of the present invention further comprise the step of selecting at least one probe for the at least one subregion. In some embodiments, the at least one probe for the at least one subregion has at least one base pair difference as compared to the sequences of the database microorganisms. In some embodiments, the methods of the present invention further comprise the step of arraying the at least one probe on a solid surface.
  • the present invention provides a set of nucleic acid probes generated by the foregoing methods.
  • the present invention provides microarrays comprising the foregoing set of nucleic acid probes.
  • the present invention provides a microarray comprising nucleic acids probes for a target organism, wherein the probes are selected by generating a maximum number of nonoverlapping probes of a predetermined length from a gene sequence derived from the target organism, comparing the sequence of each nonoverlapping probe to sequences from a plurality of database microorganisms, identifying at least one subregion in the gene sequence derived from a target microorganism, wherein the at least one subregion contains exact matches to less than a predetermined number of the database microorganisms; and generating a set of overlapping probes of a predetermined length for the at least one subregion.
  • the present invention is not limited to microarrays formed on any particular substrate.
  • the probes are arrayed on a glass slide.
  • the present invention provides methods for high throughput detection of multiple target organisms comprising a) providing: i) a microarray comprising sets of multiple discreet nucleic acid probes for at least one target organism, wherein the nucleic acid probes are complemetary to multiple amplicons from multiple target genes of the at least one target organism; ii) primers complementary to multiple target genes of the multiple target organisms; b) using the primers to amplify the multiple target genes from the multiple target organisms to produce amplicons; c) contacting the microarray with the amplicons under conditions such that the amplicons hybridize to the discreet nucleic acid probes; d) detecting the presence of amplicon binding to the discreet nucleic acid probes.
  • the methods provide a set of nucleic acid primers. In some embodiments, the methods provide a set of nucleic acid probes. In some embodiments, the methods provide sequences selected from the group consisting of SEQ ID Nos. 1-19206. In some embodiments, the methods provide sequences selected from the group consisting of SEQ ID Nos. 1-791. In some embodiments, the methods provide sequences selected from the group consisting of SEQ ID Nos. 792-11533. . In some embodiments, the methods provide sequences selected from the group consisting of SEQ ID Nos. 11534-13225. In some embodiments, the methods provide sequences selected from the group consisting of SEQ ID Nos. 13226- 19206
  • the present invention provides a microarray comprising the set of nucleic acid probes generated by methods of the present inventions.
  • the microarray comprises at least one sequence selected from the group consisting of SEQ ID Nos. 1-19206
  • the present invention provides a microarray comprising nucleic acids probes for a target organism, wherein said probes are selected by generating a maximum number of nonoverlapping oligonucleotide probes of a predetermined length from a gene sequence derived from said target organism, comparing the sequence of each nonoverlapping oligonucleotide probes to sequences from a plurality of database microorganisms, identifying at least one subregion in said gene sequence derived from a target microorganism, wherein said at least one subregion contains exact matches to less than a predetermined number of said database microorganisms; and generating a set of overlapping probes of a predetermined length for said at least one subregion.
  • the oligonucleotide probes are arrayed on a substrate selected from the group consisting of a glass slide and a serpentine chip.
  • the target organisms are selected from the group consisting of Aeromonas hydrophila, Campylobacter jejuni, Clostridium perfringens, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia enter ocolitica, E. faecalis, S. ente ⁇ ca,, C.
  • the oligonucleotide probes are complementary to virulence marker genes from said target organisms.
  • the present invention provides a microarray for high throughput detection of waterborne pathogens comprising sets of multiple discreet nucleic acid probes for at least ten target organisms, wherein said nucleic acid probes are complemetary to multiple amplicons from multiple virulence marker genes from each target organism and multiple discreet nucleic acid probes are provided for each amplicon, and wherein said at least 10 target microorganism are selected from the group consisting of Aeromonas hydrophila, Campylobacter jejuni, Clostridium perf ⁇ ngens, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Vibrio cholerae,
  • the nucleic acid probes are selected from the group consisting of oligonucleotide probes encoded by SEQ ID Nos. 1-19206 and combinations thereof.
  • the present invention provides a method for high throughput detection of multiple target organisms a) providing: i) a microarray comprising sets of multiple discreet nucleic acid probes for multiple target organisms, wherein said nucleic acid probes are complemetary to multiple amplicons from multiple target genes of said at least one target organism, and wherein multiple discreet nucleic acid probes are provided for each amplicon; ii) primers complementary to multiple target genes of said multiple target organisms; and b) using said primers to amplify said multiple target genes from said multiple target organisms to produce amplicons; c) contacting said microarray with said amplicons under conditions such that said amplicons hybridize to said discreet nucleic acid probes; and d) detecting the presence of amplicon binding to said discreet nucleic acid probes.
  • the target organisms are selected from the group consisting of Aeromonas hydrophila, Campylobacter jejuni, Clostridium perfringens, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia enterocolitica, E. faecalis, S. enterica,, C. parvum, G. intestinalis, K. pneumoniae, E. coli. and combinations thereof.
  • the probes are complementary to virulence marker genes from said target organisms.
  • the nucleic acid probes are selected from the group consisting of probes encoded by SEQ ID Nos. 1-19206 and combinations thereof.
  • the present invention provides a set of oligonucleotide probes consisting of at least 2 sequences selected from the group consisting of SEQ ID Nos. 1-19206.
  • the present invention provides a microarry consisting of at least 2 sequences selected from the group consisting of SEQ ID Nos. 1-19206.
  • the microarray is a serpentine chip.
  • FIG. 1 shows an exemplary result of testing microbial sequences obtained by compositions and methods of the present inventions for a 16s rDNA sequence.
  • Panel A Graph of 73 non-overlapping 20-mers. Each 20-mer sequence was then used to obtain the number of matches (exact) in The Ribosomal Database Project II (RDP-II) database and plotted on the y-axis.
  • Panel B For the region from 135 to 220 yielding the first and second regions with 10 matches or less (in the left panel), the 20-mer probes were tested for matches in RDP-II by walking one base at a time.
  • FIG. 2 shows an exemplary evaluation of 791 targeted and 2,034 not-targeted probes with a composite target sample containing 47 VMG amplicons (24 labeled with Cy3 and 23 labeled with Cy5).
  • A Part of the Xeotron chip after hybridization and washing up to 30 0 C, spot features are 50 ⁇ m in diameter.
  • B Distribution and boxplot of SNRs for 791 targeted (black bars and lower boxplot) and 2034 not-targeted probes (grey bars and upper boxplot).
  • C Distribution of the positive fraction for 47 targeted (black bars) and 67 not-targeted probe sets (grey bars). The dashed line shows the cut-off (0.5) for presence/absence calls.
  • D Success rate of probe design.
  • the number of probes showing positive signal (y-axis) vs. the number of designed probes (x-axis) for each VMG (black symbols: targeted VMGs, and grey symbols: not-targeted VMGs).
  • the dashed lines represent varying levels of success. The slope of these lines noted is equal to the positive fraction.
  • FIG. 3 shows an exemplary targeted probe response and characterization.
  • VMGs are sorted from bottom to top according to increasing median SNRs.
  • Probe sets are sorted from bottom to top according to increasing median signal-to-noise ratio.
  • FIG. 4 shows an exemplary success rate of probe design as function of free energy of probe-target duplex formation ( ⁇ G°d up iex)- Each symbol represents 40 probes with error bars reflecting the standard deviation among three replicates. The percent positive probes is plotted at the median ⁇ G o dup i ex of each bin. The G+C content on the upper X-axis was derived from a linear relationship between a ⁇ G°d up iex and G+C content.
  • FIG. 5 shows an exemplary performance of a probe set for a VMG biochip with DNA extracted from several types of water samples.
  • Genomic DNA (10 pg) of each pathogen was spiked into 10 ng of background DNA, yielding a relative abundance for each pathogen of 0.1%.
  • Spiked samples and unspiked background samples were labeled with Cy3 and Cy5, respectively, mixed and hybridized in duplicate.
  • 673 probes 46 VMG amplicons, marker gene targets chosen from validated probes as described herein
  • 2,034 probes 67 VMG amplicons
  • Boxplots represent the distribution of the positive fraction among the VMG amplicons.
  • the dashed line represents the selected threshold for presence/absence (positve/negative) calls.
  • FIG. 6 shows an exemplary derivation of optimal probe design criteria demonstrating a dependence of positive fraction on Gibb's free energy of duplex formation
  • the shaded area represents the ⁇ G°d up ⁇ e ⁇ and G+C content range yielding a selectivity higher than 70%.
  • FIG. 7A-0 shows an exemplary list of 20 mer probes for waterborne pathogens.
  • FIG. 8 shows an exemplary list of microorganisms and virulence marker genes for use in compositions and methods of the present inventions.
  • FIG. 9 shows an exemplary description of multiplex PCR reactions and the specificity and sensitivity achievable with the probe design of the present invention.
  • FIG. 10 shows an exemplary Serpentine Chip, developed for use in compositions and methods of the present inventions, please note the solid (nontransparent) base.
  • FIG. 11 shows an exemplary microfluidic DNA biochip with recirculation capabilities for detecting and quantifying infectious agents by hybridizing PCR amplified products to oligonucleotide probes as provided herein (see, Figures 12 and 13): (a) the exemplary chip was approximately 1 cm2, (b) a close-up view of microfluidic channels and a portion of the approximately 8,000 reactors on the chip, (c) a close-up view of 6 reactors, each with a 50 micron diameter, (d) a signal to noise ratio for 5 genes belonging to one of the 20 organisms that were tested on the chip, and (e) a laser scanned signal intensities for part of the chip experimental results (a green signal represents a positive hybridization result).
  • FIG. 12 shows an exemplary list of 50 mer oligonucleotide probes for identifying waterborne pathogens developed and used as described herein.
  • FIG. 13 shows an exemplary list of validated 50 mer oligonucleotide probes for identifying waterborne pathogens developed and used as described herein.
  • FIG. 14 shows exemplary results from spot testing 10,000 50 mer probes (see, Figure 12) attached to a Serpentine Chip, oligonucleotide probes were synthesized as described herein, hybridzation products were labeled with a SY3 dye, (A) results were visualized by a laser scanner, green areas were positive, dark spots were negative, (B) demonstrates exemplary low signal to noise ratios.
  • FIG. 15 shows an exemplary confirmation of amplification in a serpentine PCR chip demonstrating reaction products obtained from a nonleaking chip (a) microfulidic channel, (b) PCR product detectable after the 15 th cycle, and (c) demonstration of success obtaining the expected size PCR product by routine gel electrophoresis.
  • FIG. 16 shows the stability of exemplary freeze-dried PCR reagents (A) Optimization of trehalose concentration for freeze-dried Taq Polymerase and (B) Stability of freeze-dried PCR reagents with 15% Trehalose.
  • FIG. 17 shows exemplary results from a helicase-dependent isothermal amplification.
  • an agent includes a plurality of agents, including mixtures thereof.
  • serpentine chip refers to a chip with serpentine shaped microchannels.
  • the term "arraying" in reference to a primer or probe refers to the use of the primer or probe in a microarray, for example, by attaching the probe or primer to the microarray surface, adding the probe or primer to the microarry, and the like.
  • the term "transducer device” refers to a device that is capable of converting a non-electrical phenomenon into electrical information, and transmitting the information to a device that interprets the electrical signal.
  • Such devices can include, but are not limited to, devices that use photometry, fluorometry, and chemiluminescence; fiber optics and direct optical sensing (e.g., grating coupler); surface plasmon resonance; potentiometric and amperometric electrodes; field effect transistors; piezoelectric sensing; and surface acoustic wave.
  • optical transparency refers to the property of matter whereby the matter is capable of transmitting light such that the light can be observed by visual light detectors (e.g., eyes and detection equipment).
  • electrosenescence or “EL” refer to a direct conversion of electrical energy into light.
  • electrostatic sheet and “electroluminescent film” and “ELM” and “electroluminescent panel” and “electroluminescent wire” and “EL lamp” refer to a type of capacitor comprising a thin layer of light emitting phosphor located between two electrodes, wherein the first electrode is opaque and the second electrode is translucent to allow light to escape.
  • AC current 400 - 1600 Hz
  • phosphor chemical composition and dye pigments determine the brightness and color of the emitted light.
  • electroluminescent compositions include ruthenium, ruthenium polypyridyl complex, tris-bippyridine, tris(bipyridine) Ru(II) complexes, tripropylamine, Ruthenium Tris(2,2')bipyridyl/Tripropylamine, and the like.
  • phosphor refers to a substance that exhibits the phenomenon of phosphorescence, such as a natural substance, for example a transition metal compound or rare earth compounds of various types or synthetic, for example, a suitable host material, to which an activator is added such as a copper-activated zinc sulfide and the silver-activated zinc sulfide (zinc sulfide silver).
  • ACTFEL and "alternating current thin film electroluminescence” refer to emitted light following exposure to an electrical current.
  • film refers to any substance capable of coating at least a portion of a substrate surface and immobilizing capture particles. Examples of materials used to make such films include, but are not limited to, agarose, acrylamide, SEPHADEX, proteins (e.g. bovine serum albumin (BSA), polylysine, collagen, etc.), hydrogels (e.g. polyethylene oxide, polyvinyl alcohol, polyhydroxyl butylate, etc.), film forming latexes (e.g.
  • films include additional material such as plasticisers (e.g. polyethylene glycol [PEG], detergents, etc.) to improve stability and/or performance of the film.
  • plasticisers e.g. polyethylene glycol [PEG], detergents, etc.
  • a film is a material that will react with the capture particles and present them in the same focal plane.
  • a film is pre-activated with cross-linking groups such as aldehydes, or groups added after the film has been formed.
  • optical signal refers to any energy (e.g. photodetectable energy) emitted from a sample (e.g., produced from a microarray that has one or more optically excited [i.e., by electromagnetic radiation] molecules bound to its surface).
  • filter refers to a device or coating that preferentially allows light of a characteristic spectra to pass through it (e.g., the selective transmission of light beams).
  • Polychromatic or “broadband” as used herein, refers to a plurality of electromagnetic wavelengths emitted from a light source.
  • microarray refers to a substrate with a plurality of molecules (e.g., nucleotides) bound to its surface. Microarrays, for example, are described generally in Schena, "Microarray Biochip Technology,” Eaton Publishing, Natick, Mass., 2000. Additionally, the term “patterned microarrays” refers to microarray substrates with a plurality of molecules non-randomly bound to its surface.
  • optical detector or “photodetector” refers to a device that generates an output signal when irradiated with optical energy.
  • optical detector system is taken to mean a device for converting energy from one form to another for the purpose of measurement of a physical quantity or for information transfer.
  • Optical detectors include but are not limited to photomultipliers and photodiodes.
  • photomultiplier or “photomultiplier tube” refers to optical detection components that convert incident photons into electrons via the photoelectric effect and secondary electron emission.
  • photomultiplier tube is meant to include devices that contain separate dynodes for current multiplication as well as those devices which contain one or more channel electron multipliers.
  • the term "photodiode” refers to a solid-state light detector type including, but not limited to PN, PIN, APD and CCD.
  • the terms "plate reader” in reference to a “detection device” refer to a device to detect the transmission of light through or reflection of light (i.e., polarized light or non-polarized light of specific wavelengths) from the surface of an assay, that for the purposes of the present invention the assay is a "chip” and “PCR chip” or a “glass slide” comprising a PCR assay or a “plate” such as a 96-well plate and the like.
  • a microtiter plate reader measures transmittance, absorbance, or reflectance through, in, or from each well of a multitest device such as a microtiter testing plate (e.g., MicroPlate.TM. testing plates) or a miniaturized testing card (e.g., MicroCard.TM. miniaturized testing cards).
  • a microtiter testing plate e.g., MicroPlate.TM. testing plates
  • a miniaturized testing card e.g., MicroCard.TM. miniaturized testing cards.
  • thin layer refers to a very thin deposition of a colloidal substance (phosphor, dielectric, silver) onto the ITO coated glass plate.
  • ITO refers to an Indium Tin Oxide (In203:Sn02) A thin layer of indium oxide that has been doped with tin; transparent, conductive coating on glass plate.
  • phosphor refers to a powder made of a material such as zinc sulfide, doped with either copper or manganese to achieve the emission colors when exposed to an electric field.
  • dielectric layer refers to an insulating layer that serves to even out the electric field across the phosphor layer and prevents short circuits.
  • capacitor refers to an electrical device that can store energy in the electric field between a pair of closely spaced conductors or 'plates'. When current is passed through the capacitor, electric charges of equal magnitude, but opposite sign, build up on each plate.
  • electrode refers to a plate of a capacitor, for example, one front electrode, such an electrode comprising as transparent ITO and one back electrode, such as an electrode comprising silver.
  • electrostatic power supply refers to an electronic device that produces a particular DC voltage or current from a source of electricity such as a battery or wall outlet.
  • power adapter As used herein, “power adapter,” “transformer,” or “power supply” refer to an external power supply for laptop computers or portable or semi-portable electronic device As used herein, “AC adapter” refers to a rectifier to convert AC current to DC and a transformer to convert voltage from 120 down to 9, 12, 15 or whatever is required.
  • power supply refers to an electrical system that converts AC current from the wall outlet into the DC currents required by the computer circuitry.
  • a computer power supply typically generates multiple voltages. For example, 12 volts is used for drives, and either 3.3 or 5 volts is used for the electronic circuitry.
  • external AC adaptor power brick refers to an electronic device that produces AC current.
  • AC powered linear power supply refers to a transformer to convert the voltage from the wall outlet to a lower voltage.
  • An array of diodes called a diode bridge then rectifies the AC voltage to DC voltage.
  • a low-pass filter smoothes out the voltage ripple that is left after the rectification.
  • a linear regulator converts the voltage to the desired output voltage, along with other possible features such as current limiting.
  • AC current and “Alternating Current” and “AC” refers to a type of electrical current, the direction of which is reversed at regular intervals or cycles. In the United States, the standard is 120 reversals or 60 cycles per second.
  • DC current and “Direct Current” and “DC” refers to a type of electricity transmission and distribution by which electricity flows in one direction through the conductor, usually relatively low voltage and high current. For typical 120 volt or 220 volt devices, DC must be converted to alternating current.
  • battery refers to a device that stores chemical energy and makes it available in an electrical form. Batteries comprise electrochemical devices such as one or more galvanic cells, fuel cells or flow cell, examples include, lead acid, nickel cadmium, nickel metal hydride, lithium ion, lithium polymer, CMOS battery and the like.
  • CMOS battery refers to a battery that maintains the time, date, hard disk and other configuration settings in the CMOS memory.
  • frequency refers to a number of oscillations (vibrations) in one second.
  • Frequency / is the reciprocal of the time T taken to complete one cycle (the period), or MT.
  • the frequency with which earth rotates is once per 24 hours.
  • Frequency is usually expressed in units called hertz (Hz).
  • Frequency is measured in terms “hertz” or “Hz” that refer to “oscillations per second” or “cycles per second.” For example, “one hertz” or “1 Hz” is equal to one cycle per second, for example, “one kilohertz” or “kHz” is 1,000 Hz, and “one megahertz” or “MHz” is 1,000,000 Hz.
  • the alternating current in a wall outlet in the U.S. and Canada is 60Hz.
  • Electromagnetic radiation is measured in kiloHertz (kHz), megahertz (MHz) and gigahertz (GHz).
  • inverter or “rectifier” refer to a device that converts direct current electricity to alternating current either for stand-alone systems or to supply power to an electricity grid.
  • volt and V refer to a unit of electrical force equal to that amount of electromotive force that will cause a steady current of one ampere to flow through a resistance of one ohm.
  • voltage refers to an amount of electromotive force, measured in volts, that exists between two points.
  • Ohm refers to a measure of the electrical resistance of a material equal to the resistance of a circuit in which the potential difference of 1 volt produces a current of 1 ampere.
  • Ampere and amp refers to a unit of electrical current or rate of flow of electrons, such that one volt across one ohm of resistance causes a current flow of one ampere.
  • Charge-Coupled Device and “CCD” refers to an electronic memory that records the intensity of light as a variable charge.
  • storage CCDs refers to either a separate array (frame transfer) or individual photosites (interline transfer) coupled to each imaging photosite.
  • CMOS Complementary-symmetry/metal-oxide semiconductor
  • CMOS IMAGE SENSOR refers to a "CMOS-based chip” that records intensities of light as variable charges similar to a CCD chip. In one embodiment, as CMOS chip use less power than a CCD chip.
  • optical signal refers to any energy (e.g., photodetectable energy) from a sample (e.g., produced from a microarray that has one or more optically excited [i.e., by electromagnetic radiation] molecules bound to its surface).
  • filter refers to a device or coating that preferentially allows light of a characteristic spectra to pass through it (e.g., the selective transmission of light beams).
  • micromirror array refers to a plurality of individual light reflecting surfaces that are addressable (e.g., electronically addressable in any combination), such that one or more individual micromirrors can be selectively tilted, as desired.
  • optical detector and “photodetector” refers to a device that generates an output signal when exposed to optical energy.
  • optical detector system refers devices for converting energy from one form to another for the purpose of measurement of a physical quantity and/or for information transfer. Optical detectors include but are not limited to photomultipliers and photodiodes, as well as fluorescence detectors.
  • TTL Transistor-Transistor Logic
  • TTL Transistor-Transistor Logic
  • the family uses zero Volt and five Volt signals to represent logical "0" and "1" respectively.
  • dynamic range refers to the range of input energy over which a detector and data acquisition system is useful. This range encompasses the lowest level signal that is distinguishable from noise to the highest level that can be detected without distortion or saturation.
  • noise in its broadest sense refers to any undesired disturbances (i.e., signal not directly resulting from the intended detected event) within the frequency band of interest.
  • noise is the summation of unwanted or disturbing energy introduced into a system from man-made and natural sources.
  • noise may distort a signal such that the information carried by the signal becomes degraded or less reliable.
  • the term "signal-to-noise ratio” refers the ability to resolve true signal from the noise of a system.
  • One example of computing a signal-to-noise ratio is by taking the ratio of levels of the desired signal to the level of noise present with the signal.
  • phenomena affecting signal- to-noise ratio include, but are not limited to, detector noise, system noise, and background artifacts.
  • the term “detector noise” refers to undesired disturbances (i.e., signal not directly resulting from the intended detected energy) that originate within the detector.
  • Detector noise includes dark current noise and shot noise. Dark current noise in an optical detector system results from the various thermal emissions from the photodetector. Shot noise in an optical system is the product of the fundamental particle nature (i.e., Poisson-distributed energy fluctuations) of incident photons as they pass through the photodetector.
  • system noise refers to undesired disturbances that originate within the system.
  • System noise includes, but is not limited to noise contributions from signal amplifiers, electromagnetic noise that is inadvertently coupled into the signal path, and fluctuations in the power applied to certain components (e.g., a light source)
  • background artifacts include signal components caused by undesired optical emissions from the microarray. These artifacts arise from a number of sources, including: non-specific hybridization, intrinsic fluorescence of the substrate and/or reagents, incompletely attenuated fluorescent excitation light, and stray ambient light.
  • the noise of an optical detector system is determined by measuring the noise of the background region and noise of the signal from the microarray feature.
  • processor refers to a device that performs a set of steps according to a program (e.g., a digital computer).
  • processors for example, include Central Processing Units ("CPUs"), electronic devices, and systems for receiving, transmitting, storing and/or manipulating digital data under programmed control.
  • CPUs Central Processing Units
  • memory device and “computer memory” refer to any data storage device that is readable by a computer, including, but not limited to, random access memory, hard disks, magnetic (e.g., floppy) disks, zip disks, compact discs, DVDs, magnetic tape, and the like.
  • gene refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor. It is intended that the term encompass polypeptides encoded by a full length coding sequence, as well as any portion of the coding sequence, so long as the desired activity and/or functional properties (e.g., enzymatic activity, ligand binding, etc.) of the full-length or fragmented polypeptide are retained.
  • the term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA.
  • sequences that are located 5' of the coding region and which are present on the mRNA are referred to as "5' untranslated sequences.”
  • sequences that are located 3' (i.e., "downstream") of the coding region and that are present on the mRNA are referred to as “3' untranslated sequences.”
  • the term “gene” encompasses both cDNA and genomic forms of a gene.
  • a genomic form of a genetic clone contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers.
  • Introns are removed or "spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule
  • amino acid sequence and like terms, such as “polypeptide” and “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript.
  • flanking sequences or regions are referred to as “flanking" sequences or regions (these flanking sequences are located 5 ' or 3' to the non-translated sequences present on the mRNA transcript).
  • the 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene.
  • the 3' flanking region may contain sequences that direct the termination of transcription, post- transcriptional cleavage and polyadenylation.
  • nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
  • DNA molecules are said to have "5 1 ends” and "3 1 ends” because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage.
  • an end of an oligonucleotide or polynucleotide referred to as the "5 1 end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends.
  • an oligonucleotide having a nucleotide sequence encoding a gene and “polynucleotide having a nucleotide sequence encoding a gene,” means a nucleic acid sequence comprising the coding region of a gene or, in other words, the nucleic acid sequence that encodes a gene product.
  • the coding region may be present in either a cDNA, genomic DNA, or RNA form.
  • the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded.
  • Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript.
  • regulatory element refers to a genetic element that controls some aspect of the expression of nucleic acid sequences.
  • a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region.
  • Other regulatory elements include splicing signals, polyadenylation signals, termination signals, etc.
  • complementary and complementarity are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • nucleic acid strands For example, for the sequence "A-G-T,” is complementary to the sequence “T-C- A.”
  • Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification and hybridization reactions, as well as detection methods that depend upon binding between nucleic acids.
  • low stringency conditions factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
  • conditions that promote hybridization under conditions of high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
  • substantially homologous refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
  • a gene may produce multiple RNA species that are generated by differential splicing of the primary RNA transcript.
  • cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon "A” on cDNA 1 wherein cDNA 2 contains exon "B” instead). Because the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; the two splice variants are therefore substantially homologous to such a probe and to each other.
  • substantially homologous refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Those skilled in the art will recognize that “stringency” conditions may be altered by varying the parameters just described either individually or in concert. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences (e.g., hybridization under "high stringency” conditions may occur between homologs with about 85-100% identity, preferably about 70-100% identity).
  • nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (e.g., hybridization under "medium stringency” conditions may occur between homologs with about 50-70% identity).
  • intermediate stringency e.g., hybridization under "medium stringency” conditions may occur between homologs with about 50-70% identity.
  • conditions of "weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.
  • the term “selectively hybridize” means that, for particular identical sequences, a substantial portion of the particular identical sequences hybridize to a given desired sequence or sequences, and a substantial portion of the particular identical sequences do not hybridize to other undesired sequences.
  • a “substantial portion of the particular identical sequences” in each instance refers to a portion of the total number of the particular identical sequences, and it does not refer to a portion of an individual particular identical sequence.
  • "a substantial portion of the particular identical sequences” means at least 70% of the particular identical sequences.
  • "a substantial portion of the particular identical sequences” means at least 80% of the particular identical sequences.
  • "a substantial portion of the particular identical sequences” means at least 90% of the particular identical sequences.
  • "a substantial portion of the particular identical sequences” means at least 95% of the particular identical sequences.
  • Amplification is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out. Template specificity is achieved in most amplification techniques by the choice of enzyme.
  • Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid.
  • MDV-I RNA is the specific template for the replicase (Kacian et al, Proc. Natl. Acad. Sci. USA, 69:3038 [1972], herein incorporated by reference).
  • this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al. , Nature, 228:227 [1970], herein incorporated by reference).
  • T4 DNA ligase the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (Wu and Wallace, Genomics, 4:560 [1989], herein incorporated by reference).
  • Taq and Pfu polymerases by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non- target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press [1989], herein incorporated by reference).
  • amplifiable nucleic acid is used in reference to nucleic acids that may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid” will usually comprise “sample template.”
  • sample template refers to nucleic acid originating from a sample that is analyzed for the presence of “target” (defined below).
  • target defined below
  • background template is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • probe refers to a molecule (e.g., an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification), that is capable of hybridizing to another molecule of interest (e.g., another oligonucleotide).
  • probes When probes are oligonucleotides they may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular targets (e.g., gene sequences).
  • probes used in the present invention are labeled with any "reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular label.
  • enzyme e.g., ELISA, as well as enzyme-based histochemical assays
  • fluorescent, radioactive, and luminescent systems e.g., fluorescent, radioactive, and luminescent systems.
  • the term probe is used to refer to any hybridizable material that is affixed to the microarray for the purpose of detecting "target" sequences in the analyte.
  • probe element and “probe site” refer to a plurality of probe molecules (e.g., identical probe molecules) affixed to a microarray substrate. Probe elements containing different characteristic molecules are typically arranged in a two- dimensional array, for example, by microfluidic spotting techniques or by patterned photolithographic synthesis, etc.
  • the term "target,” when used in reference to hybridization assays, refers to the molecules (e.g., nucleic acid) to be detected.
  • the "target” is sought to be sorted out from other molecules (e.g., nucleic acid sequences) or is to be identified as being present in a sample through its specific interaction (e.g., hybridization) with another agent (e.g., a probe oligonucleotide).
  • a “segment” is defined as a region of nucleic acid within the target sequence.
  • a "target nucleic acid sequence” is a sequence in a sample that is not a known control gene that is added to the sample.
  • a target nucleic acid sequence serves as a template for amplification in a PCR reaction.
  • a target nucleic acid sequence is a portion of a larger nucleic acid sequence.
  • a target nucleic acid sequence is a portion of a gene.
  • PCR polymerase chain reaction
  • the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule.
  • the primers are extended with a polymerase so as to form a new pair of complementary strands.
  • the steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle”; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
  • the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
  • PCR polymerase chain reaction
  • PCR product refers to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
  • amplification reagents refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template, and the amplification enzyme.
  • amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
  • reverse-transcriptase and "RT-PCR” refer to a type of PCR where the starting material is mRNA.
  • the starting mRNA is enzymatically converted to complementary DNA or "cDNA” using a reverse transcriptase enzyme.
  • the cDNA is then used as a “template” for a "PCR” reaction.
  • a “multiplex amplification reaction” is an amplification reaction in which two or more target nucleic acid sequences are amplified in the same reaction.
  • a “multiplex polymerase chain reaction” or “multiplex PCR” is a polymerase chain reaction method in which two or more target nucleic acid sequences are amplified in the same reaction.
  • a “singleplex amplification reaction” is an amplification reaction in which only one target nucleic acid sequence is amplified in the reaction.
  • a “singleplex polymerase chain reaction” or “singleplex PCR” is a polymerase chain reaction method in which only one target nucleic acid sequence is amplified in the reaction.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • the term "recombinant DNA molecule” as used herein refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.
  • antisense is used in reference to RNA sequences that are complementary to a specific RNA sequence (e.g., mRNA). Included within this definition are antisense RNA (“asRNA”) molecules involved in gene regulation by bacteria. Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter that permits the synthesis of a coding strand. Once introduced into an embryo, this transcribed strand combines with natural mRNA produced by the embryo to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated.
  • asRNA antisense RNA
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
  • the designation (-) i.e., “negative” is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.
  • isolated when used in relation to a nucleic acid, as in “an isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA found in the state they exist in nature.
  • a given DNA sequence e.g., a gene
  • RNA sequences such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins.
  • the isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form.
  • oligonucleotide or polynucleotide When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).
  • coding region when used in reference to a structural gene refers to the nucleotide sequences that encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule.
  • the coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG” that encodes the initiator methionine and on the 3' side by one of the three triplets which specify stop ⁇ codons (i.e., TA, TAG, TGA).
  • ATG nucleotide triplet
  • purified and “to purify” refer to the removal of contaminants from a sample.
  • recombinant DNA molecule refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.
  • portion when in reference to a nucleotide sequence (as in “a portion of a given nucleotide sequence”) refers to fragments of that sequence. The fragments may range in size from four nucleotides to the entire nucleotide sequence minus one nucleotide.
  • recombinant protein and “recombinant polypeptide” as used herein refer to a protein molecule that are expressed from a recombinant DNA molecule.
  • biologically active polypeptide refers to any polypeptide which maintains a desired biological activity.
  • portion when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
  • microbe and “microbial” refer to microorganisms.
  • the microbes identified using the present invention are bacteria (i.e., eubacteria and archaea). However, it is not intended that the present invention be limited to bacteria, as other microorganisms are also encompassed within this definition, including fungi, viruses, and parasites (e.g., protozoans and helminths).
  • the term "reference DNA” refers to DNA that is obtained from a known organism (i.e., a reference strain).
  • the reference DNA comprises random genome fragments.
  • the genome fragments are of approximately 1 to 2 kb in size.
  • the reference DNA of the present invention comprises mixtures of genomes from multiple reference strains.
  • multiple reference strains refers to the use of more than one reference strains in an analysis. In some embodiments, multiple reference strains within the same species are used, while in other embodiments, “multiple reference strains” refers to the use of multiple species within the same genus, and in still further embodiments, the term refers to the use of multiple species within different genera.
  • test DNA and “sample DNA” refer to the DNA to be analyzed using the method of the present invention. In preferred embodiments, this test DNA is tested in the competitive hybridization methods of the present invention, in which reference DNA(s) from multiple reference strains is/are used.
  • sample and “specimen” in the present specification and claims are used in their broadest sense. On the one hand, they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompasses all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, as well as solid tissue. These terms also refers to swabs and other sampling devices that are commonly used to obtain samples for culture of microorganisms. Biological samples may be animal, including human, fluid or tissue, food products and ingredients such as dairy items, vegetables, meat and meat byproducts, and waste.
  • Environmental samples include environmental material such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, disposable, and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
  • the present invention relates to the design and manufacture of microarrays for use in detecting multiple organisms in a sample in a high throughput manner.
  • the present invention relates to the detection of waterborne pathogens in a sample of water.
  • the microarrays comprise nucleic acids probes for multiple target organisms.
  • the probes are selected by generating a maximum number of nonoverlapping probes of a predetermined length from a gene sequence derived from the target organism, comparing the sequence of each nonoverlapping probe to sequences from a plurality of database microorganisms, identifying at least one subregion in the gene sequence derived from the target microorganism, wherein the subregion contains exact matches to less than a predetermined number of the database microorganisms; and generating a set of overlapping probes of a predetermined length for said at least one subregion.
  • the present invention embodies, in part, an in situ synthesized oligonucleotide probe biochip developed for the detection of 12 pathogens.
  • a redundant set of 791 probes was selected targeting 47 virulence and marker genes (VMGs).
  • VMGs virulence and marker genes
  • 95% of the probes behaved as predicted based on hybridizations with a composite sample of all VMGs.
  • 673 probes (85% of the initial probe set) were selected for the detection of VMGs in environmental samples.
  • a combination of multiplex PCR and the microarray allowed the detection of the pathogens at a relative abundance level between 0.1% and 0.01% for most of the VMGs.
  • VMG biochip using an in situ synthesized microfluidic platform targeting 93 virulence and marker genes (VMGs) in the following pathogens: Aeromonas hydrophila, Campylobacter jejuni, Clostridium perfringens, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Vibrio cholerae, Vibrio par ahaemolyticus, Yersinia enter ocolitica, E. faecalis, S. enterica,, C. parvum, G. intestinalis, K. pneumoniae, E. coli.
  • VMGs virulence and marker genes
  • Each VMG was targeted by 17 probes on average. Many VMGs are targeted in more than one region. This design allows for redundancy in organism detection by targeting more than one VMG in an organism and redundancy in VMG detection by targeting VMGs in more than one region.
  • the virulence marker genes are selected from the genes listed in the table listed in Fig. 8.
  • Fig. 8 also provides the number of probes for each gene on an exemplary chip.
  • Fig. 9 presents provides a description of the which targets were amplified in multiplex PCR reactions and the specificity and sensitivity of the biochip design.
  • the biochips of the present invention were validated by spiking DNA of 12 pathogens into environmental background DNA extracted from river water, tertiary effluent, and tap water separately.
  • the VMG targets were enriched via multiplex PCR to improve the detection limit, labeled, and allowed to hybridize on the biochip.
  • the chip allowed detection 11 of the 12 pathogens presented to the biochip in environmental backgrounds.
  • the targets of the undetected organism had a relatively low G+C content. Over all the VMG biochip is robust and could be utilized for pathogen detection in environmental samples.
  • the methods of the present invention result in highly reliable pathogen detection. This reliability is accomplished by increasing the redundancy of the detection approach.
  • redundancy is achieved by using detections methods using probes to multiple genes from a single organism (e.g., a pathogen) and multiple amplicons per gene as well as using multiple probes per each Amplicon or combinations thereof.
  • high sensitivity is obtained by coupling two rounds of amplification.
  • the first round can be multiplex PCR and the second round can be isomthermal Klenow amplification.
  • probe design uses heuristic rules that were developed for solution-based hybridization and are not fully applicable to hybridizations performed on solid substrates.
  • Convention probe design also fails to consider target dangling-end influence on hybridization behavior.
  • conventional probe design can erroneously remove probes that will behave well on solid substrates, fail to remove probes that compliment problematic target fragments, and can reduce the size of probe sets.
  • the present invention provides a new strategy for probe design and validation of probes for use on high-thoughput microarray platforms.
  • the inventive strategy takes advantage of the fact that the availability of specific probes (i.e., those probes that hybridize only to the target of interest and do not cross-hybridize with non-targets) varies along the target sequence.
  • 16S rDNA probes were designed with the following steps: 1) the number of exact matches in the Ribosomal Database Project II (RDP-II) database maintained at Michigan State University (RDP Release 9), were determined for every non-overlapping 20-mer probe possible in a 16S rDNA gene for an organism of interest (generating about 70 - 75 probes for the target sequence) and 2) generating additional probes by overlapping 19 bases in the sub-region of 16S rDNA with less than about 10 to 50 exact matches.
  • the probes were validated using targets with specified dangling-ends for 18-mer probes. This demonstrated that the length and composition of target dangling-ends can significantly influence sensitivity and cross hybridization in reference and community assays can influence confidence in probe validation.
  • the specificity of the probes was examined using a single organism (Burkholderia xenovorans LB400), which was hybridized with a complex sample of DNA extracted from an anaerobic bioreactor. Since LB400 is not present in the bioreactor sample, this allowed evaluation of the specificity and sensitivity of LB400 probes. Results showed that specific signal increased as the conservation of probe sequences decreased (as determined by number of exact matches). Over 77% of perfect match probes displayed specificity, sensitivity and thermal melting curve characteristics in complex community samples, demonstrating that the extended probe design of the present invention is effective for retaining a larger number of high quality probes. Furthermore, portions of the sub-region yield greater specificity with as little as 1 bp difference and provide numerous probes for further evaluation.
  • the present invention provides a microarray comprising nucleic acids probes for a target organism, wherein the probes are selected by generating a maximum number of nonoverlapping probes of a predetermined length from a gene sequence derived from the target organism, comparing the sequence of each nonoverlapping probe to sequences from a plurality of database microorganisms, identifying at least one subregion in the gene sequence derived from a target microorganism, wherein the at least one subregion contains exact matches to less than a predetermined number of the database microorganisms; and generating a set of overlapping probes of a predetermined length for the at least one subregion.
  • the number of exact matches is determined for every non-overlapping probe of a predetermined length possible in a gene sequence from an organism of interest.
  • the predetermined length can be varied and can range from 10 bases to about 20, 30, 40, 50, 60, 70, 80, 90 or 100 bases or more. In preferred embodiments, predetermined length is about 20 bases.
  • the number of exact matches can be obtained for each probe using Blast tools and public sequence databases (i.e., database sequences for database organisms). This data is then used to identify sub-regions of the genome of the organism of interest that have exact matches to less than a predetermined number of database organisms.
  • the number of database organisms to which there are exact matches can vary from about 1 organism to about 50 organisms, preferably from about 5 to about 30 organisms, and more preferably from about 10 to about 20 organisms.
  • the sub-regions range from 10 to 300 bases in length, and are more preferably from about 10 to about 20, 50, 75, 100, 150, 200, 250 or 300 bases in length.
  • Panel A of Figure 1 provides a graphical depiction of this analysis for the 16s rDNA sequence.
  • Additional probes are then generated by overlapping sequences of a predetermined number of bases in the identified sub-regions.
  • the sub-regions range from 10 to 300 bases in length, and are more preferably from about 10 to about 20, 50, 75, 100, 150, 200, 250 or 300 bases in length.
  • the sequences used for overlapping the predetermined region are preferably from about 10 to about 50 bases in length, and in some particularly preferred embodiments are from 10 to 30 bases in length, and most preferably about 19 bases in length. Since some portions of the sub-region yield greater specificity with as little as 1 base pair difference, numerous probes are identified. See, Panel A, Figure 1.
  • the probes are then validated using PCR to ensure sufficient presence of the target.
  • the optimal length and composition of target dangling-end is determined to minimize hybridization between targets, thus reducing ambiguous hybridization behavior.
  • targets are fragmented to a predetermined length to eliminate cross hybridization in a community sample and to reduce target-target dangling end hybridization.
  • a probe may include Watson-Crick bases or modified bases.
  • Modified bases include, but are not limited to, the AEGIS bases (from Eragen Biosciences), which have been described, e.g., in U.S. Pat. Nos. 5,432,272; 5,965,364; and 6,001,983.
  • bases are joined by a natural phosphodiester bond or a different chemical linkage. Different chemical linkages include, but are not limited to, a peptide bond or an LNA linkage, which is described, e.g., in published PCT applications WO 00/56748; and WO 00/66604.
  • the means for generating the polynucleotide probes is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (Froehler et al., 1986, Nucleic Acid Res. 14:5399-5407; McBride et al., 1983, Tetrahedron Lett. 24:246-248).
  • Other methods for synthesizing probes include those described in U.S> pat. Nos. 6,965,040 and 6,426,184, both of which are incorporated herein by reference.
  • Synthetic sequences are typically between about 15 and about 600 bases in length, more typically between about 20 and about 100 bases, most preferably between about 40 and about 70 bases in length.
  • target nucleic acid sequences include RNA and DNA.
  • RNA target sequences include, but are not limited to, mRNA, rRNA, tRNA, snRNA, viral RNA, and variants of RNA, such as splicing variants.
  • DNA target sequences include, but are not limited to, genomic DNA, plasmid DNA, phage DNA, nucleolar DNA, mitochondrial DNA, chloroplast DNA, cDNA, synthetic DNA, yeast artificial chromosomal DNA ("YAC”), bacterial artificial chromosome DNA (“BAC”), other extrachromosomal DNA, and primer extension products.
  • YAC yeast artificial chromosomal DNA
  • BAC bacterial artificial chromosome DNA
  • Target nucleic acid sequences also include, but are not limited to, analogs of both RNA and DNA.
  • Exemplary nucleic acid analogs include, but are not limited to, locked nucleic acids (“LNAs”), peptide nucleic acids (“PNAs”), 8-aza-7- deazaguanine (“PPG's”), and other nucleic acid analogs.
  • Exemplary target nucleic acid sequences include, but are not limited to, chimeras of RNA and DNA.
  • isolation techniques include, but are not limited to, (1) organic extraction followed by ethanol precipitation, e.g., using a phenol/chloroform organic reagent (e.g., Ausubel et al., eds., Current Protocols in Molecular Biology Volume 1, Chapter 2, Section I, John Wiley & Sons, New York (1993), herein incorporated by reference), in certain embodiments, using an automated nucleic acid extractor, e.g., the Model 341 DNA Extractor available from Applied Biosystems (Foster City, Calif); (2) stationary phase adsorption methods (e.g., Boom et al., U.S.
  • a phenol/chloroform organic reagent e.g., Ausubel et al., eds., Current Protocols in Molecular Biology Volume 1, Chapter 2, Section I, John Wiley & Sons, New York (1993), herein incorporated by reference
  • an automated nucleic acid extractor e.g., the Model 341 DNA Extract
  • a target nucleic acid sequence may be derived from any living, or once living, organism, including but not limited to, a prokaryote, a eukaryote, a plant, an animal, and a virus.
  • a target nucleic acid sequence is derived from a human.
  • the target nucleic acid sequence may originate from a nucleus of a cell, e.g., genomic DNA, or may be extranuclear nucleic acid, e.g., originate from a plasmid, a mitochondrial nucleic acid, from various RNAs, and the like.
  • the sequence from the organism is RNA, it may be reverse-transcribed into a cDNA target nucleic acid sequence.
  • the target nucleic acid sequence may be present in a double-stranded or single-stranded form.
  • target nucleic acid sequences are amplified.
  • multiple target nucleic acid sequences can be amplified in the same reaction (e.g., in multiplex amplification reactions).
  • more than one different multiplex amplification reaction is performed.
  • 5 to 10 different multiplex amplification reactions are performed.
  • 10 to 25 different multiplex amplification reactions are performed.
  • 25 to 50 different multiplex amplification reactions are performed.
  • a sufficient number of different amplification reactions can be performed such that all of the target nucleic acid sequences together represent all of the genes in a genome.
  • the genome may be derived from any living, or once living organism including but not limited to, a prokaryote, a eukaryote, a plant, an animal, and a virus.
  • the genome is prokaryotic.
  • a sufficient number of different amplification reactions can be performed such that all of the target nucleic acid sequences together represent most of the genes in a genome.
  • a sufficient number of different amplification reactions can be performed such that all of the target nucleic acid sequences together represent all of the nucleic acids in a transcriptome. In certain embodiments, a sufficient number of different amplification reactions can be performed such that all of the target nucleic acid sequences together represent most of the nucleic acids in a transcriptome.
  • transcriptome refers to the activated genes, mRNAs, and/or transcripts found in a particular tissue at a particular time.
  • Exemplary target nucleic acid sequences include, but are not limited to, amplification products, ligation products, transcription products, reverse transcription products, primer extension products, methylated DNA, and cleavage products.
  • Exemplary amplification products include, but are not limited to, PCR and isothermal products.
  • Different target nucleic acid sequences may be different portions of a single contiguous nucleic acid or may be on different nucleic acids. Different portions of a single contiguous nucleic acid may or may not overlap.
  • nucleic acids in a sample may be subjected to a cleavage procedure.
  • such cleavage products may be target nucleic acid sequences.
  • a target nucleic acid sequence is derived from a crude cell lysate. Examples of target nucleic acid sequences include, but are not limited to, nucleic acids from buccal swabs, crude bacterial lysates, blood, skin, semen, hair, bone, mucus, saliva, cell cultures, and tissue biopsies.
  • one or more target nucleic acid samples are derived from communities of microorganisms. In some embodiments, the community of microorganisms is present in a public water source, such as a well, reservoir, river, stream, or lake.
  • target nucleic acid sequences are obtained from a cell, cell line, tissue, or organism that has undergone a treatment.
  • the treatment results in the up-regulation or down-regulation of certain target nucleic acid sequences in treated cells, cell lines, tissues, or organisms.
  • a target nucleic acid sequence is obtained from a single cell. In certain embodiments, a target nucleic acid sequence is obtained from tens of cells. In certain embodiments, a target nucleic acid sequence is extracted from hundreds of cells or more. In certain embodiments, a target nucleic acid sequence is extracted from cells of a single organism. In certain embodiments, a target nucleic acid sequence is extracted from cells of two or more different organisms. In certain embodiments, a target nucleic acid sequence concentration in a PCR reaction ranges from about 1 to about 10,000,000 molecules per reaction.
  • each primer is sufficiently long to prime the template- directed synthesis of the target nucleic acid sequence under the conditions of the amplification reaction.
  • the lengths of the primers depends on many factors, including, but not limited to, the desired hybridization temperature between the primers, the target nucleic acid sequence and the complexity of the different target nucleic acid sequences to be amplified, and other factors.
  • a primer is about 15 to about 35 nucleotides in length. In certain embodiments, a primer is fewer than 15 nucleotides in length. In certain embodiments, a primer is greater than 35 nucleotides in length.
  • a set of primers comprises at least one set of primers which comprises at least one designed portion and at least one random portion.
  • the designed portion of a primer set is at the 5' end of the primers.
  • the designed portion of a primer set is at the 3' end of the primers.
  • the designed portion of a primer set is in the center of the primers.
  • the designed portion of a primer set includes two or more designed portions.
  • the designed portions of a primer set are located in two or more portions separated by random portions.
  • a polymerase is active at 37 0 C. In certain embodiments, a polymerase is active at a temperature other than 37 0 C. In certain embodiments, a polymerase is active at a temperature greater than 37 0 C. In certain embodiments, a polymerase is active at both 37 0 C. and other temperatures.
  • thermostable polymerase remains active at a temperature greater than about 42 0 C. In certain embodiments, a thermostable polymerase remains active at a temperature greater than about 50 0 C. In certain embodiments, a thermostable polymerase remains active at a temperature greater than about 60 0 C. In certain embodiments, a thermostable polymerase remains active at a temperature greater than about 70 0 C. In certain embodiments, a thermostable polymerase remains active at a temperature greater than about 80 0 C. In certain embodiments, a thermostable polymerase remains active at a temperature greater than about 90 0 C.
  • thermostable polymerases include, but are not limited to, Thermus thermophilus HB8 (described, e.g., in U.S. Pat. No. 5,789,224); mutant Thermus thermophilus HB8, including, but not limited to, Thermus thermophilus HB8 (Dl 8A; F669Y; E683R), Thermus thermophilus HB8 (A271; F669Y; E683W), and Thermus thermophilus HB8 (D18A; F669Y); Thermus oshimai (described, e.g., in U.S. Provisional Application No. 60/334,798, filed Nov. 30, 2001, corresponding to U.S. Application No.
  • mutant Thermus oshimai including, but not limited to, Thermus oshimai (G43D; F665Y); Thermus scotoductus (described, e.g., in U.S. Provisional Application No. 60/334,489, filed Nov. 30, 2001); mutant Thermus scotoductus, including, but not limited to, Thermus scotoductus (G46D; F668Y); Thermus thermophilus 1B21
  • thermophilus 1B21 including, but not limited to, Thermus thermophilus 1B21 (G46D; F669Y); Thermus thermophilus GK24 (described, e.g., in U.S. Provisional Application No. 60/336,046, filed Nov. 30, 2001), mutant Thermits thermophilus 1B21, including, but not limited to, Thermus thermophilus 1B21 (G46D; F669Y); Thermus thermophilus GK24 (described, e.g., in U.S. Provisional Application No. 60/336,046, filed Nov.
  • mutant Thermus thermophilus GK24 including, but not limited to, Thermus thermophilus GK24 (G46D; F669Y); Thermus aquaticus polymerase; mutant Thermus aquaticus polymerase, including, but not limited to, Thermus aquaticus (G46D; F667Y) (AmpliTaq.RTM. FS or Taq (G46D; F667Y), described, e.g., in U.S. Pat. No.
  • thermostable polymerase is a mutant of a naturally-occurring polymerase.
  • non-thermostable polymerases include, but are not limited to DNA polymerase I; mutant DNA polymerase I, including, but not limited to, Klenow fragment and Klenow fragment (3'>5' exonuclease minus); T4 DNA polymerase; mutant T4 DNA polymerase; T7 DNA polymerase; mutant T7 DNA polymerase; phi29 DNA polymerase; and mutant phi29 DNA polymerase.
  • a polymerase is a processive polymerase.
  • a processive polymerase remains associated with the template for two or more nucleotide additions.
  • a non-processive polymerase disassociates from the template after the addition of each nucleotide.
  • a processive DNA polymerase has a characteristic polymerization rate.
  • a processive DNA polymerase has a polymerization rate of between 5 to 300 nucleotides per second.
  • a processive DNA polymerase has a higher processivity in the presence of accessory factors, such as one or more additives.
  • the processivity of a processive DNA polymerase may be influenced by the presence or absence of accessory single-stranded DNA-binding proteins and helicases.
  • the net polymerization rate will depend on the enzyme concentration, because at higher concentrations there are more re-initiation events and thus the net polymerization rate is increased.
  • the processive polymerase is Bst polymerase.
  • Strand displacement refers to the phenomenon in which a chemical, physical, or biological agent causes at least partial dissociation of a nucleic acid that is hybridized to its complementary strand.
  • a DNA polymerase is a strand displacement polymerase.
  • a processive DNA polymerase is also a strand displacement polymerase, which is capable of displacing a hybridized strand encountered during replication.
  • a strand displacement polymerase requires a factor that facilitates strand displacement to be capable of displacing a hybridized strand encountered during replication.
  • a strand displacement polymerase is capable of displacing a hybridized strand encountered during replication in the absence of a strand displacement factor. In certain embodiments, the strand displacement polymerase lacks 5' to 3' exonuclease activity.
  • the dissociation of a nucleic acid that is hybridized to its complementary strand occurs in a 5' to 3' direction in conjunction with replication.
  • a primer extension reaction forms a newly synthesized strand while displacing a second nucleic acid strand from the template nucleic acid strand
  • both the newly synthesized and displaced second nucleic acid strand have the same base sequence, which is complementary to the template nucleic acid strand.
  • a molecule comprises both strand displacement activity and another activity.
  • a molecule comprises both strand displacement activity and polymerase activity.
  • strand displacement activity is the only activity associated with a molecule.
  • Enzymes that possess both strand displacement activity and polymerase activity include, but are not limited to, E. coli DNA polymerase 1 , the Klenow fragment of DNA polymerase 1 , the bacteriophage T7 DNA polymerase, the bacteriophage T5 DNA polymerase, the (p29 polymerase, and the Bst polymerase.
  • Certain methods of using enzymes possessing strand displacement activity include, but are not limited to, those described by, e.g., Romberg, A., DNA Replication, W.H. Freeman & Co., San Francisco, Calif., 1980.
  • strand displacement replication refers to nucleic acid replication which involves strand displacement.
  • strand displacement is facilitated through the use of a strand displacement factor, such as a helicase.
  • a DNA polymerase that can perform a strand displacement replication in the presence of a strand displacement factor is used in strand displacement replication.
  • the DNA polymerase does not perform a strand displacement replication in the absence of such a factor.
  • Strand displacement factors useful in strand displacement replication include, but are not limited to, BMRFl polymerase accessory subunit (Tsurumi et al., J.
  • SSB single-stranded DNA binding proteins
  • Strand displacement amplification (SDA) reaction methods include, but are not limited to, those described in, e.g., Fraiser et al., U.S. Pat. No. 5,648,211; Cleuziat et al., U.S. Pat. No. 5,824,517; and Walker et al., Proc. Natl. Acad. Sci.
  • the ability of a polymerase to carry out strand displacement replication can be determined by using the polymerase in a strand displacement replication assay such as those described, e.g., in U.S. Pat. No. 6,642,034, herein incorporated by reference or in a primer-block assay described, e.g., in Kong et al., J. Biol. Chem. 268:1965-1975 (1993), herein incorporated by reference.
  • an amplification reaction comprises a blend of polymerases.
  • at least one polymerase possesses exonuclease activity.
  • none of the polymerases in an amplification reaction possess exonuclease activity.
  • Exemplary polymerases that may be used in an amplification reaction include, but are not limited to, phi.29 DNA polymerase, taq polymerase, stoffel fragment, Bst DNA polymerase.
  • E. coli DNA polymerase 1 the Klenow fragment of DNA polymerase 1 , the bacteriophage T7 DNA polymerase, the bacteriophage T5 DNA polymerase, and other polymerases known in the art.
  • a polymerase is inactive in the reaction composition and is subsequently activated at a given temperature.
  • an amplification reaction composition comprising (a) two or more target nucleic acid sequences, (b) at least one set of primers, and (c) at least one polymerase.
  • an amplification reaction composition is formed comprising two or more target nucleic acid sequences, at least one primer set, dNTPs, at least one buffering agent and at least one polymerase.
  • the amplification reaction is incubated under conditions that allow the formation of one or more amplification products.
  • the amplification reaction further includes one or more additives. In certain embodiments, no strand displacement factors are required for strand displacement.
  • an amplification reaction composition comprises strand displacement factors.
  • Exemplary strand displacement factors include, but are not limited to, helicases and single stranded DNA binding protein.
  • the temperature of the reaction affects strand displacement. In certain embodiments, a temperature of approximately 40 0 C, 45 0 C, 50 0 C, 55 0 C, 60 0 C, 65 0 C, 70 0 C, 75 0 C, 80 0 C, 85 0 C, or 9O 0 C. facilitates strand displacement by allowing segments of double stranded DNA to separate and reanneal.
  • the temperature of the amplification reaction is kept at isothermal reaction conditions.
  • isothermal reaction conditions refers to conditions wherein the temperature is kept substantially constant.
  • isothermal reaction conditions prevent the template DNA from being completely disassociated.
  • short primers can hybridize to a double stranded template maintained at an isothermal temperature.
  • the primers that strand invade and anneal to the template DNA can be extended by a strand-displacing DNA polymerase.
  • an amplification process is isothermal at 50 0 C and uses Bst DNA polymerase for strand displacement and extension.
  • an amplification reaction uses a fragment of Bst DNA polymerase with the 3'>5' exonuclease activity removed ("the large fragment of Bst DNA polymerase").
  • Certain amplification methods include, but are not limited to, Random PCR or
  • PEP-PCR Primer Extension Preamplification-PCR
  • Linker Adapter PCR Manton et al., Cytogenet. Cell Genet. 66(1): 54-57 (1994), herein incorporated by reference
  • Tagged-PCR Grothues et. al., Nuc. Acids Res. 21(5) 1321-1322 (1993), herein incorporated by reference
  • Inter-Alu-PCR Billnell et.
  • DOP-PCR Degenerate Oligonucleotide Primed-PCR
  • I-PEP-PCR Improved-Primer Extension Preamplification PCR
  • LL-DOP PCR Kettler et al., Anal. Biochem.
  • multiplex amplification is used to distinguish between target nucleic acid sequences that have single nucleotide polymorphisms ("SNP").
  • one or more multiplex amplification reactions include one or more primer sets specific for two or more target nucleic acid sequences that differ at only a single nucleotide and are present in similar abundance.
  • the one or more multiplex amplification reactions further include one or more probes with different detectable labels specific for the presence or absence of that particular single nucleotide.
  • the signal from the label in the multiplex amplification reaction is detected as an indicator of the presence of one or more SNPs.
  • multiplex amplification is used for melting curve analysis.
  • a multiplex amplification reaction includes two or more primer sets specific for two or more target nucleic acid sequences of similar abundance and also includes one or more probes that intercalate into double-stranded target nucleic acid sequences and does not bind to single-stranded target nucleic acid sequences.
  • a multiplex amplification reaction includes two or more primer sets specific for two or more target nucleic acid sequences and also includes one or more probes that bind to single-stranded target nucleic acid sequences but does not bind to double-stranded target nucleic acid sequences.
  • the one or more probes includes a detectable label, and the label is detectable only when the one or more probes interact with their target nucleic acid sequences.
  • the temperature of the reaction is modified gradually and the signal from the detectable label is monitored such that the shift of the one or more target nucleic acid sequences from single-stranded to double-stranded or from double-stranded to single-stranded as a function of temperature is recorded.
  • the signal from the detectable label is monitored using realtime PCR.
  • a multiplex amplification reaction includes two or more primer sets specific for two or more target nucleic acid sequences and also includes two or more probes that bind to single-stranded target nucleic acid sequences but do not bind to double-stranded target nucleic acid sequences.
  • the two or more probes include a detectable label, and the label is detectable only when the two or more probes interact with their target nucleic acid sequence(s).
  • the temperature of the reaction is modified gradually and the signal from the detectable label is monitored such that the shift of one or more target nucleic acid sequences from single-stranded to double stranded or from double-stranded to single-stranded as a function of temperature is recorded.
  • the signal from the detectable label is monitored using real-time PCR.
  • one or more target nucleic acid sequences undergo a treatment before being included in an amplification reaction.
  • a target nucleic acid treatment selectively modifies a target nucleic acid according to the methylation state of the target nucleic acid sequence (see, e.g., Published U.S. Patent Application No. 2004-0101843, U.S. Pat. No. 6,265,171; and U.S. Pat. No. 6,331,393, all of which are herein incorporated by reference).
  • the sample from which one or more target nucleic acid sequences is derived undergoes a treatment prior to the inclusion of the target nucleic acid sequences from that sample in a multiplex amplification reaction.
  • the amplification of one or more target nucleic acid sequences from a treated sample is compared to the amplification of one or more target nucleic acid sequences from an untreated control sample.
  • the expression of one or more genes in response to the treatment is determined.
  • the products of two or more amplification reactions are combined.
  • the products of one amplification reaction may have a different amplification profile than the products of the second amplification reaction.
  • multiplex PCR reactions are performed with nucleic acid samples from a community of microorganisms.
  • primers are designed to amplify target regions identified according to the probe design and selection methods described above.
  • the amplified target regions are designated amplicons.
  • multiple target regions from a gene of interest are amplified to produce multiple amplicons.
  • multiple target regions from multiple target genes of interest are amplified.
  • the primers have a length of from 18-30 bases, a GC content of from 40- 60%, and annealing temperature of from about 52 to about 58 degrees Celsius.
  • the primers are designed for detection of multiple waterborne pathogens.
  • the primers are designed to amplify portions of virulence-associated genes. In some embodiments, about 5, 10, 12, 15 or 20 waterborne pathogens are analyzed. In some embodiments, about 10, 20, 30, 40, 50, 60 or more genes are analyzed, for example, about 10, 20, 30, 40, 50, 60 virulence- associated genes.
  • the assay design is redundant. For each organism assayed, probes are used for multiple genes from each target organism, multiple amplicons from each gene are analyzed, and multiple discreet probes are used for each amplicon. Accordingly, in some embodiments, about 5, 10, 15, 20, 30 40, or 50 to about 100 genes are analyzed in an assay of the present invention. In further embodiments, about 1, 3, 5, 10, 15 or 20 to about 50 amplicons per gene are amplified and analyzed in an assay of the present invention. In further embodiments, about 1, 3, 5, 10, 15 or 20 to about 50 probes for each amplicon are used in assay of the present invention. In some embodiments, the probes are attached to a microarray and amplicons are hybridized to the microarray, as described in more detail below.
  • a label is attached to one or more amplicons during either a first amplification with a thermostable polymerase or a second amplification with a non-thermostable polymerase.
  • the labels are included in the amplification reaction.
  • the labels have one or more of the following properties: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g., FRET (Fluorescent Resonance Energy Transfer); (iii) stabilizes hybridization, e.g., duplex formation; and (iv) provides a member of a binding complex or affinity set, e.g., affinity, antibody/antigen, ionic complexes, hapten/ligand, e.g., biotin/avidin.
  • use of labels can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods.
  • Labels include, but are not limited to, light-emitting, light-scattering, and light- absorbing compounds which generate or quench a detectable fluorescent, chemiluminescent, or bioluminescent signal (see, e.g., Kricka, L. in Nonisotopic DNA Probe Techniques (1992), Academic Press, San Diego, pp. 3-28, and Non-Radioactive Labelling, A Practical Introduction, Garman, A. J. (1997) Academic Press, San Diego).
  • Fluorescent reporter dyes useful as labels include, but are not limited to, fluoresceins (see, e.g., U.S. Pat. Nos.
  • fluorescein dyes include, but are not limited to, 6-carboxyfluorescein; 2',4',1,4,- tetrachlorofluorescein; and 2',4',5',7',l,4-hexachlorofluorescein.
  • the fluorescent label is selected from SYBR®-green, 6-carboxyfluorescein ("FAM"), TET, ROX, VICTM, and JOE.
  • a label is a radiolabel.
  • labels are hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g. intercalators and intercalating dyes (including, but not limited to, ethidium bromide and SYBR.RTM. green), minor-groove binders, and cross-linking functional groups (see, e.g., Blackburn, G. and Gait, M. Eds. "DNA and RNA structure” in Nucleic Acids in Chemistry and Biology, 2.sup.nd Edition, (1996) Oxford University Press, pp. 15-81, herein incorporated by reference).
  • intercalators and intercalating dyes including, but not limited to, ethidium bromide and SYBR.RTM. green
  • minor-groove binders include, but not limited to, ethidium bromide and SYBR.RTM. green
  • cross-linking functional groups see, e.g., Blackburn, G. and Gait, M. Eds. "DNA and RNA structure”
  • labels effect the separation or immobilization of a molecule by specific or non-specific capture, for example biotin, digoxigenin, and other haptens (see, e.g., Andrus, A. "Chemical methods for 5' non- isotopic labeling of PCR probes and primers” (1995) in PCR 2: A Practical Approach, Oxford University Press, Oxford, pp. 39-54, herein incorporated by reference).
  • different amplicons comprise detectable and different labels that are distinguishable from one another.
  • labels are different fluorophores capable of emitting light at different, spectrally- resolvable wavelengths (e.g., 4-differently colored fluorophores); certain such labeled probes are known in the art and described above, and in, e.g., U.S. Pat. No. 6,140,054, herein incorporated by reference and Saiki et al, 1986, Nature 324:163-166, herein incorporated by reference.
  • the detectable label is a fluorescent label, e.g., by incorporation of nucleotide analogs.
  • radioactive isotopes include 32 P, 35 S, 14 C, 15 N and 125 I.
  • Fluorescent molecules suitable for the present invention include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, texas red, 5'carboxy- fluorescein (FMA), 2',7'-dimethoxy-4',5'-dichloro-6-carboxy-fIuorescein (JOE), N,N,N ⁇ N'-tetramethyl-6-carboxy-rhodamine (TAMRA), 6'carboxy-X-rhodamine (ROX), HEX, TET, IRD40, and IRD41.
  • FMA 5'carboxy- fluorescein
  • JE 2',7'-dimethoxy-4',5'-dichloro-6-carboxy-fIuorescein
  • TAMRA N,N,N ⁇ N'-tetramethyl-6-carboxy-rhodamine
  • ROX 6'carboxy-X-rhodamine
  • Fluorescent molecules that are suitable for the invention further include: cyamine dyes, including by not limited to Cy3, Cy3.5 and Cy5; BODIPY dyes including but not limited to BODIPY-FL, BODIPY-TR, BODIPY-TMR, BODIPY- 630/650, and BODIPY-650/670; and ALEXA dyes, including but not limited to ALEXA- 488, ALEXA-532, ALEXA-546, ALEXA-568, and ALEXA-594; as well as other fluorescent dyes which will be known to those who are skilled in the art.
  • Electron rich indicator molecules suitable for the present invention include, but are not limited to, ferritin, hemocyanin, and colloidal gold.
  • the target polynucleotides may be labeled by specifically complexing a first group to the polynucleotide.
  • a second group covalently linked to an indicator molecules and which has an affinity for the first group, can be used to indirectly detect the target polynucleotide.
  • compounds suitable for use as a first group include, but are not limited to, biotin and imminobiotin.
  • Compounds suitable for use as a second group include, but are not limited to, avidin and streptavidin.
  • a microarray is an array of positionally-addressable binding (e.g., hybridization) sites on a support. Each of such binding sites comprises a plurality of polynucleotide molecules of a probe bound to the predetermined region on the support.
  • Microarrays can be made in a number of ways, of which several are described herein below (see e.g., Meltzer, 2001, Curr. Opin. Genet. Dev. 11(3):258-63; Andrews et al., 2000, Genome Res. 10(12):2030-43; Abdellatif, 2000, Circ. Res.
  • microarrays share certain characteristics.
  • the arrays are preferably reproducible, allowing multiple copies of a given array to be produced and easily compared with each other.
  • the microarrays are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions.
  • both larger and smaller (e.g., 0.5 cm.sup.2 or less) arrays are also contemplated and may be preferable, e.g., for simultaneously evaluating a very large number of different probes. Any of these methods described herein may be used for making microarrays comprising the probes designed and identified as described above.
  • methods of the invention utilize polynucleotide probes synthesized directly on the support to form the array.
  • the probes are attached to a solid support or surface, which may be made, e.g., from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, gel, or other porous or nonporous material.
  • plastic e.g., polypropylene, nylon
  • polyacrylamide nitrocellulose
  • gel or other porous or nonporous material.
  • microarrays may also be used.
  • any type of array for example, dot blots on a nylon hybridization membrane (see Sambrook et al., supra) could be used.
  • very small arrays will frequently be preferred because hybridization volumes will be smaller.
  • microarrays of the invention are manufactured by means of an ink jet printing device for oligonucleotide synthesis, e.g., using the methods and systems described by Blanchard in International Patent Publication No. WO 98/41531, published Sep. 24, 1998; Blanchard et al., 1996, Biosensors and Bioelectronics 11:687- 690; Blanchard, 1998, in Synthetic DNA Arrays in Genetic Engineering, Vol. 20, J. K. Setlow, Ed, Plenum Press, New York at pages 111-123; Hughes et al., 2001, Nature BioTechnology 19:342-347; and U.S. Pat. No. 6,028,189 to Blanchard, all of which are herein incorporated by reference.
  • the oligonucleotide probes in such microarrays are preferably synthesized in arrays, e.g., on a glass slide, by serially depositing individual nucleotide bases in microdroplets of a high surface tension solvent such as propylene carbonate.
  • the microdroplets have small volumes (e.g., 100 pL or less, more preferably 50 pL or less) and are separated from each other on the microarray (e.g., by hydrophobic domains) to form circular surface tension wells which define the locations of the array elements (i.e., the different probes).
  • Polynucleotide probes are attached to the surface covalently at the 3' end of the polynucleotide.
  • sample processing is through hybridization of the amplicons obtained through multiplex PCR on a nucleotide microarray.
  • the microarray is an oligonucleotide array.
  • the microarray contains in the range of 20 to 50,000 nucleic acid probes.
  • the probes are identified by the methods described in detail above.
  • the probes can be arranged in a variety of patterns.
  • the probes can be arranged in rows and columns, polygonal (e.g., hexagonal), or circular patterns, etc.
  • the microarrays comprise a redundant set of probes.
  • the microarray comprises probes for multiple genes from each target organism.
  • the microarray further comprises probes to multiple amplicons from each the multiple genes that are analyzed.
  • the microarray comprises multiple discreet probes for each amplicon.
  • the density of probes on a microarray is about 100 different (i.e., non- identical) probes per 1 cm 2 or higher. More preferably, a microarray used in the methods of the invention will have at least 550 probes per 1 cm 2 , at least 1000 probes per 1 cm 2 , at least 1500 probes per 1 cm or at least 2000 probes per 1 cm . In a particularly preferred embodiment, the microarray is a high density array, preferably having a density of at least about 2500 different probes per 1 cm 2 .
  • microarrays used in the invention therefore preferably contain at least 2500, at least 5000, at least 10000, at least 15000, at least 20000, at least 25000, at least 50000 or at least 55000 different (i.e., non-identical) probes.
  • each probe sequence may also comprise a linker (e.g., spacer) in addition to the sequence that is complementary to its target sequence.
  • a linker refers to a chemical structure between the sequence that is complementary to its target sequence and the surface.
  • the linker need not be a nucleotide sequence.
  • the linker can be composed of a nucleotide sequence, or peptide nucleic acids, hydrocarbon chains, etc.
  • the level of hybridization to the site in the array will reflect the prevalence of the corresponding complementary sequences in the sample.
  • detectably labeled e.g., with a fluorophore
  • the site on the array corresponding to a nucleotide sequence that is not in the sample will have little or no signal (e.g., fluorescent signal), and a nucleotide sequence that is prevalent in the sample will have a relatively strong signal.
  • the relative abundance of different nucleotide sequences in a sample may be determined by the signal strength pattern of probes on a microarray.
  • the target polynucleotides may be from any source.
  • the target polynucleotide molecules may be naturally occurring nucleic acid molecules such as genomic or extragenomic DNA molecules isolated from an organism, or RNA molecules, such as mRNA molecules, isolated from an organism.
  • the polynucleotide molecules may be synthesized, including, e.g., nucleic acid molecules synthesized enzymatically in vivo or in vitro, such as cDNA molecules, or polynucleotide molecules synthesized by PCR, RNA molecules synthesized by in vitro transcription, etc.
  • the sample of target polynucleotides can comprise, e.g., molecules of DNA, RNA, or copolymers of DNA and RNA.
  • the target polynucleotides of the invention will correspond to particular genes or to particular gene transcripts (e.g., to particular mRNA sequences expressed in cells or to particular cDNA sequences derived from such mRNA sequences).
  • the target polynucleotides may correspond to particular fragments of a gene transcript.
  • the target polynucleotides may correspond to different exons of the same gene, e.g., so that different splice variants of that gene may be detected and/or analyzed.
  • the target polynucleotides to be analyzed are prepared in vitro from nucleic acids extracted from cells.
  • RNA is extracted from cells (e.g., total cellular RNA, poly(A)+ messenger RNA, fraction thereof) and messenger RNA is purified from the total extracted RNA.
  • Methods for preparing total and poly(A)+ RNA are well known in the art, and are described generally, e.g. in Sambrook et al., supra.
  • RNA is extracted from cells of the various types of interest in this invention using guanidinium thiocyanate lysis followed by CsCl centrifugation and an oligo dT purification (Chirgwin et al., 1979, Biochemistry 18:5294-5299).
  • total RNA is extracted from cells using guanidinium thiocyanate lysis followed by purification on RNeasy columns (Qiagen).
  • cDNA is then synthesized from the purified mRNA using, e.g., oligo-dT or random primers.
  • the target polynucleotides are cRNA prepared from cDNA prepared from purified mRNA or from total RNA extracted from cells.
  • cRNA can either be complementary to (anti-sense) or of the same sequence (sense) as the sample RNA.
  • the extracted RNA molecules are amplified using a process in which double-stranded cDNA molecules are synthesized from the sample RNA molecules using primers linked to an RNA polymerase promoter.
  • RNA polymerase promoters can be incorporated into either or both strands of the cDNA.
  • cRNA can be synthesized that is the same sequence as the sample RNA.
  • RNA polymerase promoter that is on the second strand of the double-stranded cDNA molecule using an RNA polymerase
  • RNA polymerase see, e.g., U.S. Pat. Nos. 5,891,636, 5,716,785; 5,545,522 and 6,132,997; see also, U.S. Pat. No. 6,271,002 and U.S. Provisional Patent Application Ser. No. 60/253,641, filed on Nov. 28, 2000, by Ziman et al., all of which are herein incorporated by reference).
  • Both oligo-dT primers U.S. Pat. Nos.
  • RNA polymerase promoter or complement thereof
  • the target polynucleotides are short and/or fragmented polynucleotide molecules which are representative of the original nucleic acid population of the cell.
  • total RNA is used as input for cRNA synthesis.
  • An oligo- dT primer containing a T7 RNA polymerase promoter sequence can be used to prime first strand cDNA synthesis.
  • random hexamers can be used to prime second strand cDNA synthesis by a reverse transcriptase. This reaction yields a double-stranded cDNA that contains the T7 RNA polymerase promoter at the 3' end. The double-stranded cDNA can then be transcribed into cRNA by T7 RNA polymerase.
  • the target nucleic acids are amplified as described in detail above.
  • the target polynucleotides to be analyzed are preferably detectably labeled.
  • cDNA can be labeled directly, e.g., with nucleotide analogs, or indirectly, e.g., by making a second, labeled cDNA strand using the first strand as a template.
  • the double-stranded cDNA can be transcribed into cRNA and labeled.
  • amplicons are labeled by including suitable labeled substrates during amplification with either thermostable or non-thermostable polymerases.
  • nucleic acid hybridization and wash conditions are chosen so that the polynucleotide molecules to be analyzed (or target polynucleotide molecules) specifically bind or specifically hybridize to the complementary polynucleotide sequences of the array, preferably to one or more specific array sites, wherein its complementary sequence is located.
  • Arrays containing double-stranded probe DNA situated thereon are preferably subjected to denaturing conditions to render the DNA single-stranded prior to contacting with the target polynucleotide molecules.
  • Arrays containing single-stranded probe DNA may need to be denatured prior to contacting with the target polynucleotide molecules, e.g., to remove hairpins or dimers which form due to self complementary sequences.
  • Optimal hybridization conditions will depend on the length (e.g., oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA, or DNA) of probe and target nucleic acids.
  • the fluorescence emissions at each site of a transcript array can be, preferably, detected by scanning confocal laser microscopy.
  • a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used.
  • a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al., 1996, Genome Res. 6:639-645, herein incorporated by reference).
  • the arrays are scanned with a laser fluorescence scanner with a computer controlled X-Y stage and a microscope objective.
  • Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser, and the emitted light is split by wavelength and detected with two photomultiplier tubes.
  • fluorescence laser scanning devices are described, e.g., in Schena et al., 1996, Genome Res. 6:639-645.
  • the fiber-optic bundle described by Ferguson et al., 1996, Nature BioTechnology 14:1681-1684, herein incorporated by reference may be used to monitor mRNA abundance levels at a large number of sites simultaneously.
  • Signals are recorded and, in a preferred embodiment, analyzed by computer, e.g., using a 12 bit or 16 bit analog to digital board.
  • the scanned image is despeckled using a graphics program (e.g., Hijaak Graphics Suite) and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site. If necessary, an experimentally determined correction for cross talk (or overlap) between the channels for the two fluors may be made. For any particular hybridization site on the transcript array, a ratio of the emission of the two fluorophores can be calculated.
  • arrays are designed for the high-thoughput analysis of multiple waterborne pathogens.
  • the assays are configured to detect the presence of from about 5, 10, 15, or 20 to about 30 different waterborne pathogens.
  • the assays of the present invention are designed to detect the presence of at least one of Aeromonas hydrophila, Campylobacter jejuni, Clostridium per fringens, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Vibrio cholerae, Vibrio parahaemolyticus, and Yersinia enter ocolitica and combinations thereof.
  • target regions of Virulence Maker Genes are amplified and then hybridized to a microarray comprising probes that hybridize to the VMGs.
  • the microarray comprises about 5, 10, 15, 20, 25, or 30 to about 100 different probes for each VMG for each organism.
  • the probes to multiple VGAs from a single organism are included on the microarray and multiple different probes per amplicon from a given target VGA are included on the microarray.
  • the probes are identified as described in detail above.
  • Figure 7 provides a list of exemplary probes identified by the methods described herein and that are suitable for use on microarrays of the present invention. Accordingly, in some embodiments, the microarrays of the present invention comprise any of probes encoded by SEQ ID Nos. 1- 791, or combinations thereof.
  • the arrays are analyzed using the system described in co- pending application entitled, Electroluminescent-Based Fluorescence Detection Device, filed November 22, 2006, incorporated herein by reference.
  • the divice described below can be utilized in combination with the primers, probes and microarrays described herein.
  • the present invention provides fluorescence detection devices comprising an electroluminescent light (EL) source that provide static and/or real-time fluorescent read-outs in a number of formats including visual and digital.
  • EL electroluminescent light
  • the present invention provides fluorescence detection devices comprising an electroluminescent light (EL) source that provides PCR assay capabilities, such as thermal cycling and/or isothermal amplification assays, computational capabilities for data read-outs, and read-out capabilities in a number of formats including visual and digital.
  • EL electroluminescent light
  • reactions include, but are not limited to, chemical and biological reactions.
  • Biological reactions include, but are not limited to mRNA transcription, nucleic acid amplification, DNA amplification, cDNA amplification, sequencing, and the like. It is also not intended that the invention be limited by the particular purpose for carrying out the biological reactions. In one diagnostic application, it may be desirable to simply detect the presence or absence of a particular pathogen. In another diagnostic application, it may be desirable to simply detect the presence or absence of specific allelic variants of pathogens in a clinical sample.
  • the present invention provides a device, comprising, a) an electroluminescent light source, b) an excitation filter, c) a biological sample holder, and d) an emission filter, wherein said biological sample holder, is disposed between said excitation filter and said emission filter and said electroluminescent light source is adjacent to said excitation filter so that light produced by said electroluminescent light source passes through said excitation filter to illuminate said biological sample holder.
  • the present invention is not limited to a particular electroluminescent light source.
  • the present invention is not limited to a particular biological sample holder. Indeed, a variety of biological sample holders may be used, including, but not limited to a biological sample holder of the present invention.
  • the biological sample holder is compatible with a PCR chip.
  • the biological sample holder is compatible with a microarray chip comprising the probes described above.
  • the biological sample holder is stationary. In one embodiment, the biological sample holder is mobile.
  • amplified target nucleic acid sequences can be used for any purpose for which nucleic acids are used.
  • Certain exemplary uses for amplification products include, but are not limited to, forensic purposes, genotyping, sequencing, detecting SNPs, detecting microsatellite DNA, detecting expression of genes, quantifying expression of genes, nucleic acid library construction, melting curve analysis, and any other purpose that involves manipulating and/or detecting nucleic acids or nucleic acid sequences.
  • amplification products may be used in any process that uses nucleic acids.
  • exemplary assays in which amplification products may be used include, but are not limited to, agarose gel electrophoresis, picogreen assays, oligonucleotide ligation assays, and assays described in U.S. Pat. Nos. 5,470,705, 5,514,543, 5,580,732, 5,624,800, 5,807,682, 6,759,202, 6,756,204, 6,734,296, 6,395,486, U.S. patent application Ser. Nos. 09/584,905 and 09/724,755, and Published U.S. Patent Application No. US 2003-0190646 Al, all of which are herein incorporated by reference.
  • amplification products are treated before they are used in a downstream process. Such treatments include, but are not limited to, heating or enzymatic digestion of amplification products prior to their use in a downstream process.
  • a high-throughput assay system includes a plurality of multiplex amplification reactions.
  • the plurality of multiplex amplification reactions is contained on one or more plates or cards, in separate reaction spaces (including, but not limited to, wells or spots).
  • each of the plurality of multiplex amplification reactions amplifies two to five target nucleic acid sequences of similar abundance.
  • each of the plurality of multiplex amplification reactions amplifies more than five target nucleic acid sequences of similar abundance.
  • each of the plurality of multiplex amplification reactions includes a sufficient number of differently-labeled probes such that the amplification product of each target nucleic acid sequence can be separately identified.
  • the amplification reaction proceeds using real-time PCR.
  • Exemplary high-throughput assay systems include, but are not limited to, an Applied Biosystems plate-reader system (using a plate with any number of wells, including, but not limited to, a 96-well plate, a-384 well plate, a 768-well plate, a 1,536- well plate, a 3,456-well plate, a 6,144-well plate, and a plate with 30,000 or more wells), the ABI 7900 Micro Fluidic Card system (using a card with any number of wells, including, but not limited to, a 384- well card), other microfluidic systems that exploit the use of TaqMan probes (including, but not limited to, systems described in WO 04083443 Al, and published U.S. Patent Application Nos.
  • an Applied Biosystems plate-reader system using a plate with any number of wells, including, but not limited to, a 96-well plate, a-384 well plate, a 768-well plate, a 1,536- well plate, a
  • a high-throughput multiplex amplification assay system is capable of analyzing most of the genes in a genome on a single plate or card. In certain embodiments, a high-throughput multiplex amplification assay system is capable of analyzing all genes in an entire genome on a single plate or card.
  • a high-throughput multiplex amplification assay system is capable of analyzing most of the nucleic acids in a transcriptome on a single plate or card. In certain embodiments, a high-throughput multiplex amplification assay system is capable of analyzing all of the nucleic acids in a transcriptome on a single plate or card.
  • an entire gene When referring to analyzing most of the genes in a genome by performing one or more amplification reactions, for each gene analyzed, either an entire gene may be amplified or a portion of an entire gene may be amplified. When referring to analyzing all of the genes in a genome by performing one or more amplification reactions, for each gene analyzed, either an entire gene may be amplified or a portion of an entire gene may be amplified. When referring to analyzing most of the nucleic acids in a transcriptome by performing one or more amplification reactions, for each nucleic acid analyzed, either an entire nucleic acid or a portion of an entire nucleic acid may be amplified. When referring to analyzing all of the nucleic acids in a transcriptome by performing one or more amplification reactions, for each nucleic acid analyzed, either an entire nucleic acid or a portion of an entire nucleic acid may be amplified.
  • N normal
  • M molar
  • mM millimolar
  • ⁇ M micromolar
  • mol molecular weight
  • mmol millimoles
  • ⁇ mol micromol
  • nmol nanomoles
  • pmol picomoles
  • g grams); mg (milligrams); ⁇ g (micrograms); ng (nanograms); pg (picograms); L and 1 (liters); ml (milliliters); ⁇ l (microliters); cm (centimeters); mm (millimeters); ⁇ m (micrometers); nm (nanometers); U (units); min (minute); s and sec (second); deg (degree); 0 C (degrees Centigrade/Celsius), polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • This Example describes the development of PCT primers and oligonucleotide probes used in a microarry assay for waterborne bacterial pathogens.
  • Oligonucleotide probe design and chip synthesis Two sets of probes were designed and included on a test microarray.
  • a total of 47 PCR primer sets were designed for these genes (Table 1), with each amplicon being targeted by 7 to 35 probes, with an average of 17 probes per amplicon.
  • VMG primers and probes designed and selected in this study were described to detect the selected pathogens in PCR-based assays.
  • Probes were selected to satisfy the following criteria i) the probes were complementary to all retrieved sequences of a given gene with species level specificity, and ii) the probes contained at least two mismatches to all other non-target sequences within the database. At least 2,825 probes were selected satisfying these criteria.
  • Each of the 47 VMG regions obtained in this study was targeted by, on average, 17 probes with a range of 7 to 35 probes per region (Table 1).
  • the designed probes were synthesized in situ on microfluidic chips using a proprietary light-directed synthesis technology developed by the University of Michigan (Gao, X.L., et al., Nucleic Acids Research, 2001. 29(22):p. 4744-4750, herein incorporated by reference) and commercialized by Xeotron (Houston, TX, now part of Invitrogen, Carlsbad, CA).
  • the microfluidic chip contained 8,000 microreactors with a diameter of 50 ⁇ m and were interconnected with flow channels (Fig. 2a).
  • the in situ synthesis technology employed conventional phosphoramidite chemistry with photogenerated acid for deprotection of the 5'-hydroxyl groups of nucleotide monomers for the synthesis of oligonucleotide probes.
  • the probes were attached to the substrate via a spacer consisting of Ts and Ci 8 with an effective length of 12 nucleotides.
  • the microarray also contained at least 22 randomly spaced control spots containing solely linker chemistry to assess background signal intensity
  • Genomic DNA from at least 12 reference bacteria was used to validate the VMG biochip.
  • Cultures of A. hydrophila (ATCC7966), Clostridium perfringens (ATTC 12916), Salmonella (ATCC13311), L monocytogenes (ATCC 15313), P. aeruginosa (ATCC 10145), V. parahaemolyticus (ATCC43996), and Y. enterocolitica (ATCC55075) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown overnight according to the provided instructions. Cells were grown overnight according to the instructions provided by ATCC for the respective organisms.
  • Genomic DNA was , extracted from 1 to 2 ml of the cultures using the DNeasy Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.
  • V. cholerae ATCC39315
  • C jejuni ATCC700819
  • H. pylori ATCC700392
  • S. aureus ATCC700699
  • L. pneumophila ATCC33152
  • Tap water, river water, and tertiary effluent from a wastewater treatment plant were collected as described herein.
  • the samples were filtered through 0.45 ⁇ m nitrocellulose filters (Millipore, Billerica, MA) upon arrival in the lab.
  • DNA was extracted from the microorganisms accumulated on the filters by cutting the filters into at least 8 pieces and processing the filter pieces using the MegaPrep UltraCleanTM Soil DNA Kit (Mo Bio Laboratories, Carlsbad, CA) according to the provided instructions. Extracts were further purified through ethanol precipitation, followed by final purification with the QIAquick PCR purification kit (Qiagen).
  • DNA quantification was performed with NanoDrop® ND- 1000 spectrophotometer using a wavelength of 260 nm (NanoDrop Technologies, Wilmington, DE). Polymerase chain reaction amplification of target genes. Forty seven primer sets were designed to flank regions of high probe density in 35 VMGs (Table 1). Primers were selected to have similar annealing temperature (two sets; one with 53 0 C and another with 58 0 C) and for covering the majority of alleles of a given VMG available in the GenBank database. Primers were synthesized and obtained from Integrated DNA Technologies (Corallville, IA).
  • primers Uniqueness of the primers was confirmed by Blast search against the GenBank database. To ensure primer specificity, mismatches to related non-target sequences were located near the 3' end of the primer.
  • primers were segregated into five primer sets with each set containing nine to ten primer pairs. Combinations of primer sets were selected so that each pathogen was targeted in at least two different multiplex PCR reactions (except for P. aeruginosa).
  • PCR reaction mixtures (25 ⁇ l) consisted of Ix PCR buffer, 2 mM MgCl 2 , 1.5 (monoplex PCR) or 3 units (multiplex PCR) of AmpliTaq Gold (Roche Molecular Systems, Switzerland), 200 ⁇ M deoxynucleoside triphosphates (Invitrogen), 500 nM each primer, 200 ng BSA (New England Biolaboratories, Beverly, MA), and a 1 ⁇ l of DNA solution.
  • PCR mixtures were purified using a QIAquick PCR purification kit (Qiagen), and amplification products quantified with a NanoDrop ND- 1000 spectrophotometer (NanoDrop Technologies). Fluorescent labeling of DNA targets (PCR products).
  • PCR products pooled from different PCR reactions were labeled according to an amino-allyl dUTP (aa-dUTP) incorporation and cyanine-dye coupling protocol as described (Stedtfeld, et al., 2007. Appl. Environ. Microbiol. 73:380-389, 58; Wick, et al., Nucleic Acids Res. 34:e26, herein incorporated by reference) with minor modifications. Klenow reactions were incubated for 2 hr instead of 90 min, and dye coupling reactions were incubated for 80 instead of 60 minutes.
  • a-dUTP amino-allyl dUTP
  • PCR products were amplified and aa-dUTP was incorporated in the amplification products with the Bioprime DNA labeling kit (Invitrogen, San Diego, CA) using a 5:1 aa-dUTP:dTTP ratio and an incubation time of 120 min.
  • Purified products were then coupled with cyanine dye by incubation for 80 min in a 1 : 1 mixture of 0.1 M sodium carbonate buffer (pH 9.3) and iV-hydroxysuccinimide ester cyanine dye (Amersham Biosciences).
  • the initial point of the melting curve profile was obtained by washing the chip with high stringency wash buffer (20 mM NaCl, 10 mM Na 2 PO 4 , 5 mM Na 2 EDTA, pH adjusted to 6.6 with HCl) for 1.4 min at 25 0 C, and imaging of the chip.
  • High stringency wash buffer was degassed under vacuum to prevent formation of air bubbles during the wash steps.
  • Subsequent development of the melting profile was performed by manual cycles of washing and scanning of the chip, repeated at 1 degree intervals until 60 0 C was reached. At the end of this series, the chip was additionally stripped for 2.5 min each at 60, 45 and 30 0 C with nuclease-free water (Sigma).
  • Hybridization signal intensities were obtained from scanned images using GenePix 5.0 (Axon Instruments, Union City, CA). Fluorescence signal intensities were extracted from the scanned images using GenePix5.0 (Axon Instruments, Union City, CA), yielding values between 0 and 65,535 arbitrary units (a.u.). The median of all pixel intensities within a spot was used as raw spot intensity. Subsequent data analysis was done with Microsoft Excel (Microsoft, Redmond, WA), and plotting with SigmaPlot 9.0 (Systat Software, Point Richmond, CA).
  • Raw spot intensities were divided by the mean intensity of 22 empty control spots to yield signal-to-noise ratios (SNRs) (Stedtfeld., et al., Appl. Environ. Microbiol. 73:380-389, herein incorporated by reference).
  • SNRs signal-to-noise ratios
  • the SNR was computed for each wash step between 30 and 45 °C.
  • An overall SNR was subsequently obtained by averaging all SNR within this interval to include variation in both signal strength and probe-target dissociation behavior. This estimation is equivalent to calculating an overall SNR based on the area under the melting curve within this temperature interval.
  • the SNR for all probes was normalized by dividing by the median SNR of the 2,034 not-targeted probes.
  • the SNR obtained after this normalization step served as the basis for positive/negative calls, with probes yielding a normalized SNR equal or greater than 3 considered positive.
  • SNR were additionally normalized to the replicate with the median SNR (for triplicate experiments), or the replicate with the lowest SNR (for duplicate experiments) using the slope of the linear regression line between replicates as normalization factor.
  • the PF was computed for each probe set by dividing the number of probes with SNR > 3 within a set by the number of probes within that set (Wilson et al., MoI. Cell. Probes 16:119-127, herein incorporated by reference).
  • a first set of samples was prepared by spiking 10 pg of genomic DNA of each pathogen (equivalent to approximately 1,400 to 5,500 genome copies depending on the pathogen) into 10 ng of DNA from the different water samples, yielding a relative abundance of 0.1% for each of the pathogens. Both spiked and unspiked samples were enriched using the multiplex PCR assays. The amplicons prepared from spiked water samples were labeled with Cy3 and the amplicons from unspiked water samples were labeled with Cy5. Equal amounts of both samples (100 pmol of each dye or ⁇ 2.5 ⁇ g of DNA) were mixed and hybridized in duplicate. For the unspiked water samples, 2,034 not-targeted probes were evaluated to address assay specificity.
  • a signal was considered positive when its intensity was at least three times the background signal intensity (SNR>3).
  • SNR background signal intensity
  • the SNR were additionally normalized to the replicate with the median SNR (for triplicate experiments), or the replicate with the lowest SNR (for duplicate experiments).
  • the metric for presence/absence calls at the VMG level was the positive fraction. See Wilson, W.J., et al., Sequence-specific identification of 18 pathogenic microorganisms using microarray technology. Molecular and Cellular Probes, 2002. 16(2): p. 119-127, herein incorporated by reference.
  • the positive fraction was calculated by dividing the number of probes designed for the VMG by the number of probes with positive signal for the VMG. VMGs with positive fractions of 0.5 or higher were scored as present. Gibbs free energy calculation.
  • a temperature of 43°C was used instead of the actual 2O 0 C hybridization temperature to account for the presence of 35% formamide in the hybridization solution. See Blake, R.D. and S. G. Delcourt, Thermodynamic effects of formamide on DNA stability. Nucleic Acids Research, 1996. 24(11): p. 2095-2103; Urakawa, H., et al., Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses. Applied and Environmental Microbiology, 2002. 68(1): p. 235-244, all of which are herein incorporated by reference). The Na+ concentration used was 1 M.
  • Probe success rate was evaluated through hybridization of a target mixture containing all 47 VMG amplicons obtained by individual PCR amplifications. Of the 47 amplicons, 24 were labeled with Cy3 and 23 were labeled with Cy5, Miller et al., An In- situ Synthesized Virulence and Marker Gene (VMG) Biochip for the Detection of Bacterial Pathogens in Water, in preparation for submission, herein incorporated by reference).
  • VMG Virulence and Marker Gene
  • a SNR of 3 was used as a threshold for positive signals.
  • the total number of probes (2,825) was divided into targeted (720) and non targeted probes (2,105).
  • Twenty four VMG targets were labeled with Cy3 and twenty three VMG targets were labeled with Cy5 (Table 1). Both samples were mixed and hybridized to the VMG chip. Using this experimental set-up, 5,650 possible hybridization events could be analyzed. Evaluations consider probes presented with target (targeted probes) and probes not presented with target (non-targeted probes) separately.
  • a positive fraction (PF) was calculated each probe set (and corresponding amplicon) by dividing the number of positive probes by the number of interrogated probes within that set.
  • a PF obtained based on the number of initially designed probes is equivalent to the success level of probe selection for individual probe sets.
  • 22 sets displayed a positive fraction of 1 , indicating that 100% of the probes in that set yielded positive signal.
  • Eighteen sets displayed a positive fraction between 1 and 0.8, and 7 sets displayed a positive fraction between 0.8 and 0.6 (Fig. 2C).
  • Wilson et al. (Wilson, W.J., et al., Sequence-specific identification of 18 pathogenic microorganisms using microarray technology.
  • the number of designed probes also demonstrates the excellent separation between the positive fractions for targeted and not-targeted probe sets (Fig. 2D). It is also evident that the positive fraction was not related to the number of designed probes. High success rates (>90%) for the design of oligonucleotide probes have been described in numerous recent studies for 16S rRNA genes and sets of functional genes, and demonstrates that current probe design approaches are adequate and successful. Substantial differences in SNR were observed among probes targeting the same
  • Multiplex amplification of VMG targets The use of multiplex PCR can greatly increase the efficiency of sample analysis, but it can also introduce bias. This bias was examined by hybridizing targets produced in multiplex PCR and comparing the VMG chip response to that obtained with individually amplified gene regions. Out of 720 probes giving positive signal in response to individually amplified targets, 673 yielded positive signals in response to multiplex amplified targets (Table 1). Twenty eight of the probe sets responded the same to both individually amplified and multiplex amplified targets. Thirteen probe sets displayed a one probe difference, three sets displayed a two probe difference, one set displayed a three probe difference, and one set displayed a five probe difference between individually and multiplex amplified targets.
  • the probes that did not respond to multiplex targets displayed relatively low SNR in response to individually amplified targets (data not shown).
  • Eighteen of 20 probes for the ctxB gene of V. cholera ctxB displayed a differential response to multiplex amplified targets. This gene regions was not amplified to an abundance detectable via the VMG chip in multiplex PCR. Only ctxB of V. cholerae was not amplified to a level detectable by the microarray, and displayed a positive fraction of 0.2 (2 positive out of 20 probes).
  • Success level of probes as function of ⁇ G°d U piex- The use of hybridization free energy ( ⁇ G0) was evaluated as a parameter to facilitate in silico probe selection. 791 targeted probes were sorted according to ⁇ G°d U pie ⁇ , binned (40 probes per bin), and the percent positive probes in each bin calculated. The latter is equivalent to the success level for probes with ⁇ G° dU pi ex within the bin range. A drastic decrease in the success rate was observed for probes with a ⁇ G° dup i ex less negative than -17 kcal/mole (Fig. 4), and a corresponding G+C content lower than of 34.4%.
  • probe hybridization ⁇ GO does have value as a selection parameter, it can be used to predict the behavior of groups of probes with accuracy (Fig. 5). Furthermore, strong hybridization signals were mainly observed by the inventors for probes with a highly negative ⁇ G°d up iex-
  • a ⁇ G°d up i ex threshold of -17 kcal/mole serves as a valuable design rule for future oligonucleotide probe (18-mers) screening exercises.
  • This recommendation is in agreement with Loy et al. who proposed a ⁇ G° dup i ex threshold of -16 kcal/mole for 18-mer probes targeting the 16S rRNA gene. The latter was suggested based on the observation that the majority of probes displaying positive signal (including cross-hybridization signals) were attributed to duplexes with a ⁇ G° du pi ex more negative than -16 kcal/mole.
  • probe design rules derived from such hybridizations may not be stringent enough to be applied to nucleic acid target mixtures obtained from environmental samples, containing low abundance target sequences in the presence of excess of non-target sequences.
  • Genomic DNA of 12 pathogens was spiked at an abundance of 0.1% into DNA obtained from three background samples: tap water (drinking water), river water and waste water treatment plant tertiary effluent. Eleven pathogens were detected in all three backgrounds, with all of the gene regions for these organisms indicating presence (Table 1). The response of S. aureus probes indicated presence in all three backgrounds, although the gene regions were in disagreement between background samples. The gene regions of C. perfringens were not detected in any of the background samples. This is likely due to the low G+C content of the C. perfringens probe sets.
  • the median positive fractions of the 46 targeted probe sets were 0.8, 0.82, and 0.79 in tap water, tertiary effluent, and river water, respectively (Fig. 6). This precision in three different background samples illustrates the robustness of the chip.
  • the median positive fraction for the 67 non-targeted gene regions were 0.03, 0.09, and 0.18 for the three backgrounds, with tap water resulting in the narrowest distribution of non-targeted fractions and river water resulting in the widest. This could be due to the relative complexity of the background samples, with a greater amount of cross hybridization expected in samples containing greater diversity of target sequence.
  • Using a positive fraction threshold of 0.5 the number of detectable gene regions was 39, 40, and 37, again illustrating the robustness of the chip. Positive fractions presented in the preceding analysis were based on the 673 probes that produced positive signals in response to multiplex amplified targets.
  • Wilson et al. was able to detect in a background of DNA obtained from air samples based on a positive fraction threshold of 0.8.
  • Other studies described lower detection limits, albeit in different matrices.
  • Pathogen templates spiked into tertiary effluent background DNA at an abundance of 0.001% resulted in no detection.
  • Probe selectivity defined as the difference between the percent for the targeted and non- targeted probes, is presented in Fig. 6 as a function of ⁇ G0. The highest selectivity of 80% was obtained for probes with a ⁇ G0 of approximately -19.3 kcal/mole. Probes with a ⁇ G0 higher or lower than this value displayed lower selectivity. More than 70% selectivity was obtained for probes with a ⁇ G0 between approximately -18.6 and -21.1 kcal/mole, which correlates to a G+C content of 47.2 ⁇ 5%.
  • region 1 with a ⁇ G0 lower than -19 kcal/mole and region 2 with a ⁇ G0 higher than -19 kcal/mole.
  • the positive bin fraction of the targeted probes is consistent at 0.9 in region 1 while the positive bin fraction of the non-targeted probes decreases with a slope of -0.07.
  • the positive bin fraction of the targeted probes decreases with a slope of -0.17 in region 2 while the positive bin fraction of the non-targeted probes remains relatively constant.
  • a probe having a more negative ⁇ G0 is more likely to give a positive signal, but it is also more prone to cross hybridization with non-targets (Held, G.A., G. Grinstein, and Y. Tu, Modeling of DNA microarray data by using physical properties of hybridization. Proceedings of the National Academy of Sciences of the United States of America, 2003. 100(13): p. 7575-7580 and Fig. 7 top panel).
  • a probe having a more positive ⁇ GO has a low tendency to hybridize with non-targets as well as a lower likelihood to give a positive signal.
  • the increase in cross hybridization positive bin fraction is less drastic than the decrease in target hybridization positive bin fraction as you move in the more positive direction. This means that if there is a choice between selecting a probe that has a ⁇ GO of -20 or -18 kcal/mole, the former is more likely to give a higher selectivity. Consequently, it is more difficult to obtain quality probes for and detect lower G+C content regions than higher G+C content regions.
  • probes were sorted according to their ⁇ GO values, and the fraction of probes with positive signal was calculated for bins of 40 targeted probes and bins of 80 non-targeted probes (Fig. 6 top panel).
  • the hybridizations using tap water as a background resulted in very little non-targeted probe signal (cross-hybridization), so the non-targeted probes from that background were not included in this analysis.
  • the targeted probes from all three background experiments were assessed.
  • Probe selectivity defined as the capability of a probe to detect intended targets with the exclusion of non-targets, is the parameter that needs to be optimized during probe selection. Probe selectivity was accordingly calculated as the percent of targeted probes giving positive signal minus the percent of non-targeted probes yielding positive signals.
  • a peak shaped profile was observed when plotting the selectivity as function of ⁇ GO (Fig. 5B).
  • the highest selectivity (approximately 80%) was observed for probes with a ⁇ GO of -19.3 kcal/mole. Probes with a ⁇ GO different from this optimum displayed lower selectvities. Higher than 70% selectivity was observed for probes with a ⁇ GO between -18.6 and -21.1 kcal/mole.
  • this amounts to a G+C content range between 43.4 and 56.3%, with an optimum at 47.2%.
  • Hybridization free energy was shown to be a valuable parameter for the selection of promising probes.
  • Probe selectivity was highly depended on ⁇ GO, and a maxium was observed for probes with a ⁇ GO of -19.3 kcal/mole.
  • the capability of theoretical (solution-based) models to predict hybridization signals on microarrays, and their applicability in facilitating probe selection remains in debate. See Loy, A., et al., 16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales". Applied and Environmental Microbiology, 2005. 71(3): p.
  • ⁇ G°d up ⁇ e ⁇ is a valuable parameter that can be used to increase the success rate of in silico probe selection.
  • the inventors developed and validated a coupled format of multiplex PCR and an in situ synthesized DNA biochip for the detection of 12 bacterial pathogens in various water samples. By using redundant probe sets targeting various VMGs and inference of presence/absence calls based on the PF, false-positive calls could be eliminated. Pathogens could be detected at a relative abundance level of 0.1% to 0.01%, depending on the pathogen and VMG.
  • Genomic DNA from 17 bacterial pathogens (Table 1) was used as the source of virulence and marker genes.
  • A. hydrophila, C. perfringens, E. faecalis, L. monocytogenes, L. pneumophila, P. aeruginosa, S. enterica, V. parahaemolyticus, Y. enterocolitica type strains were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and grown as per the protocol provided.
  • ATCC American Type Culture Collection
  • VA Manassas, VA
  • H. pylori, C. jejuni, C. parvum, G. intestinalis, K. pneumoniae, S. aureus, V. cholerae only genomic DNA was obtained from the ATCC.
  • E. coli was kindly provided by Dr. Thomas Whittam at Michigan State University. DNA from pure cultures was extracted using Promega Wizard DNA Extraction Kit (Promega, Madison, WI).
  • Genomic DNA Background Samples Tap water (drinking water) (40 L), river water (10 L) from the Red Cedar River in East Lansing (MI), and tertiary effluent (20 L) from a local wastewater treatment plant (East Lansing, MI). Samples were filtered through 0.45 ⁇ m nitrocellulose filters (Millepore, Billerica, MA) immediately after collection. Genomic DNA was extracted from the filters as instructed with the MegaPrep UltraCleanTM Soil DNA Kit (Mo Bio Laboratories, Carlsbad, CA). Genomic DNA was purified further through ethanol precipitation, followed by final purification with the QIAquick PCR purification kit (Qiagen).
  • PCR Primer Design Ninety-six virulence and marker genes (VMG) from 20 human pathogens were targeted. Primer design consisted of the following: i. Kodon (Applied Maths, Austin, TX) was used to generate 96 consensus sequences for each VMG (>4 genes for each organism), ii. Primer Express (Applied Biosystems, Foster City, CA) was used to design primers from the consensus sequence.
  • amplicons had a maximum length of 150 bases and Tm of 59 0 C. Some genes required longer amplicons ( ⁇ 250 bases) for generating acceptable primer pairs, iii.
  • Designed primers were BLASTed against Genbank database using a script that automatically highlighted unspecific primers. Specificity was based on the extent of 3 ' end perfect matches to other bacterial sequences. Sequences were selected manually based on results of BLAST output, iv. When available, primers described in the literature 5 for successful quantitative PCR were also used. Overall, 110 primers pairs were designed and extracted from the literature.
  • PCR reaction mixtures (10 ⁇ l per sample or 5 ⁇ l for each sample array) consisted of 1 x LightCycler Fast Start DNA Master SYBR Green I Kit (Roche Applied Sciences, Indianapolis, IN), 1.6 x SYBR Green I, 0.5% Glycerol, 0.2% Pluronic F-68, 1 mg per ml BSA (New England Biolaboratories, Beverly, MA), 2.5 mM MgCl 2 , 8% formamide, and a DNA mixture.
  • Genomic DNA from 17 pathogenic organisms was tested individually (6 ng in total sample or 20 pg per reaction well). 2) Genomic DNA from 4, 8, and 17 pathogenic organisms was mixed and tested simultaneously (20 pg per reaction well per organism). 3) Genomic DNA from 17 pathogens was mixed together and spiked at
  • Aeromonas hydrophila alt, exeF, tapA, arcV 5 7966 Burkholderia mallei, pseudomallei pilA, pilD, orfl 3, orfl l, 4
  • Giardia intestinalis lamblia 5-giardin, VSPH71, VSP4177, 30888
  • This example describes developmental stages of microfluidics systems for use in detecting pathogens using PCR primers, 20 mer and 50 mer PCR oligonucleotide probes 5 designed and used according to methods of the present invention. Further, this example demonstrates the use of these oligonucleotide probes in combination with microfluidic and serpentine chips (for example, see, Figures 10 and 11) for PCR reactions, (Hashsham, et al., Microbe, Volume 2, Number 11, 2007, herein incorporated by reference) ( Figure 14).
  • Microfluidics-based assays were used for detecting and quantifying infectious agents by hybridizing PCR amplified products onto oligonucleotide probes.
  • the inventors developed and validated a chip (Fig. 11) containing 8,000 microreactors, each with a diameter of 50 microns. Each reactor had oligonucleotide probes synthesized in situ using a low-cost, light-directed DNA synthesis technology. The chip was used to
  • This example describes stability of freeze dried Taq polymerase and optimization of Trehalose concentrations for use in compositions and methods of the present inventions.
  • PCR microarry
  • the inventors contemplate chips with primers and reagents already dispensed in them.
  • the primers/polymerase/reagents must be made stable at room temperature or even under hot climates.
  • a common practice to obtain freeze-dried reagents is to add sugar (e.g., Trehalose) at the time of freeze- drying. Optimization of the trehalose concentration and stability of the freeze-dried reagents for long periods (6 to 12 months) are two key aspects.
  • a trehalose concentration of 15% has generally been reported as optimal in literature and confirmed in the inventors lab ( Figure 4), although lower concentrations seem to work as well.
  • the reagents were stable for at least one month (FIG.16).
  • Example 5 Example 5
  • This example describes isothermal amplification using a helicase enzyme and primers of the present inventions for use in compositions and methods of the present inventions.
  • Helicase-dependent amplification is isothermal (at around 60 0 C) and does not require temperature cycling. The inventors assessed the performance of this enzyme under 21 different conditions that indicated that less than 10 min. was needed for the signals to cross the background threshold. This experiment was conducted at high target concentration (-10,000 copies). Further test are needed to evaluate the detection limit, replication, and primer design.
  • Helicase BioHelix Corporation, Beverly, Massachusetts, www. biohelix.com/

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Abstract

La présente invention porte sur la mise au point et la fabrication de micro-réseaux pour une utilisation dans la détection de multiples organismes dans un échantillon de façon à haut débit. En particulier, la présente invention porte sur la détection de pathogènes hydriques dans un échantillon d'eau.
PCT/US2007/024291 2006-11-22 2007-11-21 Criblage haut débit à l'aide de micro-réseaux Ceased WO2008140494A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2557178A4 (fr) * 2010-03-31 2013-11-13 Yamaguchi Tech Licensing Procédé pour détecter des bactéries causant la pneumonie en utilisant la chromatographie d'acide nucléique
WO2015152703A3 (fr) * 2014-04-01 2016-01-14 Universidad Nacional Autónoma de México Macropuce pour la détection dans des échantillons environnementaux et biologiques de microorganismes entéropathogènes
CN107142328A (zh) * 2017-07-07 2017-09-08 中国水产科学研究院东海水产研究所 基于纳米金变色响应等温核酸扩增的副溶血性弧菌检测法
CN115251876A (zh) * 2018-06-04 2022-11-01 原相科技股份有限公司 生理检测装置
CN117385477A (zh) * 2023-03-10 2024-01-12 深圳赛陆医疗科技有限公司 用于空间转录组学测序的芯片及其制备方法、空间转录组学测序方法

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US6251588B1 (en) * 1998-02-10 2001-06-26 Agilent Technologies, Inc. Method for evaluating oligonucleotide probe sequences
DE50012114D1 (de) * 2000-09-05 2006-04-13 Zeltz Patrick Verfahren zur spezifischen Bestimmung von DNA-Sequenzen mittels paralleler Amplifikation
US20040259124A1 (en) * 2003-02-19 2004-12-23 Affymetrix, Inc. Methods for oligonucleotide probe design
US8032310B2 (en) * 2004-07-02 2011-10-04 The United States Of America As Represented By The Secretary Of The Navy Computer-implemented method, computer readable storage medium, and apparatus for identification of a biological sequence

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2557178A4 (fr) * 2010-03-31 2013-11-13 Yamaguchi Tech Licensing Procédé pour détecter des bactéries causant la pneumonie en utilisant la chromatographie d'acide nucléique
US9347944B2 (en) 2010-03-31 2016-05-24 Yamaguchi Technology Licensing Organization, Ltd. Method for detecting pneumonia causative bacteria using nucleic acid chromatography
WO2015152703A3 (fr) * 2014-04-01 2016-01-14 Universidad Nacional Autónoma de México Macropuce pour la détection dans des échantillons environnementaux et biologiques de microorganismes entéropathogènes
CN107142328A (zh) * 2017-07-07 2017-09-08 中国水产科学研究院东海水产研究所 基于纳米金变色响应等温核酸扩增的副溶血性弧菌检测法
CN115251876A (zh) * 2018-06-04 2022-11-01 原相科技股份有限公司 生理检测装置
CN117385477A (zh) * 2023-03-10 2024-01-12 深圳赛陆医疗科技有限公司 用于空间转录组学测序的芯片及其制备方法、空间转录组学测序方法

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