WO1999024621A2 - Methodes et compositions de detection de sequences nucleotidiques specifiques - Google Patents

Methodes et compositions de detection de sequences nucleotidiques specifiques Download PDF

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Publication number
WO1999024621A2
WO1999024621A2 PCT/US1998/024226 US9824226W WO9924621A2 WO 1999024621 A2 WO1999024621 A2 WO 1999024621A2 US 9824226 W US9824226 W US 9824226W WO 9924621 A2 WO9924621 A2 WO 9924621A2
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Prior art keywords
target
probe
nucleic acid
acid sequence
dna
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PCT/US1998/024226
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English (en)
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WO1999024621A9 (fr
WO1999024621A3 (fr
Inventor
Elliot R. Ramberg
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Cygene, Inc.
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Priority to MXPA00004675A priority Critical patent/MXPA00004675A/es
Priority to JP2000519613A priority patent/JP2001522588A/ja
Priority to AU15237/99A priority patent/AU1523799A/en
Priority to CA002309861A priority patent/CA2309861A1/fr
Priority to KR1020007005156A priority patent/KR20010032036A/ko
Priority to EP98959441A priority patent/EP1030936A2/fr
Publication of WO1999024621A2 publication Critical patent/WO1999024621A2/fr
Publication of WO1999024621A3 publication Critical patent/WO1999024621A3/fr
Publication of WO1999024621A9 publication Critical patent/WO1999024621A9/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
    • C12Q1/682—Signal amplification
    • 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
    • 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

Definitions

  • the present invention comprises methods and compositions for detecting nucleic acid sequences. More particularly, the present invention comprises methods and compositions for detection of specific genetic sequences using differing nucleic acid target protection and recovery strategies. Additionally, the present invention comprises novel methods for nucleic acid cleavage.
  • Molecular biological techniques have provided many accurate, rapid tests for determining, identifying or detecting, DNA and RNA sequences. Unfortunately, most of these tests depend on PCR amplification of DNA as one or several of the steps involved in the tests. The amplification of the target nucleic acid with PCR may lead to amplification of a nucleic acid sequence other than the desired target nucleic acid sequence.
  • RNA sequence-based amplification NASBA Org.anon Teknika
  • Becton Dickinson transcription-based amplification system
  • Geneprobe transcription mediated amplification
  • PCR polymerase chain reaction
  • LCR/ Abbott ligase chain reaction
  • q-beta bacteriophaqe replicase ligase chain reaction
  • Target amplification methods suffer from amplicon and other forms of sample contamination as well as problems relating to specificity. Such problems are inherent in the limitations of their technology bases and consistent with their lack of proper experimental (kit) designs, which result in less than high specificities and sensitivities. Additionally, these technologies lack the ability to directly screen for specific RNA targets, which places severe limitations on these technologies to support advances in RNA viral, cancer, and other infectious disease diagnoses and therapy management.
  • the inability to screen more than microgram amounts of nucleic acids for a unique target further acts to decrease the specificity and sensitivity of these methods.
  • the singular truth in infectious disease diagnosis is that the earlier in the infectious time course that the detection is necessary, the larger the sample of host
  • DNA (milligram amounts) that is required for analysis.
  • Non-specific high background signals and inappropriate levels of sensitivity due to a total inability to recognize a target down to a single copy, coupled with the inability to quantify the target, offer little chance for a diagnostic breakthrough.
  • Bioluminescence currently the most sensitive assay in single gene copy detection, is only able to detect a minimum of hundreds of thousands of protected targets. What is needed is a method of increasing the sensitivity of target detection to detect a single gene.
  • nucleic acid amplification methods allow for the detection of relatively small quantities of target nucleic acid molecules present in a small sample (microgram amounts of DNA), there is also a need for the ability to detect target nucleic acid molecules in a shorter amount of time with less background interference.
  • Problems inherent in PCR and other amplification techniques include, sample' contamination during the collection procedures and the presence of amplicons (amplified DNA) which contaminate DNA specimens to provide false positive results.
  • amplicons amplified DNA
  • PCR results must often be confirmed and validated by other techniques such as DNA-Southern blotting, RNA blotting and probe hybridization, or in situ hybridization. Additionally, PCR and amplification techniques can only be used with very small amounts of starting sample DNA, in the range of a maximum of 1 microgram. This negates use of PCR techniques for the detection of low copy number nucleic acid targets in a large volume of total/nonspecific nucleic acids and for early infectious time- course diagnostics.
  • RNA analysis includes Northern blots and Ribonuclease Protection Assay.
  • Northern blots first denature the RNA molecule and make sure it is unfolded in a linear form. The RNA is then subjected to gel electrophoresis, transferred to a membrane, hybridized with a labeled probe and subjected to a visualization method. This procedure is both qualitative and quantitative. This lengthy, high cost, procedure has little or no relevance in RNA diagnostics due to all the downsides in its design which are similar to the problems with use of Southern blotting in DNA diagnostics. These include time consumption, high costs/material and labor, lack of sensitivity in detecting targets of relative low abundance, and inability to be sensitive down to a single target copy, thus a late infectious time-course assay.
  • the Ribonuclease Protection Assay includes binding a probe to an RNA molecule, treatment with SI nuclease to remove non-specific RNA and single stranded RNA regions, and analysis on an electrophoretic gel. This procedure is only qualitative and lacks sensitivity for diagnostic technology due to the amount of RNA needed for visualization in the assay.
  • RT-PCR Reverse transcriptase-PCR
  • cDNA DNA copy
  • quantitative PCR is performed.
  • RT-PCR is expensive, has lower specificity and lower sensitivity, requires extreme standardization of all steps to provide reproducible results and is extremely labor intensive. Also, RT-PCR cannot be used in early infectious time-course nucleic acid diagnostics.
  • methods and compositions are needed that are capable of detecting specific nucleic acid sequences. Especially needed are methods and compositions that provide the flexibility that would allow for isolation of nucleic acid sequences using a desired level of specificity.
  • RNA including, but not limited to, mRNA
  • RNA sequences isolated from a variety of cell and/or tissue sources.
  • the methods are inexpensive, specific with multiple levels of specificity, sensitive, able to assay a wide range of amounts of nucleic acids, provide reproducible results, and require minimum labor.
  • the methods and compositions of the present invention can be used in diagnostics and therapeutics, such as the detection of microorganisms, such as viruses and other microorganisms and pathogens of humans, animals and plants; diagnosis of infectious diseases, cancer and metastisis in humans, animals and plants, assays of blood products, and for genetic analysis for use in such areas as early detection of tumors, forensics, paternity determinations, transplantation of tissues or organs and genetic disease determinations. These assays can also be used for detection of contamination of food, soil, water, blood products and air quality testing.
  • Two embodiments of the present invention comprise the Restriction Fragment Target Assay (RFTA) and the Target Protection Assay (TPA).
  • the present invention also comprises a novel technique for site-directed cutting of nucleic acids, comprising cutting nucleic acids with cutter probes (CPs) having a reactive group, such as halogenated nucleotide derivatives, preferably pyrimidine or purine analogues.
  • RFTA comprises selective restriction cleavage of nucleic acids and detection using specialized probes.
  • TPA comprises protection of the target sequence and detection with specialized probes.
  • Cutter probes comprise novel methods and compositions for cutting nucleic acids and can be used in any known assays requiring cleavage of nucleic acids.
  • Preferred methods comprise oligonucleotide probe sequences complementary to the target sequence to be cut, in which a reactive group, such as a halogenated nucleotide derivative, is incorporated in the probe in a position juxtaposed to the position to be cleaved in the target.
  • a reactive group such as a halogenated nucleotide derivative
  • BU substituted bromouridine
  • the cutter probes only cleave the DNA or
  • RNA at the specified site When BU is substituted for some of the thymidines, other thymidine bases existing in the cutter probes along with the other three bases form the basis for specificity of placement and cutting by the base cutter probe.
  • the cutter probes have an anti-parallel sequence to the target strand.
  • CPA cutter probe assay
  • Other uses of the cutter probes include destruction of a specific section of a target nucleic acid and detection of known point mutations.
  • the BU, the reactive group in the CP is activated by the appropriate form of specific energy, light energy or chemical agent and the target sequence is cleaved.
  • the CP may be used in the novel assays of the present invention. Additionally, once the target sequence is bound by the cutter probe the target/probe complex is resistant to specific nuclease degradation and forms a PNAS, a protected target nucleic acid structure (PNAS) in TPA and a partial duplex target probe complex (PDTP) in RFTA. Cutter probes are disclosed in U.S. Provisional Patent Applications 60/075,812, filed February 24, 1998, and 60/076,872, filed March 5, 1998, both of which are incorporated herein in their respective entirety. Restriction Fragment Target Assay (RFTA)
  • RFTA may be used to detect single-stranded or double-stranded RNA or DNA targets.
  • the RFTA methods and compositions comprise at least one primary probe and at least one secondary probe.
  • the probes are preferably single- stranded.
  • the primary probe may have at least two sections. At least one section of the primary probe is complementary to the target and at least one other section of the primary probe is complementary to a secondary probe.
  • the secondary probes are not complementary to the target, but only to the section of the primary probe that is complementary to the secondary probe. Additional sections may be added to the probes.
  • RFTA RFTA is significantly faster and more convenient to perform than membrane hybridization, and requires less technical skill and specialized equipment, such as electro- or vacuum-transfer systems, UV cross-linkers or vacuum ovens, and hybridization ovens and water baths.
  • RFTA is also substantially cheaper to perform than standard RFLP analysis in that much less probe is utilized for the hybridization in a small volume prior to electrophoresis as opposed to hybridization in a relatively large volume, with proportionally slower hybridization kinetics, utilized with membrane hybridization.
  • RFTA electrophoresis system
  • the available detection systems all work as well, if not better, on fixed or dried gels as they do on membranes.
  • RFTA may be more sensitive than membrane hybridization because it does not require nucleic acid transfer or membrane cross linking, both of which can result in loss of specific signal due to damage or inefficient transfers.
  • RFTA is not prone to the high rates of non-specific signal often seen with PCR-based testing because RFTA does not utilize DNA amplification.
  • RFTA has the advantage of multiplexing, in that several probes can be tested with a single sample at the same time on any given electrophoresis gel, eliminating the need to perform replicate isolation steps, such as running several gels and/or stripping already hybridized membranes for subsequent re-probing, both of which are expensive and time-consuming procedures.
  • One example of a preferred RFTA method of the present invention employs the following steps: 1) Isolation and purification of the sample nucleic acid with the target nucleic acid; 2) Cleavage of the sample nucleic acid to excise target nucleic acid; 3) Denaturation of ds nucleic acid sample into single strands; 4) Combining the purified nucleic acid sample with at least one labeled, target specific primary probe complementary to at least part of the desired target sequence.
  • the probe is added in molar excess amounts under conditions allowing for spontaneous hybridization of the probe to the target nucleic acid; 5) Isolation of the target nucleic acid-probe complexes; and 6) Detection of the target nucleic acid-probe complexes.
  • Target Protection Assay CTPA ' Target Protection Assay CTPA '
  • the methods and compositions comprising the TPA embodiments may be used to detect single-stranded RNA with a double-stranded hairpin DNA probe.
  • the methods and compositions of the TPA embodiments can be used with components and methods commonly used in molecular biological techniques. Additionally, TPA embodiments can be used with novel nucleic acid cleavage techniques such as CP.
  • TPA RNA-TPA
  • in vitro construction of a double- stranded hairpin DNA probe of variable length preferably having sequences that are polypurine or polypyrimidine rich that fold in the center and form a duplex DNA structure.
  • the dsDNA hairpin capture probe is characterized by having two sections: the 3' end is a polypurine rich region, the 5' end is a polypyrimidine rich region, both regions are joined in the middle by stretch that forms the loop of the hairpin. The hairpin capture probe folds back on itself to form a hairpin. Also, the 3' end should be conjugated with a biochemical hook (digoxigenin or DIG) close to but not at the 3' end. 1.
  • DIG biochemical hook
  • RNA target site will be located between the 3' poly A of the mRNA and the 5' mRNA end (closer to the 5' end). This is the first level of specificity.
  • the PNAS is then isolated.
  • the PNAS is attached to a solid substrate using methods known to those skilled in the art. Such methods include, but are not limited to, attaching the probe/target complex to magnetic beads by use of a biochemical hook or specific binding pairs such as DIG (digoxigenin) and BIOTIN and anti-DIG, and streptavidin coated magnetic beads.
  • the length of the target can be variable, preferably in a range between 8 and 25 nucleotide bases to kilobase length segments.
  • the presence of target is identified by a reporter probe molecule, preferably a poly (dt) oligo, preferably of approximately 25 nucleotides, that will hybridize to the 3' adenine mRNA molecules.
  • a reporter probe molecule preferably a poly (dt) oligo, preferably of approximately 25 nucleotides, that will hybridize to the 3' adenine mRNA molecules.
  • Each of the reporter probe molecules has at least one member of a binding group, associated with the reporter probe molecule.
  • compositions of the present inventions comprising the embodiments of CP, RFTA and TPA, include compositions necessary to practice the methods taught herein.
  • a composition used in the methods for RFTA may comprise a primary probe with one or more sections of nucleotides complementary to the target sequence and one or more sections of nucleotides complementary to secondary probe sequences, and labeled secondary probes.
  • Compositions or kits comprising selected primary and secondary probes, along with nucleases and buffers are included in the present invention. It is to be understood that the individual molecules, probes and components can also be provided individually.
  • an object of the present invention to provide methods and compositions to detect specific genetic sequences in humans, plants and animals, preferably with variable levels of specificity. Still another object of the present invention is to provide novel methods and compositions for specific nucleic acid cleavage that comprises cutter probes. It is yet another object of the present invention to provide methods and compositions for detecting DNA sequences involving specifically designed probes, including primary and secondary probes.
  • Another object of the present invention is to provide methods and compositions for detecting nucleic acid sequences involving formation of triplex structures.
  • a further object of the present invention is to provide methods and compositions for detecting nucleic acid sequences for the determination of the identity of microorganisms or pathogens in humans, plants and animals. Another object of the present invention to provide methods and compositions for detecting nucleic acid sequences for the determination of a genetic relationship, such as paternity or species identification, or for the determination of potential donors of organs or tissues.
  • Figure 1 is an embodiment of the cutter probes being used to cut a single-stranded nucleic acid.
  • Figure 2 is an embodiment of the cutter probes being used to excise target nucleic acid from a sample.
  • Figure 3 is an embodiment of the cutter probes being used in an assay to retrieve excised target nucleic acid from a sample.
  • Figure 4 is an embodiment of a single-stranded cutter probe (a triplex forming oligonucleotide, TFO) being used to cut a double-stranded target.
  • Figure 5 is an embodiment of a single-stranded cutter probe (TFO) being used to destroy a double-stranded gene target.
  • TFO single-stranded cutter probe
  • FIG. 6 is an embodiment of the Restriction Fragment Target Assay (RFTA) wherein the target/probe complex is isolated by size separation.
  • RFTA Restriction Fragment Target Assay
  • Figure 7 is an embodiment of RFTA wherein the target/probe complex is isolated by the test-tube format.
  • Figure 8 are alternative embodiments of RFTA primary probes for use with target/probe isolation by size separation.
  • Figures 81 and 811 primary probes having hairpin loops.
  • Figure 9 are alternative embodiments of RFTA primary probes for use with target/probe isolation by the test-tube format.
  • Figure 10 is an embodiment of the RNA/RFTA assay for gel based isolation featuring the cutter probes.
  • Figure 11 is an embodiment of an RFTA/RNA application.
  • Figure 12 is an embodiment of an TPA/RNA capture assay.
  • Figure 13 is an embodiment of signal amplification for RNA/TPA tube assay.
  • Figure 14 is an embodiment of TPA/RNA.
  • Figures 15A and B are an embodiment of TPA/RNA with signal amplification.
  • Figure 16 is an embodiment of the triplex lock.
  • the present invention comprises methods and compositions for the detection of target nucleic acid sequences in a sample suspected of containing the target.
  • the methods and compositions include compositions and methods necessary for direct detection of both RNA and DNA sequences.
  • the methods and compositions are capable of detecting target sequences in a range of nucleic acid amounts, preferably from nanogram to milligram amounts of nucleic acid. Steps which improve the specificity of the assay are described in the order in which they are performed and are called levels of specificity.
  • the methods and compositions of the present invention can be used in diagnostics and therapeutics, such as the detection of microorganisms, such as viruses and other microorganisms and pathogens of humans, animals and plants; diagnosis of infectious diseases in humans, animals and plants, assays of blood products, and for genetic analysis for use in such areas as early detection of tumors, forensics, paternity determinations, transplantation of tissues or organs and genetic disease determinations.
  • diagnostics and therapeutics such as the detection of microorganisms, such as viruses and other microorganisms and pathogens of humans, animals and plants; diagnosis of infectious diseases in humans, animals and plants, assays of blood products, and for genetic analysis for use in such areas as early detection of tumors, forensics, paternity determinations, transplantation of tissues or organs and genetic disease determinations.
  • These assays can also be used for detection of contamination of food, soil, water, blood products and air quality testing.
  • An additional specificity level includes effectively increasing the size of the target by resizing the target as a result of probe hybridizations. Such a level is found in a preferred embodiment of RFTA using the Gel Format.
  • the methods and compositions of the present invention comprising CP site specific non-enzymatic cleavage of the target nucleic acid have the benefit of providing two levels of specificity to an assay.
  • the present invention employs methods and compositions that are well known molecular biological techniques.
  • the present invention contemplates combinations of nucleases, probes, hybridization schemes, capture elution methods, various methods of size detection or elution with detection.
  • isolation of nucleic acids used herein can be performed by any techniques known to those skilled in the art. Methods of isolation and purification can be found in the well-known laboratory manual of Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, New York (1989) (which is incorporated by reference herein).
  • labels are called reporter molecules, and some probes containing labels are called reporter probes.
  • the labels used can be any of a variety currently available, and can be direct, such as radioactivity, or fluorescent molecules; indirect, such as biotin/avidin or didoxigenin; an enzyme such as alkaline phosphatase or peroxidase coupled with a calorimetric and/or florescent substrate; a bioluminescent molecule, or a combination of more than one system. These examples are not intended to be limiting as the only methods of detection. Any method of labeling nucleic acids or probes can be employed in the present invention. The method of detection will be dependent on the label system which is chosen in the design of each individual test.
  • labeled nucleic acids may use fluorescence and may be achieved by placing multiple FITC molecules (Fluorescein isothiocyanate) on the nucleic acid, or any other fluorescent molecule known to those skilled in the art.
  • FITC molecules Fluorescein isothiocyanate
  • chemiluminescence, bioluminescence, or chemifluorescence (all enzyme mediated), radioactivity or other labels known to those skilled in the art may be used where signal amplification is required to increase specificity of detection.
  • Gel visualization may employ the use of radioactivity for autoradiography visualization or chemifluorescence or anything else known to those skilled in the art.
  • One method requires placing a gel, after running it ,in an ATTOPHOS 3 solution (Boehringer Mannheim).
  • Alkaline phosphatase the label on the nucleic acid, reacts with ATTOPHOS ⁇ E to produce fluorescence.
  • Another method of direct detection of specific sequences is with Aequorin bioluminescence (SeaLite Sciences, Inc.).
  • the present invention contemplates technologies that claim the ability to load as many as 100 signal amplification molecules on a single target known by those skilled in the art.
  • the target sequence may be determined by isolation of the target from the assay.
  • Two well known techniques involved size separation techniques or removal of the target by binding to a capture molecule. These two techniques are referred to herein as gel formats and test tube formats, respectively.
  • a gel format involves isolation by size separation and does not use biochemical hooks and solid substrates like the capture methods of the test tube format. Instead, the target/probe complex is isolated by sizing the target/probe complexes in any feasible manner.
  • a preferred method is gel electrophoresis, though other sizing techniques such as chromatography or differential centrifugation are contemplated by the present invention.
  • the target/probe complex(es) are size separated from the unhybridized probe, usually by, but not limited to, polyacrylamide or agarose gel electrophoresis, or capillary gel electrophoresis. Specific target/probe molecules are identified according to the label placed on the specific nucleic acid probe.
  • the expected fragment size of the target/probe complex and thus, the position to where it will migrate on the gel, can be calculated. Descriptions of calculations are found in many standard references, such as Sambrook et al.
  • the expected migration distance of a fragment of known size after a certain amount of time can be calculated by taking into consideration the type of gel and amount of current used to run the gel. This migration reflects the retardation factor (Rf) of the desired labeled moiety. Rf is defined as the distance, from the loading point, to a migration point on a gel of the desired complex, divided by the migration of the smallest molecule not retarded by the gel (usually the dye front).
  • the Rf of the probe/target complex is defined as the distance of migration of the probe/target complex divided by the migration distance of the dye front. Since the Rf is generally a constant number, due to the fact that the dye front migrates identically under identical conditions, then the Rf of the complex is proportional to the distance of migration of the target/probe complex.
  • Target sequences with the labeled probe can be detected at a specific size without interference in detection by other, unlabeled, nontarget nucleic acid sequences that are of similar size and run at the same position on the gel.
  • the target/probe complex is visualized in the gel by methods known in the art.
  • the test tube format uses specific binding pairs such as DIG or biotin streptavidin, enzyme/substrates, or antibody/antigen. These binding pairs are also referred to as biochemical hooks.
  • One of the binding pair is bound to a probe, usually referred to as the capture probe, or a portion of a probe that binds to another probe or the target sequence.
  • the other binding pair member is attached to a solid support surface or to a surface that can be used to separate the captured molecules from the solution, such as a magnetic bead.
  • Solid supports include, but are not limited to, plastic plates, siliconized plates, and plastic beads from which the target can be cleaved or eluted for further analysis. This allows for the analysis of more than one target in a single assay.
  • Two preferred embodiments of the present invention comprise the Restriction
  • each embodiment may be used with the novel methods and compositions for cleavage of nucleic acids with cutter probes (CP) having a reactive group, such as a halogenated nucleotide.
  • CP cutter probes
  • the methods and compositions of CP can also be used in any assay wherein the cleavage of nucleic acids is desired.
  • CP refers to a nucleic acid probe with a reactive group that forms a free radical when activated.
  • the reactive group is a halogen-substituted nucleotide derivative, preferably bromine or chlorine, most preferably a halogen- substituted pyrimidine derivative, though any nucleotide may be halogen-substituted.
  • functionally equivalent molecules that is, those characterized by post- energizing production of two free radicals that at high efficiency, and which cleave the sugar-phosphate backbones of nucleic acid strands, are contemplated as comprised by the term CP.
  • the present invention also comprises molecules that generate free radicals sufficient to cleave phosphate sugar backbones of nucleotides in multi-stranded complexes.
  • BU bromouracil
  • CP Other possible reactive groups that are contemplated by the term BU or CP include chlorouracil, bromocytosine and chlorocytosine, which are known to produce free radicals upon activation.
  • An example of the use of such as probe comprises energizing a 5' bromouracil (BU) containing probe.
  • BU 5' bromouracil
  • a uracil free radical is formed that would, at high frequency, restrict the nucleic acid strand juxtaposed to the BU
  • the bromine free radical will at low frequency restrict the opposite nucleic acid strand.
  • addition of an intercalating dye causes the bromouracil activation to produce a bromine free radical that will cleave the opposite strand at high frequency.
  • the methods and compositions of the present invention comprise cleavage of target nucleic acids with cutter probes.
  • the target nucleic acids may be either single- stranded (ss) or double-stranded (ds).
  • the CP is single-stranded, though other forms or associated structures are contemplated in the present invention.
  • the novel cutter probes can be used in a Cutter Probe Assay (CPA) to detect the presence of a target nucleic acid.
  • CUA Cutter Probe Assay
  • Other uses of the cutter probes include destruction of a specific section of a target nucleic acid and detection of known point mutations.
  • restriction endonucleases which cuts the nucleic acid at a sequence that is specific for the enzyme used and only where the restriction sites are located throughout the Genome. Restricting the nucleic acid in this fashion is limited because the restriction sequences may not always be found in desired positions in a conserved nucleic acid target region. Furthermore, many restriction enzymes have unique requirements, such as requiring methylation of bases for cleavage. The method of the present invention does not have such constraints.
  • the present invention includes use of novel nucleotides which are altered to contain reactive groups that, when activated, for example, by being acted on by a specific energy or chemical agent, cause a specific break in nucleic acid strands.
  • the present invention contemplates use of any UV energy or other frequency that is capable of causing such a double-stranded break.
  • the present invention contemplates the use of any light reactive compound, including but not limited to bromine and chlorine, that is capable of incorporation into or conjugation to nucleic acid sequences, and causes a break in one or both strands of the nucleic acid upon exposure to light or other stimulus.
  • the methods .and compositions of CP employ probe sequences complementary to the target sequence to be cut.
  • a reactive group such as a halogenated nucleotide derivative
  • a reactive group such as a halogenated nucleotide derivative
  • bromouridine is incorporated in place of thymidine in the CP where a complementary adenine is to be cleaved in the target.
  • the cutter probes only cleave the DNA or RNA at the desired site.
  • BU is substituted for some of the thymines
  • the complementary sequence of the probe molecule forms the basis for specificity of placement on the target and specific site cleavage by the cutter probe.
  • a preferred embodiment uses bromouridine.
  • the activity of a CP with BU is based on the photosensitization of bromouracil-substituted DNA.
  • BU substituted DNA can be cleaved by light ranging from short wavelength UV 254 nm to long wavelength UV 313 nm and even into the high intensity visible light region of greater than 313 nm, and including x-ray and gamma radiation.
  • energy of the above types causes the halogenated pyrimidine (BU) to convert to two free radicals, a bromine free radical and a uracilyl free radical.
  • the uracilyl radical breaks the sugar phosphate backbone on the strand the BU is incorporated into and the two adjacent bases upstream and downstream, as well as the bromine free radical breaking the target strand's sugar phosphate backbone.
  • the double- stranded break effect at the bromouridine bases is pronounced at 254 nm.
  • thymine dimers tend to form as an auxiliary reaction.
  • a preferred embodiment uses the 313 nm UV light wavelength. In an in vitro system, if thymine dimers offer no detriment to the application, then approximately 254nm UV exposure is preferred.
  • base cutter probes The advantages of using base cutter probes is to restrict the target at any desired site on a DNA or RNA molecule. This allows cutting of a sample nucleic acid suspected of containing the target at the exact size of the target and to determine the margins of the target. Such cutting results in a blunt ended target without 5' or 3' overhangs.
  • BU replaces the thymines in the probes (54,56), that will be juxtaposed to the adenines to be cleaved in the target.
  • the BU interact with the adenine in the targets (50).
  • the BU-containing probes (54,56) hybridize to a specific region on a target nucleic acid strand (50), the target/cutter probe complex is exposed to UV light.
  • the light exposure results in a strand break at both the BU insertion site on the probe (54,56), and at the juxtaposed site on the opposite target nucleic acid strand (50).
  • the BU molecules in the probe can be located next to each other or scattered throughout the probe at any position. Using this technique, one can selectively cut nucleic acid at any site. Cutting is not restricted to sites that are recognized by restriction endonucleases that only cut at specific sequences. With this novel method, non- enzymatic, yet sequence-specific nucleic acid cutting is possible for both ends of the target.
  • a CP may be complementary to the target and activation of the CP yields excision of a ds target/probe complex.
  • the present invention is not limited to use of just one or two BU sites, and complete cutting may include more than two adjacent BU molecules.
  • double- stranded DNA samples (64) suspected of containing target nucleic acid sequences (50) can be used with a CP (54,56).
  • the sample sequences (64) are separated by denaturation, and then hybridization with the CP (54,56).
  • Activation of the CP yields restriction of the entire target at restriction site 1 and restriction site 2.
  • the CPs are on either side of the target yielding an excised ss target.
  • CPA Cutter Probe Assay
  • a preferred CPA three-step method is:
  • Isolation of the target may be accomplished by any known techniques, preferably either a test-tube format or by size separation.
  • the CPA employing a test-tube format for target/probe complex isolation is shown in Fig. 3 and can be used for RNA or DNA.
  • One or more sequence specific CP can be used in any assay.
  • the sample nucleic acid (64) to be tested is isolated and purified.
  • the sample is a DNA (64).
  • the sample may also be RNA, which is usually ss.
  • step B CP oligos (54, 56) are designed and produced based on target sequence and desired cleavages site or sites.
  • a CP-anchor (56) has a biochemical hook conjugated to it in any position.
  • a CP-reporter (54) has a reporter molecule conjugated to it at any position to aid in target/probe complex visualization.
  • step C the sample nucleic acid suspected of containing the target is denatured if it is ds.
  • step D the following components are mixed under conditions allowing for hybridization: denatured sample nucleic acid; cutter probes pair; and a fixed substrate for binding the CP anchor/target complex, for example, magnetic beads (72) coated with anti-DIG (88).
  • the label on the CP reporter (54) is denoted by asterisks. UV irradiation in vitro at approximately 313 nm restricting the target (50).
  • Step E of Fig. 3 shows anchoring the complex to a solid substrate (72). Once bound, the restricted target/probe complex is easily separated from the high molecular weight genomic DNA, ssDNA (denatured) or RNA target. For example, the magnetic beads (8) having the target probe complex attached via the anchor capture molecule (88) are washed extensively. The target (50) with the two probes (54,56) is the PNAS (90).
  • each assay employs two cutter probes, referred to as the cutter probe-anchor pair (CP-anchor pair, CP1), and two reporter probes, referred to as the cutter probe-reporter pair (CP-reporter pair, CP2).
  • the cutter probe-anchor pair is characterized as being able to bind only to part of a target sequence.
  • the DNA anchor probe (56) shown in Fig. 3 has a bromouracil base at the 5' end and a Digoxigenin (DIG) or anchor molecule conjugated to another base in the probe.
  • the anchor capture molecule (88) is anti-DIG.
  • the cutter probe-reporter pair (54) is characterized by being able to bind the remaining part of the ssDNA target, a sequence to which the CP-anchor pair can not bind.
  • Either CP pair binds to a complementary ssDNA target or a denatured strand of a double stranded DNA or RNA target or an RNA target.
  • the specific elements shown are used for example and it is to be understood that the invention is not limited to such specific examples.
  • the above probes can vary in length. Also, as shown in Fig. IB and IC, if additional levels of specificity are needed in the CPA assay, more cutter probes (58, 60, 62) can be added, with each probe contributing an additional level of specificity.
  • the CPA has multiple levels of specificity built into it, including:
  • target sequence (50) itself is the nucleic acid segment that links both these probes, provides for a highly specific assay with a low background.
  • the sensitivity of detection of single-str.anded target DNA (denatured-ssDNA) or RNA target can be improved by the treatment of the now double-stranded target/probe complex and the other ssDNA/genomic DNA with an enzyme such as SI or Mung bean nuclease, or any other enzyme with similar function, for degrading ssDNA and ssRNA in a 3' to 5' direction.
  • an enzyme such as SI or Mung bean nuclease, or any other enzyme with similar function, for degrading ssDNA and ssRNA in a 3' to 5' direction.
  • the target/probe complex becomes a protected Nucleic Acid Structure (PNAS), and is resistant to degradation by these enzymes.
  • PNAS Nucleic Acid Structure
  • This preferred embodiment of a gel assay format differs from the test-tube format at step F of Fig. 3
  • the gel assay format is configured with the following levels of specificity. 1. Binding of the CP-1, anchor molecule, DIG, etc. need not be present, however, the probe (56) will protect and restrict the target (50).
  • Figure 1 illustrates this probe fragmentation (58,60,62).
  • the target sequence (50) is double-stranded and the probe (68) is ss.
  • the probe (68) is hybridized to the target sequence the probe (68) forms a triplex structure.
  • Such a probe is called a triplex forming oligonucleotide (TFO) and can be used to excise of a specific double-stranded genetic segment.
  • TFO triplex forming oligonucleotide
  • the probe can associate with (non covalent) either strand of the ds-target.
  • This embodiment can be used for excision of genes in vivo and in vitro.
  • dsDNA double-stranded genetic segment
  • Non-enzymatic restriction of DNA in these methods is both novel and beneficial over use of endonuclease restriction of DNA.
  • Endonuclease restriction requires a four to eight base restriction/recognition site by the restriction endonucleases (RE) which may not be located where one wishes to cut the sequence.
  • RE restriction endonucleases
  • bleomycin Large electronegative molecules such as bleomycin, EDTA and others have been attached to nucleic acids and used to provide a method for generating a specific rare-base cutter by attachment to an oligonucleotide which would be hybridized to a DNA (ssDNA) and the close proximity of the bleomycin or EDTA to the ssDNA would cause scision of the DNA strand.
  • ssDNA DNA
  • Novel methods and compositions of the present invention for example, for gene excision in vivo and in vitro, is achieved by the use of the TFO, a form of CP.
  • TFO a form of CP.
  • the TFO specific for a unique dsDNA segment is added in multifold excess to hybridize with the DNA.
  • the TFO lies in the major groove of the helix, where it is bound by
  • TFO is an oligonucleotide having the two end thymine bases substituted with BU, called a BU-TFO.
  • a larger dsDNA segment can be excised by using two BU-TFOs.
  • the flanking regions of a large section of DNA are hybridized to CPs or TFOs, the entire section of DNA is protected from nucleic acid degradation.
  • BU-TFO cutter probe In vivo application of the BU-TFO cutter probe requires transfection of the cell with a BU-TFO composed of altered bases that would not be recognized by the cell membrane bound DNA nucleases. Binding of the two forms of the BU-TFOs in vivo to a chromosomal site is followed by radiation with a more penetrative form of energy, for example, low doses of x-ray which have been also shown to participate in the BU sensitization effect.
  • restriction methods and cutter probe compositions are also used for gene destruction in vivo and in vitro, wherein binding of the TFO forms is similar to the binding in the gene excision process.
  • This embodiment is shown in Fig. 5.
  • Numerous and closely spaced reactive groups, such as BU are present throughout the TFO resulting, post-irradiation, in obliteration of the unwanted nucleic acid sequences, and usually cell destruction.
  • Another embodiment of the present invention is to target the cellular mRNA or other RNAs with the BU substituted molecule. This results in the binding of the BU-
  • BU-CP has advantages over antisense DNA in that with BU-CP probes the RNA is eliminated, whereas with antisense DNA, the RNA still exists and is merely bound to the DNA. The binding conditions may change and the RNA would still be present for translation or other functions.
  • compositions and methods of the cutter probes is the detection of documented point mutations.
  • Examples of using the CPs for detecting point mutations are shown in Table 1.
  • One embodiment of this method comprises denaturing and restricting the sample DNA suspected of containing the point mutation to obtain the target sequence. This restriction is accomplished using any known restriction methods or more preferably, using two CPs to isolate a smaller segment of DNA suspected of containing the point mutation site. A third CP is used to hybridize to the target sequence.
  • the irradiation of the third CP causes cleavage in the target sequence and CP complex. If no point mutation is present, no cleavage occurs. The sample is then run on a gel and visualized to determine if the complex was cleaved.
  • One method of visualizing the presence or absence of the point mutation is to label the CP that binds to the target sequence.
  • a T Does not cleave the Watson strand
  • a C Does not cleave the Watson strand
  • a C Does not cleave the Watson strand
  • RNA U A Cleaves the RNA strand
  • the Restriction Fragment Target Assay (RFTA) of the present invention directly detects both RNA or DNA targets.
  • the targets may be single- or double-stranded.
  • the RFTA method employs at least one primary probe and at least one secondary probe. All probes are single-stranded.
  • the primary probe has at least two sections. At least one section of the primary probe is complementary to the target and at least one section of the primary probe is complementary to a secondary probe.
  • the secondary probes are not complementary to the target, only to the section of the primary probe designed to be complementary to the secondary probe. Additional sections may be added to the primary probe. The additional sections may be either complementary to the target or to a secondary probe.
  • the probe/target complex can be isolated by size separation or by biochemical hooks and linking to a solid support in a test-tube format. Both isolation methods are described above.
  • Technologies, such as the TPA and RFTA can analyze from milligram amounts of nucleic acid, to nanogram, picogram and even smaller amounts of target (to femtamoles).
  • the embodiments of this invention also identify low copy number nucleic acid or other targets in a very small sample.
  • the ability to process such large amounts of nucleic acid is a radical departure from the current microgram analysis technologies, thus greatly improving DNA diagnostics.
  • RFTA can be used in a wide variety of DNA diagnostic applications ranging from simple detection of DNA sequences for the identification of infectious agents, to more complicated applications in which fine DNA sequence analysis is desired, such as with genotyping for histocompatibility antigens, viral strain typing, or genetic disease testing. Minute DNA sequence differences can be determined either using the sequence specific probe itself, or in the selection of the restriction endonucleases used in the initial digestion step, or a combination of both, with the latter method resulting in a change in size of the hybridized restriction fragment. RFTA can also be used to determine differences in variable number of tandem repeat (VNTR), as well as small tandem repeat (STR) markers between samples using this modification of the restriction fragment length polymorphism (RFLP) procedure.
  • VNTR variable number of tandem repeat
  • STR small tandem repeat
  • the RFLP procedure currently uses membranes for hybridization to perform forensic and paternity testing as well as to assess the success of bone marrow transplantation.
  • the disclosure below is directed to applications of methods and compositions for DNA, though RNA is also contemplated, and modifications for RNA applications are given below.
  • FIG. 6 shows Steps A-G: Step A, Isolation and purification of the sample DNA; Step B, release of the target sequence 50 from the sample DNA, Step C, denaturation of the double- stranded nucleic acid sample sequences; Step D, hybridization of the target sequence 50 with a labeled primary probe 74; Step E, hybridization with a secondary probe 76; Step F, hybridization with another secondary probe 78; and step G, isolation by size separation.
  • the resulting structure is then applied to an analytical gel, preferably using gel electrophoresis, and the Rf of the labeled band determined.
  • the levels of specificity as described in RFTA when the target/probe complexes are isolated by gel electrophoresis are:l) 5' Target excising mechanism, 2) 3' Target excising mechanism (an additional level of specificity if different from the first target excising mechanism); 3) Primary probe with label (74); 4) Secondary probe (76); 5) Secondary probe (78); and 6) Predictable Migration (Rf of the complex). Additional probes can be fragmented and added in specific sequence to further increase specificity levels.
  • Step A Sample DNA sources: Isolation and purification of the DNA
  • the Restriction Fragment Target Assay (RFTA) of the present invention detects a target DNA sequence present in a sample.
  • the method employs purifying a DNA sample from a source suspected of containing the target.
  • Sources include, but are not limited to, cells, tissue, organelles, serum, water, and other fluids.
  • the host or sample DNA may be in the form of high molecular weight genomic DNA. Purification may be by any one of a variety of methods known to those skilled in the art or using amplified DNA meant for such a purpose.
  • Step B Excision of the target nucleic acid of DNA RFTA
  • the purified sample DNA is then cleaved to excise target nucleic acid sequences if they are present in the sample.
  • Excision of the target nucleic acid is the first level of specificity in the RFTA method.
  • Excision can be accomplished in any one of several different ways.
  • One method includes digestion with one or more sequence-specific restriction endonucleases, whose restriction sites are known to border the target nucleic acid.
  • An additional level of specificity can be added by using a second endonuclease that excises the target at a site different from the first restriction endonuclease.
  • nucleases such as non-specific endonucleases
  • the nucleic acid could be cleaved using methods employed by ribozymes, ribonucleases, or other methods to cleave nucleic acids.
  • the choice of one or more endonucleases or other types of nucleases depends on the target sequence and the design of the individual test being performed.
  • Step C Denaturation in DNA RFTA
  • Step D Hybridization of Primary Probefs in DNA RFTA
  • the sample DNA is combined with probes under conditions allowing for hybridization.
  • three probes, a primary probe and two secondary probes are employed in the RFTA method.
  • the primary probes are specially designed to exclusively bind the target, therefore, only if the target is present in the sample, the primary probes will bind.
  • the present invention contemplates that the target and probe size are variable.
  • a) Designing Primary Probes A consideration in designing the primary probe(s) (74) is that either sample strand (known as the Watson or Crick strands, and abbreviated as W or C in the figures) or both sample strands may be used as the target strand (50) in the present invention. Use of both strands yields a two-fold amplification of signal. The latter embodiment requires use of strand specific primary probes (74) that are specific for the particular target nucleic acid strand (50).
  • the primary probe (74) may have one or more sections. In a preferred embodiment, the primary probe has three sections.
  • the first section (80) is capable of binding, by sequence homology or other means, to a secondary probe (76) that may or may not be labeled.
  • This first section (80) of the primary probe (74) is not capable, by sequence homology or other means, of binding to the target nucleic acid sequence (50) and will bind the initial secondary probe (74).
  • a second section (82) generally near the middle of the primary probe (74), is capable by sequence homology or other means, of binding to the target sequence (50).
  • a third section (84) of the primary probe (74) is capable of binding, by sequence homology or other means, to another secondary probe (78) that may or may not be labeled.
  • the secondary probes are not capable of binding to the target sequence.
  • either or both of the first (80) and third (84) sections of the primary probe (74) has a reporter molecule, such as a label, attached.
  • the reporter may be lengthened or shortened to improve the signal amplification.
  • probes For analyses where more than one sequence identification is desired, multiple primary probes, with different sequences for binding to different target regions, are used in the reaction mixture simultaneously (multiplexing). These probes are not complementary to each other. The number of probes used are limited only by the possible methods of capture-detection, separation and detection that are known to those skilled in the .art.
  • the primary probe is the third level of specificity in the RFTA method.
  • b) Labeling Probes Probes may be labeled with detection molecules using radioactivity, fluorescence, or any other detection method known to those skilled in the art. In one embodiment, the primary probe is labeled along its entire length, regardless of how many different sections comprise the primary probe.
  • the primary probes (74) are placed with the denatured sample DNA in molar excess amounts to ensure spontaneous reaction. The single-stranded sample DNA and the probes are then hybridized. Hybridization may be by known procedures such as slow cooling, ionic adjustments, or pH neutralization. This is the third level of specificity of the assay.
  • Step E Hybridization of the First Secondary Probefs ' ) in DNA RFTA
  • the first secondary probe is then hybridized to the primary probe.
  • Secondary probes (76) are used to report the target and thus, are labeled and are reporter probes.
  • Secondary probes are comprised of an oligonucleotide (ssDNA) sequence usually, but not limited to, alO-25 mer probe. In a gel format the secondary probes may be extensively labeled.
  • ssDNA oligonucleotide
  • the secondary probes may be extensively labeled.
  • Each of the secondary probes adds another level of specificity to the RFTA method. In a preferred embodiment, two secondary probes are used, which are the fourth and fifth levels of specificity, respectively.
  • the secondary probes (76) and primary probes can be added at the same time and hybridized with the denatured sample DNA in molar excess amounts. Alternatively, the secondary probes may be added after the primary probes have hybridized. Hybridization may be done by known procedures. This is the fourth level of specificity of the assay. Step F: Hybridization of Another Secondary Probe in DNA RFTA
  • Step G Isolation and Detection in DNA RFTA If the target (50) is present in the sample, the resulting complex of primary probe with the attached secondary probe or probes and target sequence is a double-stranded structure (as shown in step F of Fig. 6). The complexes are then isolated from the rest of the sample DNA and detected. Two embodiments of isolation are shown (a) size separation depicted in Fig. 6 and (b) capture probes depicted in Fig. 7. After gel electrophoreses, the detection of labeled bands at the expected gel position (Rf) indicates the presence of the nucleic acid target with six levels of specificity.
  • the target may exist in numbers lower than the primary probe and secondary probe concentration.
  • primary probes and secondary probes may hybridize together without binding to a target and provide false positives on the gel.
  • cutter probes such as BU base cutters, having sequence specificity for the second part of the primary probe (the section that is complementary to the target) may be hybridized to the target/probe complex after the hybridization of the primary and secondary probes. Thereafter, treatment of the hybridized sample complexes with UV (approximately 313 nm) results in cleavage of the probe/target complex into two smaller complexes. The two smaller complexes will be easily distinguished by computation of their Rf on a sizing gel from the probe complexes without a target.
  • the 3' end of target/probe complex may be rendered nuclease resistant by capping the free deoxyribose hydroxyl group (i.e. a hydroxyl that is not bonded to another nucelotide) at the 3' end by removing the 3' end and designing the target and secondary probe sequences such that a gap does not exist between them. Similarly, capping at the 5' end is achieved by removal of the free 5' hydroxyl or phosphate. Nuclease resistance, although unnecessary in RFTA, may be helpful in increasing the sensitivity of the assay for a specific application.
  • FIG. 7 Another embodiment of the RFTA invention wherein the target/probe complexes •are isolated by size separation is shown in Figure 7. Note that probe/target complexes are shown binding to either or both of the nucleic acid strands. This invention contemplates the use of such sets of probes to provide an amplification of the signal for detection of the target sequence.
  • Figure 8-1 shows an embodiment of RFTA wherein three different probes are used.
  • the secondary probes, (76) and (78) bind and form the complex that is then isolated by gel electrophoresis. There are unbound ends to the complex that may be treated by exonucleases if desired.
  • One or more of the probes may be labeled. Either strand, Watson or Crick, may be used as the target sequence with use of probes having corresponding binding ability. This configuration provides five levels of specificity.
  • Figure 8-II shows another embodiment using a probe (74) which forms hairpin loops on both of its ends and binding with the target sequence (50) internally to the hairpins.
  • This probe/target complex is resistant to exonuclease digestion. After formation of this probe/target complex, the exonuclease may be added to the sample and all unbound target and probe, and any other nucleic acid present, are digested away.
  • the hairpin probes may be labeled in the ways previously described. This configuration provides four levels of specificity.
  • Figure 8-III shows a preferred embodiment which uses two primary probes, (74) and (86), each capable of forming one hairpin loop on one end and the other end capable of binding to a portion of the target sequence (50).
  • the probes 7.4 and (86)
  • the hairpin loops may be of any size and form any shape that has the effect of a hairpin, a structure where an end binds to an internal sequence of the same strand.
  • Such structures include hairpins, cloverleafs, branched structures and others known to those skilled in the art.
  • any unbound or incompletely bound probes (74, 86) or targets (50) is digested by exonuclease treatment.
  • the two probes (74, 86) do not bind to each other and are only visualized in the gel if the target (50) is joining the two probes (74, 86), thus greatly reducing background signal.
  • any non-desirable complexes would not migrate to the predicted Rf, and thus, would be distinguishable from the desired target. Thus, complexes without desired targets would not generate bands on the gel at the expected Rf.
  • Another method contemplated by the present invention is the use of tube assays and capture probes to isolate probe/target complexes, instead of size separation (Fig. 7).
  • the following is an example of a preferred embodiment of the RFTA method for a target sequence of 15 nucleotides (can vary) employing a capture probe for isolation of target/probe complexes from the sample nucleic acids.
  • the sample suspected of having the target sequence is hybridized with three probes. Two of these probes are modified from the probes previously described in the RFTA method employing size separation to isolate the probe/target complex.
  • Probe Configuration 1) In one embodiment of the test-tube format, the primary probe may have approximately 55 nucleotide bases wherein a section of approximately 15 nucleotide bases are complementary to the target sequence.
  • Such a section may be placed in or near the center of the primary probe, with another section of approximately 15 nucleotide bases on one end for binding to a secondary probe and a third section of approximately 25 nucleotide bases on the other end for binding to another secondary probe.
  • a first secondary probe is at least 15 nucleotide bases and is complementary to one section of the primary probe but not complementary to the target sequence.
  • a second secondary probe is at least 25 nucleotide bases and is complementary to the end of the primary probe not bound by the first secondary probe and not complementary to the target sequence.
  • the secondary probes need not be labeled; only the primary probe was required to be labeled. However, in these test-tube or tube-based applications, only one secondary probe is labeled with any labels known in the art.
  • test-tube format of RFTA uses one secondary probe as a capture probe, (conjugated with DIG or any other biochemical "hook” known to the art) to attach the target/primary probe complex to a solid support.
  • Figure 7 shows a preferred embodiment of this method: the isolation of sample DNA, and restriction nuclease release of the target sequence from the long sample DNA strands.
  • the target sequence, 1 is denatured and hybridized with a primary probe, Probe, 2.
  • the secondary probe, 4, a capture probe has a capture molecule attached to it.
  • the capture molecule is shown to be digoxigenin (DIG), 3.
  • DIG digoxigenin
  • a magnetic bead, 5 with antibodies to DIG is added to the test-tube to capture the target/probe complex.
  • Step G of Figure 7 shows an embodiment wherein a third probe, a reporter probe, 6, preferably labeled, is added to the target/probe complex above. By binding such a labeled probe, the entire structure can be easily detected (visualized) within the test-tube container.
  • the capture and reporter probes, both secondary probes can be added in any order.
  • reporter probe may be lengthened or shortened to yield optimal signal.
  • the secondary reporter probe is the only one labeled in the test-tube assay and can be labeled using any method known to those skilled in the art; however, another label may be used also if necessary.
  • the reporter probe signal is important for detection.
  • reporter signal should only be attached to the solid substrate when the target is present to bridge the gap between capture and reporter probes.
  • Figure 9 presents tube applications and variations in the target probe complex
  • the tube based assay can be used as a preprocessing tool, for example, for concentrating all the targets from milligram quantities of nucleic acids down to microgram quantities, for DNA chip, PCR, and other nucleic acid or signal amplification technologies.
  • the signal amplification can be any method known to the art.
  • target/primary probe/capture probe/reporter probe complex The existence of the target/primary probe/capture probe/reporter probe complex is directly dependent on the presence of target sequence to attach all the probes together. Absence of the target sequence should result in essentially no non-specific signal (background).
  • PDTP target/probe
  • RFTA test-tube assay format are shown in Figure 9. Use of either or both strands of the nucleic acid, the Watson or Crick strand, is contemplated in these diagrams. Hairpin structures are shown as example only in Figure 9 and are not intended to be limiting in the type of structure formed.
  • Figure 9-1 shows the target sequence 1 bound to the primary probe 2, that has a capture molecule 3 attached directly to capture probe 4.
  • a labeled reporter probe 5 is added for detection of the structure.
  • This embodiment has 5 levels of specificity and is not exonuclease resistant, unless the ends are capped by addition of hinge/loop regions or other methods.
  • Figure 9-II shows an embodiment wherein the secondary probes form hairpin or other similar structures.
  • Target sequence 1 is bound by primary probe 2.
  • a reporter probe 5 is labeled using methods known to those skilled in the art.
  • a capture probe 4, with a capture molecule 3 attached, is also bound to the target/probe structure. This embodiment has 5 levels of specificity and is exonu
  • Figure 9-III shows the use of two probes that form hairpin loops, the capture primary probe 2 and the reporter probe 4.
  • the part of each of the probes is bound to the target sequence 1.
  • the primary probe 2 has a capture molecule 3 attached to it to provide for capture of the target/probe complex.
  • This embodiment has four levels of specificity and is resistant to exonuclease digestion.
  • Figure 9-IV shows the use of a reporter probe 4 that forms a hairpin loop.
  • the target sequence 1 is bound to primary probe 2.
  • Primary probe 2 is bound to the reporter probe 4, which forms a hairpin structure.
  • Primary probe 2 is also bound to a capture probe 5 that has a capture molecule 3 attached.
  • This embodiment has five levels of specificity and is only partially resistant to exonuclease digestion unless the ends are capped.
  • This embodiment of RFTA may allow signal to be generated in the absence of target sequence. In this case, care must be taken to unhook the reporter and capture region.
  • Figure 9- V shows the use of a primary probe 2 bound to the target sequence 1.
  • a reporter probe 5 and a capture probe 4 with a capture molecule 3 are bound to the primary probe 2.
  • This embodiment has five levels of specificity .and can be digested by exonuclease unless capping methods are employed. Additional levels of specificity can be achieved by adding multiple labels to probes, or by creating one or two reporter probes.
  • Table 2 characterizes multiple probes in tube-based assays. Additional probes can be fragmented and added in specific sequence to further increase specificity levels.
  • another embodiment of the target/probe construct includes using hairpin loops to reduce the number of probes from 3 to 2.
  • a sequence nuclease resistant although not necessary, may be used for reducing background or increasing sensitivity in a particular assay situation.
  • Designing the probes such that no gaps are formed between the probes and the target sequences render the protected target sequence, or protected target nucleic acid sequence (PNAS), resistant to nucleases, such as Exo III.
  • PNAS protected target nucleic acid sequence
  • 3' probe ends would need to be capped, or dehyroxylized or modified in some manner known to those skilled in the art to render protection from nucleases.
  • Other known means of modification include but are not limited to, the presence of DIG, biotin, avidin, or hydroxylase reactions. Such modifications can be used with any of the above structures.
  • elongated hairpin reporter probe allows for the addition of more reporter molecules, and enhances visualization of target.
  • RNA target sequences For detection of RNA target sequences, methods and compositions similar to the ones of the above described DNA methods and compositions, with modifications for RNA sequences, can be used.
  • the initial step is the purification of the sample RNA by any of a variety of methods known to those skilled in the art. In its simplest form, detection and characterization of specific RNA sequences are accomplished using hybridization of the specific labeled nucleic acid probe followed by size separation and detection of a specific target as described in the previous section. Generally, target excision from the sample RNA, is not necessary because many
  • RNA target sequences are found as discretely sized RNA molecules such as tRNA or mRNA.
  • the target RNA can be excised in the present invention.
  • an RNA molecule can be cut to a desired size for isolating the target sequence by binding a complementary probe, either DNA or RNA, of the correct size, and using a single-strand nuclease to remove the single-stranded regions.
  • Other known methods of enzyme and RNA manipulation can be used to size the RNA molecule, such as ribozymes and the CP cutter probes.
  • RNA target sequences are useful for applications such as gene expression analysis, detection of RNA viruses (such as HCV, hepatitis C virus), or discriminating active versus latent viral infections (i.e. CMV, cytomegalo virus).
  • RNA viruses such as HCV, hepatitis C virus
  • CMV cytomegalo virus
  • the present invention may also be designed to be quantitative for any of these applications.
  • a reverse transcription step may be performed initially to produce single- or double-stranded cDNA. This would allow the use of restriction fragment analysis coupled with RFTA in much the same way as was described for DNA applications.
  • RNA virus genotyping HCV
  • An additional level of sequence specificity may be added to RFTA by synthesizing the cDNA with a sequence specific primer instead of a relatively non-specific primer, such as random hexamers or an oligo- dT primer, to yield a runoff reverse transcript of a defined size which then could be subjected to RFTA analysis.
  • sequence specific primer instead of a relatively non-specific primer, such as random hexamers or an oligo- dT primer, to yield a runoff reverse transcript of a defined size which then could be subjected to RFTA analysis.
  • RNA targets such as mRNA, viral RNA, tRNA and others, can be directly detected by RFTA both in the gel and test-tube formats. Therefore, there is no need to perform a reverse transcriptase reaction to convert the RNA to a cDNA for subsequent detection.
  • RNA RFTA invention A preferred embodiment of the present RNA RFTA invention is depicted in Fig.
  • RNA RFTA Gel Assay It is also contemplated that size separation can be accomplished by test tube methods, using capture molecules on probes or other appropriate molecules. Additionally, hairpin structures, as shown for DNA RFTA, can be used where applicable.
  • Step I mRNA is isolated
  • Step II A target site on the mRNA (polypyrimidine) is selected and the primary probe is designed.
  • Step III Hybridization of primary probe to the mRNA target
  • Step IV Treatment with exoribonuclease (SI and Ming Bean nuclease)
  • Step V Generation of the target probe complex (PDTP complex)
  • Step VI Begin signal amplification by adding the secondary reporter probe poly dT (biotinylated) to hybridize to the poly A 3' end of the triplex.
  • Step VII Add the avidin-enzyme complex to complex with the conjugated biotin molecules.
  • Step VIII Electrophoresis to a predictable R f Step IX: Place gel in chromogenic substrate solution
  • This assay provides four levels of specificity.
  • Step I Isolate mRNA Step II: Hybridize with BU cutter probe Step III: Irradiate with UV light to complete restriction
  • Step IV Hybridize with primary probe
  • Step V Hybridize with secondary reporter probe
  • Step VI Run complex on gel to a predictable R f
  • FIG. 11 An example of this method is shown in FIG. 11.
  • the isolated total mRNA, some of which contains the target sequence, is present in the sample.
  • a probe 2 containing BU cutter site is bound to the target sequence 1.
  • the target is cut using UV light.
  • a labeled probe 3 is added that binds to the single-stranded portion of target sequence.
  • a third probe that binds to the labeled probe 3, is added.
  • the entire double-stranded structure of probes/target (PDTP) is run on a gel to a specific Rf. Cutting the RNA ⁇ by hybridization of the target with a base cutter probe 2, herein representing a fragment of the primary probe, is not necessary in every case, as described earlier.
  • the target may be hybridized with a primary probe 3, that has two regions that hybridize to the RNA target and the third section with a 5' end or a 3' end that does not hybridize to the target.
  • the full length of this probe is labeled as previously discussed.
  • One variation in the RNA embodiments is to increase specificity by adding a second primary probe, acting as a base cutter probe 2 that could be fully labeled or unlabeled, and would bind close in tandem to the first primary probe. Portions of the nucleic acid probe that would hybridize to the RNA target can be fragmented. Thus, for each additional probe piece produced, another level of specificity is added.
  • the fragmented probe could be an oligonucleotide easily substituted with the base cutter probe molecule as has been discussed throughout all the RFTA formats described herein.
  • the SI nuclease treatment or any known to those skilled in the art, is an optional step in the procedure with the presence of a base cutter probe and necessary in the absence of the cutter probe. Removing the non-hybridized mRNA will yield a predictable Rf value for the expected target/probe complex. If the ssRNA tail is not removed, then the absolute Rf value of the tail/target/probe complex will be slightly modified to a different, but also predictable, Rf value. The size of the final complex is not important. The ability to detect the target relies on the predictability and reproducibility of the Rf migration of the complex.
  • cleave the non-hybridized ssRNA tail include conjugating an EDTA molecule or bleomycin to the end of the probe adjacent to the ssRNA tail.
  • Hybridization of the probe having any other similar molecule known to those skilled in the art, with the RNA will result in the target
  • RNA being cleaved and the ssRNA tail(s) being removed.
  • Another approach to cleave the non-hybridized ssRNA tail is to end the probe adjacent to the mRNA tail with one or more thymine bases. If these are substituted for BU, then exposure of the hybridized complex to UV, preferably long wavelength, approximately 313 nm, will ' cause the scision of the mRNA tail, non-enzymatically.
  • the target/probe complex When the target/probe complex is run on polyacrylamide or agarose gels, the double-stranded target/probe complex is visualized and the Rf is calculated by comparison to the Rf of nucleic acid size standards. Obtaining expected Rf values for the target/probe complex confirms (with four levels of specificity) that the desired RNA target is present in the sample.
  • RFTA/RNA Gel Assays Levels of Specificity (4)
  • Omitting the exoribonuclease treatment changes the levels of specificity to 3. However, substituting base cutter probes for exoribonuclease increases the three levels of specificity by two levels, to a total of five levels of specificity.
  • the target complexes may also be detected by gel formats, which involve attachment of capture molecules to the probes.
  • Capture and reporter probes are not connected themselves, only to a common target
  • DNA-chip technology can also detect the target/probe complex
  • Either (w) or (c) strands can be detected, independently or together.
  • RNA RFTA Multiple specificity levels
  • Capture and reporter probes are not connected themselves (don't bind directly to each other), only to a common target.
  • the 5' poly A region approximately 200 to 250 mer can be used for signal amplification
  • RNA can be directly analyzed with undergoing a Reverse Transcriptase (RT) step.
  • RT Reverse Transcriptase
  • the present invention including the RFTA embodiments, for both DNA and RNA applications holds several advantages over conventional DNA or RNA blotting procedures which utilize membrane hybridization after tr.ansfer of the target nucleic acid following electrophoretic separation.
  • RFTA is significantly faster and more convenient to perform than membrane hybridization, and requires less technical skill and specialized equipment, such as electro- or vacuum-transfer systems, UV cross linkers or vacuum ovens, and hybridization ovens and water baths.
  • RFTA is also substantially cheaper to perform than standard RFLP analysis in that much less probe is utilized for the hybridization in a small volume prior to electrophoresis as opposed to hybridization in a relatively large volume, with proportionally slower hybridization kinetics, utilized with membrane hybridization.
  • RFTA electrophoresis system
  • the available detection systems all work as well, if not better, in situ or on native gels with analysis as the gel runs, or on fixed or dried gels as they do on membranes.
  • RFTA may be more sensitive than membrane hybridization because it does not require nucleic acid transfer or membrane cross linking, both of which can result in loss of specific signal due to damage or inefficient transfers. Because RFTA does not utilize
  • RFTA has the advantage that several probes can be utilized on replicate samples at one time, or with several probes in the same sample, on any given electrophoresis lane or gel, eliminating the need to run replicate gels and/or stripping already hybridized membranes for subsequent re-probing, both of which are expensive and time-consuming procedures.
  • the methods of the present invention can be used with all DNA analysis and diagnostic technologies, whether amplification (PCR, etc.) or non amplification based, and even chip based.
  • An embodiment of a mRNA RFTA Test-Tube Assay is presented.
  • Step I Isolation of mRNA
  • Step II A target site on the mRNA (polypyrimidine) is selected and the primary probe is designed.
  • Step III Hybridization of primary probe to the mRNA target.
  • Step IV Hybridize with a secondary capture probe
  • Step V Treatment with exoribonuclease (S 1 and Mung Bean nucleases).
  • Step VI Resulting PDTP complex produced
  • Step VII Add secondary reporter biotinylated amplification probe
  • Step IX Bind PDTP to a solid substrate.
  • Step X Bind PDTP to a solid substrate
  • Step X Wash
  • Step XI Add chromogenic substrate and inspect for color development.
  • Step I Isolate mRNA
  • Step II Select mRNA Target and design primary probe (BUCP)
  • Step III Hybridize BUCP with the mRNA target region (BUCP- 1 , capture)
  • Step V Capture the PDTP complex
  • Step VI Wash
  • Step VII Hybridize with secondary reporter probe and determine presence of label This has three levels of specificity.
  • compositions of the present invention should result in the DNA chip and PCR techniques functioning with increased sensitivity and specificity, due to the fact that they are now not limited in the size of the nucleic acid sample that can be analyzed in a single tube, nor will there be a large amount of possible non-specific contamination.
  • the methods of the present invention can pre-process genomic or other large quantities of DNA (milligram quantities of non-specific nucleic acids to ⁇ g quantities enriched with targets present in sample) to allow previously known DNA analysis technologies to specifically and accurately determine the presence of selected target nucleic acid sequences.
  • TPA Target Protection Assay
  • TPA is disclosed in U.S. Patent
  • RNA targets sensitive enough to detect a single DNA (gene) copy.
  • the TPA invention also detects both ds and ss DNA. mRNA/TPA
  • the methods and compositions of the present invention have the ability for processing a wide range of amounts of RNA, which imparts the sensitivity necessary for the development of those applications previously discussed and unattainable by preexisting technologies. This allows for the TPA process to be used as an early infectious time-course diagnostic technology.
  • the importance of detecting RNA targets early in an infectious time-course is based on the fact that replicating RNA viruses, tumor development .and infectious disease progression all require protein synthesis in the host, but more importantly, the production of specific mRNA species.
  • One embodiment contemplated by the present invention involves indirect single gene copy detection. For each activated gene, tens of thousands of mRNA molecules are produced. When a single gene is activated, 20,000 specific mRNA targets will be present in the cell and tissue and capable of being identified.
  • a preferred embodiment of the present method invention includes the following steps. First, a sequence specific target region on the RNA is chosen. Second, the RNA sample suspected of having the target sequence is isolated by any of the methods known to those skilled in the art, resulting in the acquisition of denatured and linear RNA. Sample RNA is purified from a viable source or using amplified DNA meant for such a purpose. Excision of the target from the sample nucleic acids is unnecessary due to the fact that the mRNA is already predictably sized. In the third step, the specific single-stranded RNA target is protected by hybridization with a duplex DNA structure resulting in the formation of a partial triplex molecule called a protected target nucleic acid structure (PNAS). An embodiment of triplex formation is shown in Figure 12..
  • PNAS protected target nucleic acid structure
  • the reporter probes are DNA; the double-stranded DNA capture molecule length is variable; and the biochemical hook is any molecule known to those skilled in the art for this purpose, such as DIG.
  • This DNA capture molecule can be a hairpin structure that forms a ds molecule that functions as the ds structure in Figure 12.
  • the TFO Hairpin Capture Probe In mRNA TPA Regions must be identified in the mRNA that are polypyrimidine rich. This is necessary due to the requirement for a pyrimidine-purine-pyrimidine motif for the triplex formation.
  • the ds DNA hairpin capture probe is characterized by having two sections:
  • the 5' end is a polypyrimidine rich region of variable length • Both regions are joined n the middle by a six base stretch that forms the loop of the hairpin.
  • the hairpin capture probe is a DNA molecule that folds back on itself to form a hairpin.
  • the 3' end should be conjugated with a biochemical hook, close to but not at the 3' end.
  • the target is hybridized to a dsDNA hairpin probe with the RNA strand orientations.
  • An additional embodiment of the invention comprises addition of poly dT labeling probes to a captured mRNA molecule. This enhances the reporting signal.
  • mRNA TPA gel-based assay comprises the following steps: Step I: Isolation of mRNA
  • Step II Selection of target region and design of a DNA capture probe.
  • Step III Hybridization of the mRNA Target and the dsDNA probe.
  • Step IV Exonuclease treatment to destroy any non-mRNA complexed dsDNA probes to increase assay specificity.
  • Step V Attachment of the PNAS to a solid support.
  • Step VII Signal amplify by hybridizing PNAS with poly dT reporter probes
  • Step IX Add the avidin-enzyme conjugate to the target solution which binds to the Biotin molecules on the conjugated poly dT amplification probe.
  • Step X Wash
  • Step XI Dissiate the PNAS from the magnetic bead without compromising the PNAS structure integrity.
  • Step XII Run the soluble PNAS on an electrophoresis gel to a predictable
  • Step XIII Place the gel in a chromogenic substrate solution and incubate to allow color development. This has five levels of specificity.
  • Target region sizes and probe lengths may vary in size from short to long, and are dependent solely on the type of target and format the assay will assume. Hybridization is accomplished in the same manner as for the RFTA method described earlier with a differently designed probe.
  • the DNA probe used here can be a hairpin or any other structure that forms a double-stranded structure. A hairpin structure is a preferred embodiment.
  • Formation of the PNAS is the first level of specificity of the methods and compositions of the present invention. This level of specificity is lower due to the necessity to work in a polypurine or polypyrimidine rich region in the DNA duplex protection molecule and some non-specific RNA species may bind to the dsDNA protection molecule.
  • One tactic to circumvent this problem is the addition of the recA protein, which enables the use of a target protection molecule (dsDNA) that varies over the four normal bases, and not the two bases in the polyrich region.
  • dsDNA target protection molecule
  • An example of the above preferred embodiment has a target sequence that has a
  • FIG. 14 An embodiment of mRNA TPA gel-based assay is presented in Fig. 14. An embodiment of mRNA TPA test tube or capture assay is shown in Figure 15.
  • Step I Isolate mRNA
  • Step II Selection of target site and design of capture probe.
  • Step III Hybridization of Target and probe.
  • Step IV Exo III treatment to remove any dsDNA capture probes that have not been protected by the target.
  • Step V Attach PNAS to solid support
  • Step VII Hybridize with reporter probe (poly dT, biotinylated and 25 mer)
  • Step VIII Wash
  • Step X Wash
  • Step XI Dissociate PNAS from the magnetic bead without dissociating
  • Step XII Gel electrophorese to a specific R f
  • Step XIII Place the gel in a chromogenic substrate and visualize bands on gel (calculate R f )
  • the DNA probe used here can be a hairpin or any other structure that forms a double-stranded structure.
  • a hairpin structure is a preferred embodiment.
  • RNA/TPA PNAS target/probe complex
  • TPA tumor-associated protein
  • RNA/TPA test-tube application offers very high specificity because the assay is mediated by at least five levels of specificity.
  • the assay allows direct analysis of large quantities of RNA.
  • Exo III treatment is necessary to eliminate non-specific DNA which is hybridized by the capture probe.
  • An example of a mRNA TPA tube-based assay using DNA hairpin structure is described..
  • Step I Isolate mRNA
  • Step II Selection of target site and design of hairpin capture probe.
  • Step III Hybridization of Target and hairpin probe.
  • Step IV Exonuclease III treatment to remove any dsDNA hairpin capture probes that have not been protected by the target.
  • Step V Attach PNAS to solid support
  • Step VII Hybridization with reporter probe (poly dT, biotinylated and 25 mer)
  • Step VIII Wash Step IX: Add avidin-enzyme conjugate
  • Step X Wash
  • Step XI Add chromogenic substrate for color development.
  • Step I Isolate mRNA
  • Step III Anchor PNAS to a fixed substrate
  • Step IV Run gel and determine R f by band signal visualization.
  • Figure 18 represents the mRNA CPA test-tube format assay.
  • Step I Isolate mRNA
  • Step III Anchor PNAS to a fixed substrate
  • Step IV Wash Step V: PNAS detected by signal amplification.
  • triplex lock Another embodiment that can be used with triplex formations is the triplex lock.
  • exposure of a ds DNA segment to EXO III may degrade the DNA on the W and C strands in a 3' -. 5' direction.
  • Binding of a triplex forming oligonucleotide (TFO) to the DNA's duplex protects the triplex from being degraded by EXOIII for a short time.
  • Figure 16 depicts an mRNA molecule forming a triplex with a ds DNA captive probe, which is a hairpin structure, designed to produce a major groove for the mRNA to lie in. This hairpin also eliminates, on of the 3' DNA duplex ends, a possible site of EXO III attack.
  • RNA/TPA The main advantage for the use of RNA/TPA is that it enables all the signal amplification technologies as well as DNA on a chip and PCR to achieve target detection, as sensitive as single gene copy detection, by use of the methods and compositions of the present invention.
  • This provides protection, concentration, and reduction of milligram amounts of nucleic acids in a patient specimen, to a single microgram of nucleic acids i.e. also containing all the targets in the original sample. This is presented in Figure . Viral load issues in HIV therapy have been heretofore unsuccessfully addressed.
  • the methods and compositions of the present invention are used for viral load diagnosis by allowing one to follow mRNA levels throughout the HIV treatment regimen period.
  • protease inhibitor therapy suffers from difficulty in ability of PCR to determine the point at which therapy needs to cease, the point at which viral load diminishes.
  • PCR has inherent flaws which affect its sensitivity and specificity, the most important of which preclude its identification of low copy number targets (DNA) in a vast excess of genomic DNA and a total lack of ability to directly analyze RNA targets.
  • the present invention solves such problems by identifying low abundance targets in a vast excess of DNA/RNA by direct analysis down to a single gene or RNA target copy.
  • TPA can deliver for analysis of viral load.
  • parameters that TPA can deliver for analysis of viral load will include monitoring the numbers of the following nucleic acid forms:
  • DNA or RNA targets by indirect evaluation of mRNA analysis. As bioluminescence and other signal amplification techniques are improved, mRNA targets can be directly evaluated at a single messenger copy level in a large abundance of non-specific nucleic acids.
  • Another use of the present invention is direct mRNA analysis.
  • a particular use of direct mRNA analysis is nucleic acid based cancer metastasis assays.
  • a lymph node harboring a single tumor cell would present itself as a minimum of 10,000 to 20,000 mRNA targets specific for a tumor cell surface marker, receptor protein, that could be detected by isolation of all the RNA in the entire lymph node (milligram amounts) and its one-time analysis.
  • Another use of the invention is direct identification of Hepatitis C RNA and mRNA. This direct detection allows early detection of the infectious virus and helps secure the safety of the blood and plasma supplies world- wide.
  • Another use of the present invention is that many therapeutic modalities can be monitored by mRNA target analysis specific to the abnormality being treated. Diagnosis of hormone abnormalities (hypo- and hyper- states) can be monitored by similar mRNA analysis. Growth Factor Therapy can also be monitored by direct mRNA inspection as is the case for gene regulation problems. Lastly, along with the myriad of human medical applications, there exists an equally or even more impressive list of agricultural and veterinary applications.
  • compositions of the present invention include compositions with the components necessary to practice the methods taught herein.
  • a composition comprising a primary probe with sections of nucleotides complementary to a specific target sequence and one or more secondary probe sequences, and labeled secondary probes.
  • Compositions or kits comprising selected primary and secondary probes, along with nucleases, ribozymes and buffers are included in the present invention. It is to be understood that the individual molecules, probes and components can also be provided individually or in combinations.
  • the following protocol is a representative procedure for the rapid isolation of DNA from large amounts of whole blood: 150 mL of blood collected in venipuncture tubes (heparin, ACD or EDTA) is pooled together and diluted with 150 ml Isoton II (Coulter Diagnostics) in a 500 ml centrifuge bottle. 30 ml of 10% Triton X-100 is added and mixed vigorously for 3 seconds. Cell nuclei are pelleted at maximum speed (12,000 x g) for 5 minutes. After removal of the supernatant, the pellet is resuspended in 10 ml PK mixture (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% Tween 20, 0.5% NP-40, and
  • RNA preparative technique 2.5 mg/ml Protease K
  • the sample is then transferred to a centrifuge tube and spun at 12,000 x g for 10 minutes.
  • the supernatant is recovered and the DNA is pelleted with the addition of 0.2 volumes of 10M ammonium acetate and 2 volumes of ethanol.
  • the precipitated DNA is pelleted at 5,000 x g for 10 minutes, washed twice with 70% ethanol, and then resuspended in 0.5 ml sterile water. Mild sonication or shearing may be required to obtain complete dissolution of the pellet.
  • Approximately 1 mg of total genomic DNA should be recovered from 150 ml whole blood (approx. 150 million nucleated cells).
  • An RNA preparative technique can also be applied.
  • Exo HI 40 ⁇ l of a 100,000u/ml stock. Incubate the reaction at 37°C for an additional 50 min, followed by inactivation of the enzymes by a biochemical or biophysical method. The sample is now ready for the next step in the procedure.
  • the anisotropy is then remeasured compared to the initial value for analysis.
  • HIV-1 Human immunodeficiency virus type 1
  • serologic assays which detect the presence of anti-HIV antibodies are used to screen blood and blood products. While generally reliable, these tests will occasionally produce false positive results due to cross reactive antibodies or false negative results if the infection is at an early stage before the onset of a measurable immune response. It is in the latter case that alternative methods such as TPA may be particularly useful, since large .amounts of sample DNA may be processed and tested in a single assay tube.
  • a direct assay for the virus using co-cultivation with a susceptible cell line does exist, however this method is labor intensive and requires several days to complete. The following example will describe the extraction of a large amount of blood for the worst case: that of a recently infected individual with low levels of infected CD4 positive cells.
  • HIV-1 Capture probe 5' - ACT GCC ATT TGTACT GCT GT-DIG - 3'
  • HIV-1 reporter probe 5' - GAA TAG TAG ACA TAA TAG TA - FITC - 3'
  • the replication of the filamentous bacteriophages occurs in harmony within the E. Coli pili containing host bacterium and the infected cells are not lysed while producing several hundred virus particles per cell per generation, released to the supernate.
  • the titer of bacteriophage in a culture of infected bacteria can reach 10 12 pfu per milliliter.
  • the extracellular infecting strand is the M13(+) strand a single-strand circular DNA molecule
  • the system that will be used to demonstrate BU Cutter Probe DNA restriction utilizes the ability of the BU Cutter Probe to hybridize to a specific sequence on the circular M13mpl8 bacteriophage.
  • the following is a sequence of base numbers 2500 to 2525.
  • the hybridization temperature should be around 37°C in a IM salt hybridization buffer.
  • a high stringency wash is added at 37°C at a lower salt concentration to remove BUCP probes that bind to non-specific regions.
  • the circular DNA with the attached probe is then irradiated in a petri dish with long wavelength UV (313 nm) at a dose rate of 14.6W m "2 .
  • UV ultraviolet
  • Hoechst Dye #33258 Prior to irradiation, however, Hoechst Dye #33258 must be added to insure high frequency cutting by the Bromine free radical of the target (opposite strand sugar-phosphate backbone of the Ml 3 mp 18 bacteriophage
  • the restriction of the bacteriophage DNA is visualized by performing Agarose (1%) Gel Electrophoresis, which demonstrates breakage induced in the probe strand as well as the Ml 3 DNA strand.
  • the BU Cutter Probe has at least one similar fluorescent molecule on both sides of the BU molecule.
  • the M13mpl8 bacteriophage DNA has another fluorescent dye, fluorescing at a different wavelength from the first, which upon gel analysis will confirm the R f of the circularized (non-restricted target) and the linearized forms (target restriction). These fluorescent dyes should be near infrared in spectrum to minimize experimental background. Analysis of the gel in a Molecular Dynamic Fluorescence Analyzer will yield the profiles.
  • the Ml 3 remains a circular intact piece of DNA migrating in the gel at a specific R f Restriction of the target will shift the gel R f to another position evidencing production of a cut and linearized bacteriophage.
  • Reaction conditions can be quality controlled by inspection using denaturing gels of other smaller bands representing (2) the Cutter Probe cleavage (if experimental conditions were not met then .an intact Cutter Probe band would be observed).
  • the target selected is a sense strand (w) sequence of the toxin production gene of Bacillus Anthracis. Presence of the target in the DNA sample indicates presence of the bacteria and infection.
  • a large serum sample 50-100 ml. is centrifugal at 12,000 x g at 4°C. 2. Resuspended pellet in 567 ⁇ l TE buffer by repeated pipetting. Add 30 ⁇ l of 10%
  • Anthracis toxin production gene 3' (541) actttgagtg gtccgtctt tatccccctt gtacagggg cgggcggtca tggtgatgta
  • the DNA must be denatured to release the ss DNA target.
  • the DNA After restriction, the DNA must be denatured by heating to 94 °C for a minimum of one minute (at 94-C) or alkalai treatment (o.4N No. OH plus 25 mM EDTA).
  • the primary probe is hybridized to a part of the target sequence. If hybridization conditions are similar the capture probe may be simultaneously added to the mixture (10 nmoles of DIG labeled probed) and binds to another section of the target region.
  • the temperature of hybridization is usually 20° C less than the melting temperature of the ds nucleic acid. Usual hybridization conditions require 10-fold less of each probe, 50°C incubation for 20-60 mins., and high salt (5x55PE) buffer at neutral OH.
  • the reporter probe (25mer) is incubated at 50oC in 5XSSPE for 20-60 mins. and a stringent wash is introduced to remove unbound probe by washing at 45oC with a 1XSSPE buffer. All tube washers containing magnetic beads are performed in a magnet tube holder to prevent bead/target loss.
  • the next step is initiation of signal development by adding the avidin-horse raddish peroxidase conjugate. The conjugate is added in 10 fold excess in the tube containing the target captured beads. The beads are similarly washed to remove unbound conjugate and the chromogenic substrate tetramethyl benzidine is added which upon contact with the peroxidase generates a soluble color.
  • the intensity of the color is proportionate to the numbers of targets present.

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Abstract

La présente invention concerne des méthodes et des compositions de détection de séquences nucléotidiques. Plus particulièrement, la présente invention concerne des méthodes et des compositions de détection de séquences génétiques spécifiques utilisant différentes stratégies de protection et de récupération de cibles nucléotidiques. De plus, la présente invention concerne de nouvelles méthodes de clivage d'acide nucléique. En outre, l'invention concerne des méthodes de détection de séquences nucléotidiques, lesquelles emploient des sondes de coupe non spécifiques de séquence, utilisent des formations triplex impliquant des structures en épingle à cheveux, et des méthodes d'amplification de signal.
PCT/US1998/024226 1997-11-12 1998-11-12 Methodes et compositions de detection de sequences nucleotidiques specifiques WO1999024621A2 (fr)

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MXPA00004675A MXPA00004675A (es) 1997-11-12 1998-11-12 Metodos y composiciones para la deteccion de secuencias nucleotidicas especificas.
JP2000519613A JP2001522588A (ja) 1997-11-12 1998-11-12 特異的ヌクレオチド配列を検出するための方法および組成物
AU15237/99A AU1523799A (en) 1997-11-12 1998-11-12 Methods and compositions for detection of specific nucleotide sequences
CA002309861A CA2309861A1 (fr) 1997-11-12 1998-11-12 Methodes et compositions de detection de sequences nucleotidiques specifiques
KR1020007005156A KR20010032036A (ko) 1997-11-12 1998-11-12 특이적 뉴클레오타이드 서열의 검출방법 및 조성물
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