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WO1999036573A1 - Detection d'informations genetiques - Google Patents

Detection d'informations genetiques Download PDF

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Publication number
WO1999036573A1
WO1999036573A1 PCT/US1999/001017 US9901017W WO9936573A1 WO 1999036573 A1 WO1999036573 A1 WO 1999036573A1 US 9901017 W US9901017 W US 9901017W WO 9936573 A1 WO9936573 A1 WO 9936573A1
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Prior art keywords
electron
nucleic acid
anchor
transfer
substrate
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PCT/US1999/001017
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English (en)
Inventor
Steven A. Schichman
Bruce Parkinson
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Schichman Steven A
Bruce Parkinson
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Publication date
Application filed by Schichman Steven A, Bruce Parkinson filed Critical Schichman Steven A
Priority to AU25595/99A priority Critical patent/AU2559599A/en
Publication of WO1999036573A1 publication Critical patent/WO1999036573A1/fr

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • the invention provides for the detection of genetic information, e.g.. for use within the fields of molecular genetics, molecular oncology, and molecular infectious disease, to provide for the detection of genetic deficiencies and abnormalities and the detection of patterns of gene expression.
  • DNA' deoxynbonucleic acid
  • Indirect methods for the detection of gene sequences usually involve the use of nucleic acid probes which specifically bind to target sequences in selected genes. Nucleotide probes bind to target sequences through a process known as hybridization.
  • Hybridization refers to the noncovalent interaction through hydrogen bonding between two single-stranded nucleic acid molecules. Hydrogen bonding occurs between so-called complementary nucleotide bases.
  • base adenine
  • CT base thymidine CT
  • G base guanosine
  • C base cytosme
  • an oiigonucleotide with the sequence 5'-ATTTGGCATCAT-3' is perfectly complementary to the oiigonucleotide 3'-TAAACCGTAGTA-5', where 5 and 3' refer to the orientation of the 5 and 3' hydroxyl groups of the deoxynbose/phosphate backbone of the linear oiigonucleotide.
  • 5 and 3' refer to the orientation of the 5 and 3' hydroxyl groups of the deoxynbose/phosphate backbone of the linear oiigonucleotide.
  • the strands of a double-stranded DNA molecule will dissociate from one another when heated above a temperature that is referred to as the "melting temperature” or melting point,” (“Tm").
  • Tm melting temperature
  • the Tm of an oiigonucleotide in aqueous solution is determined by the ionic strength and pH of the solution, and by the length and base composition of the oiigonucleotide.
  • the Tm increases with the length of the oiigonucleotide.
  • the Tm also increases with the percentage of G+C base composition.
  • a probe and a target DNA are mixed together m a buffered aqueous or non-aqueous solution, heated to a temperature above the Tm. and then slowly cooled to a temperature below the Tm. As the temperature is slowly lowered, complementary sequences "anneal" to one another as their Tm is reached.
  • Tm a buffered aqueous or non-aqueous solution
  • conditions of temperature and ionic strength can be chosen that permit discrimination between perfectly complementary oligonucleotides and those with bases that are non-complementary at one or more positions.
  • oiigonucleotide hybridization has been used to detect single-base mismatches in gene sequences (so-called allele- specific oiigonucleotide hybridization).
  • Specific genes may differ from their normal or consensus sequences for several reasons.
  • natural alleles occur, such as the hemoglobin allele that gives rise to sickle cell disease.
  • RNA ribonucleic acid
  • template DNA
  • U base uridine
  • Nucleic acid probes are labeled so that a detection scheme can be employed to determine the presence or absence of a target sequence to which a probe is complementary.
  • Detection schemes currently in use employ either radioactive or non-radioactive labels. Emissions from radioactive labels, such as 32 P, 33 P, and 35 S, are detected with x-ray film or phosphor image analyzers.
  • Non-radioactive detection schemes utilize fluorescent or chemiluminescent labels. The magnitude of a signal produced by a bound probe is often increased by forming further complexes such as streptavidin/biotin complexes, or digoxigenin/anti-digoxigenin complexes. Different detection schemes are suitable for different applications.
  • the first widely used system to detect specific gene sequences was the Southern blot, developed in the mid-1970's by Edwin Southern.
  • the Southern blot is a detection system in which target DNA, immobilized on a solid support, is hybridized with a labeled nucleic acid probe.
  • Target DNA is first cleaved into pieces by the use of restriction enzymes that cleave the DNA at the location of certain specific nucleotide sequences.
  • the DNA fragments are then separated according to size by electrophoresis through a gel matrix.
  • the separated DNA fragments are then denatured and transferred from the gel to a solid support, typically nitrocellulose or nylon filter membranes.
  • the size of the blot is determined by the size of the electrophoresis gel. Commonly used gels are 20 cm x 20 cm, and so-called "minigels" are as small as 5 cm x 5 cm.
  • the DNA is transferred, or "blotted," by capillary action.
  • the DNA is then covalently linked to the solid support, usually by heat treatment or ultraviolet irradiation.
  • Target DNA bound to a membrane is placed in contact with a liquid containing labeled probes, and any probe that is not specifically bound to the target DNA is removed by washing the blot.
  • the location of the bound probe on the membrane is revealed with a detection system appropriate for the type of label used.
  • the label may be radioactive or non-radioactive.
  • a dot blot consists of target DNA (or RNA) as a pattern of spots or dots directly fixed on a solid support membrane made of nitrocellulose or nylon. Dot blot systems do not use a gel and do not use electrophoresis to separate DNA fragments by size. Thus, the name “dot blot" is somewhat of a misnomer, because the DNA is not blotted onto the membrane from a gel.
  • the target nucleic acid on a dot blot may be DNA or RNA, and it may be genomic DNA, recombinant DNA cloned in a vector, specific segments of DNA amplified by the polymerase chain reaction ("PCR") or another in vitro amplification technique, or any other kind of hybridizable nucleic acid molecule.
  • PCR polymerase chain reaction
  • dot blots are prepared with target DNA from various different sources, e.g., from different people or from different recombinant clones.
  • Dots of target DNA are arrayed in rows and columns on a single piece of membrane; this arrangement is called a "DNA array.”
  • the spots or dots of nucleic acid in a DNA array of this sort are usually about 0.5 cm in diameter, and the filter array may contain about 100 samples on a membrane that measures approximately 12 cm x 15 cm.
  • dot blots are arrayed in a single row on a strip of membrane filter.
  • Dot blots are hybridized in a manner similar to Southern blots with probes labeled radioactively or non-radioactively.
  • Dot blots typically yield qualitative data with regard to the presence or absence of the target nucleic acid for which the probe is complementary. It is possible, however, to quantify the signals from dot blots if the signal varies linearly with the amount of target DNA in each sample. Allele-specific oiigonucleotide hybridization (see above) is usually done in a dot blot format. Thus, one example of the use of dot blots is to examine genomic DNA from many different people for the presence of a point mutation in a specific target gene (i.e. many different samples are arrayed on one filter and examined with a single labeled probe).
  • reverse dot blot refers to a system in which the oiigonucleotide probes are attached to a membrane filter. Different probes are typically arrayed in rows and columns on a single-membrane filter. Sometimes different probes are arrayed in a single row on a filter strip. The oiigonucleotide probes, which are not labeled, are covalently attached to the membrane by heat, UV treatment, or chemical bonding. In a reverse dot blot system, the probe array on the filter is hybridized with labeled target DNA isolated from a single source, such as genomic DNA from one person.
  • this system is the "reverse" of the dot blot system described above.
  • a reverse dot blot may be used, for example, to examine the DNA from one person for many different mutations.
  • probes for a series of different mutations are arrayed on one filter, and labeled DNA from an individual is in the hybridization fluid applied to the filter.
  • the dimensions and array density of a reverse dot blot are typically the same as a standard dot blot.
  • Microarrays with oiigonucleotide probes synthesized directly onto the surface are often referred to as "DNA biochips.” Localized oiigonucleotide synthesis is accomplished in part through the use of masking techniques derived from the microelectronics industry or chemjet technology derived from the ink-jet printer industry. Hybridization of microarrays with labeled target DNA is done in a small volume of liquid on the surface of the DNA biochip. Biochips may also be enclosed in a small flow chamber that is accessed and managed by microfluidic engineering.
  • Fluorescence-, chemiluminescence-, and radioactivity-based detection systems are adaptable to biochip arrays.
  • fluorescence-based detection systems are in common use.
  • a fluorescent labeled DNA is used in currently employed systems in conjunction with a fluorescence detector that scans the surface of the chip.
  • Chemistries for labeling DNA with fluorescent tags are well developed and commercially available.
  • Sensitive, high resolution fluorescence scanning detectors have been developed, but these devices are bulky, very expensive and relatively slow. It is reported that chemiluminescence detectors for biochips are under development, but their performance characteristics are presently unknown. Radioactivity- based detection systems present health hazards and regulatory problems because of the potentially harmful emissions from the isotopes used.
  • the invention disclosed herein provides a semiconductor detector device used to detect DNA hybridization.
  • One embodiment of the novel semiconductor detection system of the present invention comprises a semiconductor substrate with specific single-stranded DNA molecules attached thereto. Attached to each single-stranded DNA molecule is one or more chemical labels that are either electron-donors or electron-acceptors, as described with more particularity below. Complementary single-stranded target polynucleotides hybridize to the specific single-stranded DNA molecules to form double-stranded DNA.
  • the semiconductor substrate allows the detection of electrons which are conducted through the double-stranded DNA.
  • Another embodiment of the novel semiconductor detection system of the present invention comprises a semiconductor substrate with specific single-stranded DNA molecules attached thereto.
  • one or more chemical labels that are either electron-donors or electron-acceptors are attached to second single-stranded DNA molecules, as described with more particularity below.
  • Complementary single-stranded target polynucleotides hybridize to the attached single-stranded molecule and the probe-bearing single- stranded molecule to form double-stranded DNA.
  • the semiconductor substrate allows the detection of electrons which are conducted through double-stranded DNA.
  • the novel semiconductor detection system detects electrons which are conducted to the substrate through double-stranded DNA from an electron donor. In other embodiments of the invention, electrons are conducted from the substrate to chemical labels that are electron acceptors.
  • an “anchor-substrate” is a single structural semiconductor substrate that performs three functions. First, it functions as the substrate to which an “anchor- probe” is attached; second, it forms the hybridization platform, and third, it acts as part of the detector.
  • a single-stranded DNA "electron-transfer probe" is complementary to a target DNA and may be an anchor-probe.
  • the single-stranded DNA probe is an anchor-probe
  • such a probe is attached to an anchor-substrate at its 3' end and has an electron-donor or an electron-acceptor moiety attached at its 5' end.
  • double-stranded DNA is formed, and detectable electron flow occurs to or from the anchor- substrate.
  • the present invention uses semiconductor chip material as one element in an electron transfer system.
  • the semiconductor material forms part of the detector device.
  • Either p-type or n-type semiconductors may be used in embodiments of the present invention.
  • electron transfer occurs from an electron- donor group on the specific DNA strand into the conduction band of the semiconductor (so-called
  • electron injection occurs from the valence band of the semiconductor into an electron-acceptor group on the specific DNA strand (so called "hole injection”).
  • the anchor-probe is an electron-transfer probe that is directly coupled to the anchor-substrate.
  • the electron-transfer probe bears one or more chemical labels which are electron-donor or electron-acceptor groups.
  • electrons may be conducted from the chemical label to the anchor-substrate.
  • Double-stranded DNA is formed by hybridization of a single-stranded target nucleotide and the oiigonucleotide anchor-probe.
  • the present invention also provides novel electron-transfer probes, namely probes that have electron-transfer moieties coupled to them.
  • electron-transfer moieties are electron- donor moieties or electron-acceptor moieties.
  • the electron-transfer probe when excited, generates electrons. The electrons are then conducted through the double-stranded DNA that is formed by hybridization. Such electrons are detectable by the semiconductor material that serves as a detector device.
  • the electron-transfer probe when excited, accepts electrons. The electrons are then conducted away from the substrate through the double-stranded DNA that is formed by hybridization, and the current thus established is detectable through the semiconductor material that serves as a detector device.
  • an “anchor-probe” is directly coupled to the anchor-substrate to form an anchor-probe-substrate.
  • the probe on such an anchor-probe- substrate does not bear a chemical moiety.
  • an electron- transfer probe bears a chemical moiety that is an electron-donor or an electron-acceptor, and the anchor-probe and the electron-transfer probe are contiguously complementary to a target "V ⁇ /jD3 57 j 3 PCT/US99/01017
  • Double-stranded DNA is formed by hybridization of the anchor-probe, the electron- transfer probe and the target nucleotide.
  • the chemical labels of the electron-transfer probe when excited, generate electrons which are then conducted through the double-stranded DNA to the semiconductor material that serves as a detector device.
  • the chemical labels of the electron-transfer probe when excited, can accept electrons. Electrons are conducted through the double-stranded DNA from the semiconductor material.
  • a further embodiment of the present invention is an electron-transfer probe composition including a single-stranded nucleic acid and one or multiple electron-transfer moieties in which at least one of the electron-transfer moieties is a transition-metal complex, where the transition metal is a member of the group Cadmium, Magnesium, Copper, Cobalt, Osmium, Iridium, Platinum, Palladium, Zinc, Iron or Ruthenium.
  • the single-stranded nucleic acid is attached to an electrically responsive anchor-substrate, and the transition-metal complexes are covalently attached to a sugar of the sugar-phosphate backbone of a nucleic acid.
  • the nucleic acid in certain embodiments is RNA and in other embodiments of the present invention is DNA.
  • an electrode is an electron-transfer moiety. Electron-transfer moieties of the present invention are attached to nucleic acids at the 3' position of deoxyribose, when the nucleic acid is DNA, and at either the 2' or 3' position of ribose when the nucleic acid is RNA.
  • an electron-transfer probe composition including a single-stranded nucleic acid and one or multiple electron-transfer moieties in which at least one of the electron-transfer moieties is a dye, where the dye is an organic dyes such as those from the rhodamine, coumarin, methine or thionin families of dyes.
  • Such dyes are excited by using a monochromatic light source such as a laser, or a filtered or unfiltered polychromatic light source.
  • Suitable dyes for used in the present invention are cresyl violet, methylene blue and ruthenium trisbipyridine.
  • composition-of-matter comprising: a first hybridizable single-stranded oiigonucleotide electron-transfer probe, and a second hybridizable single-stranded oiigonucleotide anchor-probe.
  • the electron-transfer probe is complementary to a first portion of a target nucleotide sequence
  • the anchor-probe is complementary to a second portion of a target nucleotide sequence.
  • the first portion of the target nucleotide sequence and the second portion of the target nucleotide sequence are contiguous to one another.
  • the two sequences are complementary to adjacent sequences on the target nucleotide strand which have no bases between them.
  • the probe of the anchor-probe-substrate has an electron-transfer group coupled to it, and the substrate is a semiconductor that forms part of the electronic detection system
  • the aforementioned embodiment of the present invention can be targeted to any known gene sequence and used in a process to qualitatively analyze gene sequences or to quantitatively analyze gene expression
  • This process also part of the present invention, permits detection of specific mutations present m genes associated with different types of cancer and permits detection of mutations m genes known to be associated with different types of genetic disease
  • the process of the present invention is also used to detect genes in heterozygous individuals that are earners," 1 e those who are not affected by the mutation they carry
  • the process also can also be used to detect specific patterns of gene expression m normal and abnormal tissues, including neoplastic disease tissue
  • the process of the present invention may also be used to detect gene sequences denved from micro-organisms (e g. bacteria, fungi, and viruses) that may cause infectious diseases in humans and/or other animals
  • micro-organisms e g. bacteria, fungi, and viruses
  • the process can also be used to screen combinatorial libraries
  • Figure 1 is a diagram of the process of a first embodiment of the present invention
  • Figure 2 is a diagram of the process of a second embodiment of the present invention
  • Figure 3(a) illustrates an embodiment of the present invention with an electron-donor chemical moiety
  • figure 3(b) shows an embodiment of the present invention with an electron-acceptor chemical moiety
  • Figure 4 is a diagram of a device of the present invention showing double-stranded nucleic acid molecules on the surface of a semiconductor substrate
  • Figure 5 is a diagram of a multi-segmented device of the present invention
  • the invention disclosed herein provides a novel semiconductor detector device to detect DNA hybridization that is a unitary structural semiconductor substrate, 1 e , an anchor-substrate," that functions (1) as the surface to which an anchor-probe is attached, (2) as a hybridization platform, and (3) as a detector
  • the novel detection system detects the electrons which are conducted through double-stranded DNA from electron- generating chemical labels or to electron-accepting labels Double-stranded DNA is formed by DNA hybridization with oiigonucleotide probes that bear chemical 'electron-transfer labels that can be excited by light to generate or accept electrons
  • Electrons conducted through the DNA activate an anchor-substrate" semiconductor mate ⁇ al that is a novel detector device
  • the present invention also provides a process that utilizes a semiconductor detector to detect electrons transferred through DNA from an electron-generating label, or to an electron-accepting label Photographic grains may also be used as detectors Electrons are conducted very easily along double-stranded DNA, whereas electrons conducted poorly
  • Devices of the present invention are formed in an embodiment such as a matrix, or grid, composing numerous different DNA anchor-probes
  • Such an embodiment of the present invention enables simultaneously tesung for multiple target DNAs Results from such a matrix embodiment of the present invention can be readout from the device by scanning the device with a focussed light beam or by electrical multiplexing, for example, by using individually patterned diodes or transistors which correspond to each pixel
  • a particular embodiment of the present invention comprises an array of different single-stranded DNA oligomer molecules arranged in a grid on the semiconductor surface Arrays are envisaged that be a single field, or up to 1000 x 1000 pixels, or even more Each pixel of a grid of the present invention bears an oiigonucleotide that is characteristic of a specific gene, gene mutation, or microorganism
  • a preferred embodiment of the present invention has a substrate that is a semiconductor material selected from the following amorphous silicon ( ⁇ -Si), the III-IV semiconductor class, such as gallium arsenide, gallium phosphide, gallium nitride or indium phosphide, the II- VI semiconductor class such as cadmium sulfide, zinc selenide or zinc sulfide, the I-VII materials such as silver chlo ⁇ de, silver bromide or silver iodide, or the oxide semiconductors such as titanium dioxide, strontium titanate or zinc oxide
  • ⁇ -Si amorphous silicon
  • the III-IV semiconductor class such as gallium arsenide, gallium phosphide, gallium nitride or indium phosphide
  • the II- VI semiconductor class such as cadmium sulfide, zinc selenide or zinc sulfide
  • the I-VII materials such as silver chlo ⁇ de, silver bromide or
  • the semiconductor substrate may be monocrystalline, polycrystalline, amorphous or nanometer-sized semiconductor particles formed into a thin film of semiconducting material
  • the semiconductor materials may be of the doped n-type for detection of photoexcited electrons injected into the conduction band of the semiconductor
  • Embodiments of the present invention that are devices which detect electrons transferred from electron-donor groups, use n-type semiconductors as the anchor-substrate P-type semiconductors are used in embodiments of the present invention for the detection of photoexcited holes injected into the valence band
  • Embodiments of the present invention that are devices which detect electrons transferred to electron-acceptor groups use p-type semiconductors as the anchor-substrate Semiconductors used for anchor-substrates in embodiments of the present invention mav be also undoped, as would be the case for nanoscale semiconductor particle films
  • An anchor-probe of the present invention is a single-stranded DNA molecule that is complementary to a target nucleotide sequence of interest
  • An anchor-probe complementary to any target nucleotide of interest may be used in the present invention
  • Anchor-probes are bound to the surface of the semiconductor by chemical forces such as a covalent bond, or by electrostatic binding, or by hydrophobic interactions
  • covalent bonds include, but are not limited to, alkvl thiol bonds, amine condensation links, phosphate, silane, thiol, ester, amide or carboxylate links or links formed by free radical reactions with alkenes
  • Other means of linking DNA to solid substrates will be known to those of skill in the art
  • Linkages may be formed directly or by means of bifunctional linker molecules between the 3' hydroxyl group of the anchor-probe DNA and the anchor-substrate
  • anchor-probes range from 14 to 30 nucleotides in length Longer or shorter oligomer lengths are also functional in the present invention as will be known to those of skill in the art In preferred embodiments of arrays of the present invention, a different anchor-probe is used for each pixel
  • the presence of a specific segment of target nucleic acid is signaled by electrons that are conducted through double-stranded DNA which is formed by the process of the invention
  • the double-stranded DNA is assembled on the surface of the semiconductor detector device by the hybridization of two complementary nucleotide sequences The two pieces are ( 1 ) an anchor-probe which is an electron-transfer probe that composes a DNA segment to which the chemical label that tiansfers electrons is attached, and (2) DNA derived from the sample to be tested
  • the double-stranded DNA is assembled on the surface of the semiconductor detector device by the hybridization of three complementary nucleotide sequences
  • the three pieces are ( 1 ) an anchor-probe that composes a DNA segment that is attached, or anchored, to the detector surface, (2) DNA derived from the sample to be tested, and (3) an electron-transfer probe that composes a DNA segment to which the chemical label that transfers electrons is attached
  • Electron-transfer probes are single-stranded DNA oligonucleotides, typically less than 15 nucleotides in length
  • the sequence of an electron-transfer probe is complementary to the sequence of the sample DNA to be tested
  • the sequence of the electron-transfer probe is selected to be complementary to a sequence in the target DNA that is contiguous to the sequence that is complementary to the anchor-probe
  • Electron-transfer probes of the present invention are populations of single-stranded DNA molecules with electron-transfer groups attached at the 5' end.
  • the electron-transfer groups that are used in embodiments of the present invention are organic dyes such as those from the rhodamine, coumarin, methine or thionin families of dyes. However, the present invention is not limited to these dyes.
  • the electron-transfer group has a function similar to that of a photographic sensitizer and could also be from the class of dyes that includes cyanine dyes.
  • Other suitable electron-transfer groups are metal complexes such as ruthenium trisbipyodine or other chromophoric metal complexes.
  • Anchor-probes may also be synthesized directly on the surface of the anchor-substrate by techniques that are well known to those of skill in the art.
  • the hybridization process is first performed, and one then detects binding of the target DNA sequences by directing a photon excitation source, such as a laser, a polychromatic light source, or a filtered polychromatic source, at the surface array.
  • a photon excitation source such as a laser, a polychromatic light source, or a filtered polychromatic source
  • Dyes and corresponding wavelengths of light suitable for used in the present invention are: cresyl violet with 633 nm light from a helium-neon laser, methylene blue and 680 nm light from a red diode laser, and ruthenium trisbipyridine and 574.5 nm light from an argon laser.
  • the wavelength of the light source is selected so that photoexcitation of the chemical label of the electron-transfer probe is achieved.
  • the wavelength of light used in the present invention to excite the chemical label is selected to be of an energy that does not directly excite the semiconductor.
  • the intensity of the light is modulated at a convenient frequency to allow for phase detection of the signal. This technique selectively increases the detectability of DNA-specific photo-initiated electron-transfer, above any background dark-electron-transfer processes.
  • a reducing agent is added to the solution covering the array in order to reduce photooxidized electron-transfer probe molecules so that they can be repeatedly photooxidized. This technique causes each attached double-stranded DNA molecule to provide many electrons for detection. Addition of a reducing agent to the solution used in the system chemically reduces the oxidized electron-transfer group, thereby altering the state of the group so that it may again be activated by the exciting light, thus enabling another cycle of electron transfer.
  • the excitation light is scanned sequentially over the array, and detection of a photocu ⁇ ent is accomplished with a single electrical contact to the back of the array.
  • the location of pixels to which electron-transfer probes have hybridized is determined from the position of the scanning excitation beam
  • the entire arrav is bathed in light and individual grid points on the arrav are elect ⁇ callv addressed via multiplexing electronics
  • the signal-to- noise ratio with either method can be increased by integrating the photocurrent over time to accumulate photocurrent signal
  • the hyb ⁇ dization process of the present invention involves the interaction of either two or three single-stranded DNA molecules These molecules have sequences which hybodize to form a contiguous double-stranded DNA only when the molecules are complementary to one another
  • the process comprises the following steps ( 1 ) the test DNA is incubated under hybodizmg conditions on an anchor-probe-substrate of the present invention that bears an electron-transfer moiety Test DNA that contains a nucleotide sequence complementary to the probe hybodizes and becomes bound,
  • the substrate is irradiated with light to photoexcite the electron-transfer moiety, and the signal generated bv the photoexcitation is electronicallv detected as described above
  • Another process of the present invention to detect the presence of a target DNA comprises the following steps
  • test DNA and an electron-transfer probe are incubated under hybridizing conditions on an anchor-probe-substrate of the present invention that does not bear an electron-transfer moiety
  • test DNA that contains a nucleotide sequence contiguously-complementary to the electron-transfer probe and the anchor-probe hybodizes and becomes bound
  • the formed complexes are treated with DNA ligase to hgate adjacent oligonucleotides
  • the ligation step is omitted
  • the substrate is irradiated with light to photoexcite the electron-transfer group on the electron- transfer probe, and the signal generated by the photoexcitation is electronically detected
  • the sample DNA to be tested could be from any source, such as a cell line, the cells of a human, or any other organism
  • specific regions of the sample DNA are amplified, e g , by a technique such as PCR
  • PCR a technique such as PCR
  • linear or exponential amplification is used The amount of amplification required is that needed to achieve a good signal-to-noise ratio by the detector
  • RNA is obtained from a source such as a cell line or the cells of a human or other animal
  • the RNA is first converted into complementarv DNA (cDNA")
  • the cDNA can be used without amplification as the sample in an assay of the present invention
  • a specific region of the cDNA can be further amplified, e g , by a linear or exponential amplification technique, so as to achieve a concentration that will yield a detectable signal
  • composition-of-matter with two components, namely the electron-transfer probe and the anchor-probe
  • the electron-transfer probe is complementary to a first target nucleotide sequence
  • the anchor-probe is complementary to a second target nucleotide sequence that is contiguous with, and adjacent to, the first target nucleotide sequence
  • the electron-transfer probe has an electron-transfer group coupled to it
  • the anchor-probe is coupled to a semiconductor that forms part of an electronic detection system
  • the process of detecting a specific DNA sequence with the present invention may comprise a step wise hybridization process in which three pieces of single-stranded DNA are assembled to form double-stranded DNA anchored to a semiconductor at one end and containing an electron-transfer group at the other end A step wise process is earned out in four steps
  • Step 1 consists of hybodizing a sample DNA on an anchor-probe-substrate of the present invention
  • Sample DNA that is complementary to the anchor-probe will hybridize with the anchor-probe on the anchor- probe-substrate and the anchor-substrate is then washed to remove unbound sample DNA
  • Step 2 consists of hybridizing an electron-transfer probe to the complex formed in step 1 on the anchor-probe-substrate
  • a mixture of electron-transfer probes is used, with each individual probe specific for an individual anchor-probe DNA
  • a single electron-transfer probe is used, such a probe being complementary to a segment of DNA added onto the sample DNA
  • Electron-transfer probes will hybridize with complementary sample DNA sequences that are, in turn, hybridized to the anchor-probes attached to the anchor-substrate by the previously described physical or chemical means
  • Electron-transfer probe sequences are designed so as to be contiguous with the anchor-probe sequences
  • Step 3 consists of a hgation step catalyzed by a DNA ligase enzyme Specific hybridization of the anchor-probe and electron-transfer probe with a complementary sample DNA will precisely align the 5' end of the anchor-probe DNA with the 3' end of the electron-transfer probe DNA such that the ends can be hgated together to form a single contiguous molecule
  • the DNA ends may be hgated together with T4 DNA Ligase, provided an adjacent 5'-phosphate and a 3 -hydroxyl are present
  • Step 4 consists of a washing step performed with an aqueous solution ot salt and detergent under conditions that will wash off electron-transfer probes that are not hgated to anchor-probes Because the unhgated probes are short sequences, the washing will be quantitative
  • ligation of the adjacent ends is not necessary for efficient conduction of electrons through the double- stranded DNA
  • ligation of adjacent anchor-probe and electron-transfer probe oligonucleotides permits more stringent washing to be used in step 4
  • the sample DNA contains a mismatch at or very close to the adjacent ends of the anchor-probe and the electron-transfer probe, then ligation of the anchor-probe and electron-transfer probe will be inhibited
  • the ligation step enables discrimination between normal and mutant sequences
  • Another embodiment of the process uses conventional silver halide emulsions as the substrate, with the hybodized DNA segments acting as the sensitizer of the silver halide grains, as is rou ⁇ nely accomplished with the similar organic dyes in conventional photography
  • the results of the assay are ascertained by developing the film sheet and examining or scanning the film for exposed regions indicating a positive DNA match
  • FIG. 1 diagrammatically illustrates the process of the simplest embodiment of the present invention
  • Figure 1(a) shows a single-stranded DNA anchor-probe 101 with a terminally attached electron-donor chemical label 102 attached to anchor-substrate 103
  • Figure 1(b) shows a single-stranded target nucleotide 104 adjacent to the DNA anchor-probe 101 attached to anchor-substrate 103 with terminally attached electron-donor chemical label 102 attached to DNA anchor-probe 101
  • Figure 1(c) shows single-stranded target nucleotide 104 hybridized to the single-stranded DNA anchor-probe 101
  • Arrow 105 in figure 1(c) indicates the direction of the flow of electrons from the terminally attached electron-donor chemical label 102 to the anchor-substrate 103 through the double-stranded DNA structure 1 1 1 upon excitation of electron-donor label 102 by exciting light 1 10
  • Figure 2 diagrammatically illustrates the process of an embodiment ot the present invention
  • Figure 2(a) shows an unlabeled single-stranded DNA anchor-probe 201 attached to anchor-substrate 203 with a single- stranded target nucleotide 204 adjacent thereto
  • Figure 2(b) shows the single-stranded target nucleotide 204 hybodized to the unlabeled single-stranded DNA anchor-probe 201 which is attached to anchor-substrate 203
  • Figure 2(c) shows a hybridizable single-stranded electron-transfer probe 206 with a terminally attached chemical electron-donor label 202 adjacent to the single-stranded DNA anchor-probe 201 with the single- stranded target nucleotide 204 hybridized thereto
  • Figure 2(d) shows the single-stranded electron-transfer probe 206 with a terminally attached electron-donor chemical label 202 hybridized to the single-stranded DNA single- strand
  • Figure 3 shows two embodiments of the present invention
  • Figure 3(a) shows a single-stranded target nucleotide 304 hybridized to a single-stranded DNA anchor-probe 301 with an electron-donor probe 302 attached thereto
  • Arrow 305 in figure 3(a) indicates the direction of the flow of electrons from the terminally attached electron-donor chemical label 302 to the anchor-substrate 303 through the double-stranded DNA structure 31 1 upon excitation of electron-donor chemical label 302 by exciting light
  • Figure 3(b) shows a single-stranded target nucleotide 304 hybridized to a single-stranded DNA anchor-probe 301 with an electron- acceptor probe 308 attached thereto
  • Arrow 309 in figure 3(b) indicates the direction of the flow of electrons to the terminally attached electron-acceptor chemical label 308 from the anchor-substrate 303 through the double- stranded DNA structure 311 upon excitauon of electron-acceptor chemical label 308 by exciting
  • Figure 4 is a diagram of a device of the present invention that shows double-stranded nucleic acid molecules 411 assembled on the surface of semiconductor substrate 412 Upon excitauon of terminally attached electron-donor chemical label 402 with exciting light 410, electrons are conducted to the semiconductor anchor-substrate 412 through the double-stranded nucleotide structure 411 upon excitation of electron-donor label 402 by exciting light 410 An electronic detection system 413 reads out the signal from semiconductor anchor-substrate 412
  • Figure 5 is a diagram of a multi-segmented device of the present invention showing different double- stranded nucleic acid molecules, (DNA 1, DNA 2, DNA 3, etc ,) assembled on the surface of semiconductor substrate 512 Upon excitation of terminally attached electron-donor chemical labels electrons are conducted to the semiconductor anchor-substrate 512 through formed double-stranded nucleotides and electronic detection system 513 reads out signals from the semiconductor anchor-substrate 512 through a series of connectors 514

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Abstract

L'information se rapporte à un dispositif de détection à semi-conducteurs, utilisé pour détecter l'hybridation d'ADN. Le dispositif permet d'obtenir un système de détection comprenant un substrat semi-conducteur qui constitue une plate-forme sur laquelle on effectue l'hybridation, et un site de fixation de molécules d'ADN spécifiques à simple brin qui y sont liées. Le dispositif détecte les électrons qui sont transmis par conduction depuis des indicateurs chimiques à travers l'ADN double brin entre les nucléotides de test complémentaires à simple brin et les polynucléotides cibles. L'invention décrit des configurations donneur d'électrons et récepteur d'électrons, ainsi que des indicateurs chimiques à transfert d'électrons, fixés aux molécules d'ADN à simple brin.
PCT/US1999/001017 1998-01-20 1999-01-19 Detection d'informations genetiques WO1999036573A1 (fr)

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US7056748B1 (en) 1998-10-27 2006-06-06 Technion Research And Development Foundation Ltd. Method for gold deposition
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6946675B2 (en) 1997-07-14 2005-09-20 Technion Research & Development Foundation Ltd. Microelectronic components and electronic networks comprising DNA
US7851149B2 (en) 1998-10-27 2010-12-14 Erez Braun Method for gold deposition
US7056748B1 (en) 1998-10-27 2006-06-06 Technion Research And Development Foundation Ltd. Method for gold deposition
US6593090B2 (en) 1999-04-07 2003-07-15 Integrated Nano-Technologies, Llc High resolution DNA detection methods and devices
US6399303B1 (en) 1999-04-07 2002-06-04 Integrated Nano-Technologies, Llc High resolution DNA detection methods and devices
US7364920B2 (en) 1999-10-27 2008-04-29 Technion Research And Development Foundation Ltd. Method for gold deposition
US8546168B2 (en) 2000-04-24 2013-10-01 Life Technologies Corporation Method and apparatus for detecting nucleotides
US6413792B1 (en) 2000-04-24 2002-07-02 Eagle Research Development, Llc Ultra-fast nucleic acid sequencing device and a method for making and using the same
US8232582B2 (en) 2000-04-24 2012-07-31 Life Technologies Corporation Ultra-fast nucleic acid sequencing device and a method for making and using the same
US7001792B2 (en) 2000-04-24 2006-02-21 Eagle Research & Development, Llc Ultra-fast nucleic acid sequencing device and a method for making and using the same
US8426232B2 (en) 2000-04-24 2013-04-23 Life Technologies Corporation Ultra-fast nucleic acid sequencing device and a method for making and using the same
WO2001081896A1 (fr) * 2000-04-24 2001-11-01 Eagle Research & Development, Llc Dispositif de sequencage d'acide nucleique ultra-rapide et procede de fabrication et d'utilisation
US9063081B2 (en) 2000-04-24 2015-06-23 Life Technologies Corporation Ultra-fast nucleic acid sequencing device and a method for making and using the same
US9228976B2 (en) 2000-04-24 2016-01-05 Life Technologies Corporation Method and apparatus for detecting nucleotides
US9410923B2 (en) 2000-04-24 2016-08-09 Life Technologies Corporation Ultra-fast nucleic acid sequencing device and a method for making and using the same
US9758824B2 (en) 2000-04-24 2017-09-12 Life Technologies Corporation Ultra-fast nucleic acid sequencing device and a method for making and using the same

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