KR20010032036A - Methods and compositions for detection of specific nucleotide sequences - Google Patents

Abstract

The present invention includes nucleic acid sequence detection methods and compositions. More specifically, the present invention includes methods and compositions for detecting specific gene sequences using different nucleic acid target protection and recovery strategies. The present invention also encompasses nucleic acid cleavage novel methods. Disclosed herein are nucleic acid sequence detection methods using cleavage probes that are not sequence specific, use of triple helix formation, triple helix formation with hairpin structure, and signal amplification methods.

Description

METHODS AND COMPOSITIONS FOR DETECTION OF SPECIFIC NUCLEOTIDE SEQUENCES

Background of the Invention

Molecular biology techniques have provided a number of accurate and rapid tests for the determination, identification or detection of DNA and RNA sequences. Unfortunately, most of these tests rely on PCR amplification of DNA as one or several steps involved in the test. Amplification of a target nucleic acid by PCR may result in amplification of a nucleic acid sequence other than the target nucleic acid sequence of interest.

Although a number of target and signal amplification methods have been described in the literature, it is speculated that none of them provide high specificity, simplicity and speed in parallel. Nucleic acid detection technology has recently come into close scrutiny for the development of diagnostic technology for the 21st century. Nucleic acid sequence-based amplification (NASBA Organon Teknika), standard-substituted amplification (Becton Dickinson), transcription-based amplification system (TAS), transcription-mediated amplification (Genprobe), polymerase chain reaction (PCR, F. Hoffmann la Roche), And a number of different amplification-based techniques, such as target amplification of in situ PCR.

Other amplification-based techniques include signal (probe) amplification, such as ligase chain reaction (LCR / Abbot), q-beta bacteriophage replicas (Genetrak systems), cycling probe technology, (ID Biomedical of Vancouver), b DNA (Chiron), in situ hybridization (ligase hybridization), and genome amplification (GAWTS) using transcript sequences.

All of these techniques are unsatisfactory. Targeted amplification has problems with amplicons and other forms of sample contamination, and specificity. This problem is inherent in the constraints of these technical foundations and is accompanied by a lack of suitable experimental (kit) designs, resulting in a lack of high specificity and sensitivity. In addition, these technologies lack direct screening ability for specific RNA targets, placing severe restrictions on these technologies to support the progress of RNA virus, cancer, and other infectious disease diagnosis and treatment management.

In addition, the inability to screen nucleic acids above the microgram amount for the native target also serves to reduce the specificity and sensitivity of these methods. In fact, the only truth in infectious diseases is that the faster the infectious time course that requires detection, the more samples of host DNA required for analysis (milligram amount).

Signal amplification techniques also present unique problems. Inappropriate levels of non-specific high background signal and sensitivity are coupled with target incapacity due to the total inability of the target to be reduced to a single copy, providing little chance of successful diagnostic success.

Bioluminescence, currently the most sensitive assay for single gene copy detection, is able to detect at least hundreds of thousands of protected targets. What is needed is a method of increasing the sensitivity of target detection for single gene detection.

While currently available nucleic acid amplification methods allow the detection of relatively small amounts of target nucleic acid molecules (microgram amounts of DNA) present in a small amount of sample, the ability to detect target nucleic acid molecules in a shorter time with less background interference is required. Problems inherent in PCR and other amplification techniques include sample contamination during collection and the presence of amplicons (amplified DNA) that contaminate DNA specimens to provide false positive results. There are problems associated with the generation of non-specific amplification and primer dimers mediated by closely related sequences. There is also a poor regulation of specificity that leads to a false positive response, and a poor regulation of sensitivity that leads to a pseudo-negative response. PCR results should often be confirmed and validated by other techniques such as DNA-South blotting, RNA blotting and probe hybridization, or in situ hybridization. In addition, PCR and amplification techniques can only be used with very small amounts of starting sample DNA in the range of up to 1 microgram. This cancels PCR techniques for the detection of low copy number nucleic acid targets in large amounts of whole / non-specific nucleic acids and for the diagnosis of early infection time course.

Techniques for providing direct RNA analysis include northern blot and ribonuclease assays. Northern blots first denature the RNA molecule and ensure it is linearly released. RNA is then transferred to the membrane by gel electrophoresis, hybridized with labeled probes, and subjected to visualization. This procedure is qualitative and quantitative. This long and costly procedure is almost unsuitable for RNA diagnostics due to all of its downsides similar to the problems associated with the use of Southern blotting in design in DNA diagnostics. These include time consuming, high cost / materials and labor, lack of sensitivity in relatively inadequate detection of targets, and inability to become sensitive as it decreases to a single target copy.

Ribonuclease protection assays include binding of probes and RNA molecules, S1 treatment to remove non-specific RNA and single-stranded RNA regions, and separation on electrophoretic gels. This procedure is only qualitative and lacks sensitivity to diagnostic techniques due to the amount of DNA required for visualization in the assay.

Reverse transcriptase-PCR (RT-PCR), the only currently available process with some potential for RNA diagnostics, can only achieve indirect RNA analysis. RNA is converted into DNA copies (cDNA) and quantitative PCR is performed. RT-PCR is expensive, relatively low in specificity, relatively low in sensitivity and extremely labor intensive, requiring extreme standardization at all levels to provide reproducible results. In addition, RT-PCR cannot be used for early infectious time course nucleic acid diagnostics.

Accordingly, what is needed are methods and compositions that can detect specific nucleic acid sequences. There is a particular need for methods and compositions that provide the flexibility to allow separation of nucleic acid sequences using the desired level of specificity. In addition, methods that permit the separation of specific target sequences from any amount, particularly large amounts of sample nucleic acid, without the use of nucleic acid amplification techniques, and are flexible to achieve separation at different levels of specificity depending on the level of specificity desired. This is necessary. There is a particular need for methods and compositions that can detect target sequences using RNA, including but not limited to mRNA as a nucleic acid target sequence source.

Summary of the Invention

Methods and compositions for qualitative and quantitative detection and analysis of target deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences isolated from various cells and / or tissue sources are described. The method is low cost, specific to various levels of specificity, sensitive, capable of analyzing a wide range of nucleic acids, provides reproducible results, and requires minimal effort. The methods and compositions of the present invention include diagnostics and treatments such as detection of microorganisms such as viruses and other microorganisms, and pathogens of humans, animals and plants; Can be used for the diagnosis of infectious diseases, cancers and metastases in humans, animals and plants, for the analysis of blood products and for genetic analysis for use in fields such as tumors, forensics, paternity determination, tissue or organ transplantation and genetic disease determination have. These assays can also be used to detect contamination of food, soil, water and blood products and to check air quality.

Two aspects of the invention include restriction fragment target assays (RFTA) and target protection assays (TPA). The invention also encompasses new site directed cleavage techniques of nucleic acids, including cleavage probes (CP) having a reactive group such as halogenated nucleotide derivatives, preferably pyrimidines or purine homologs, and cleavage nucleic acids. RFTA includes selective restriction cleavage of nucleic acids and detection using special probes. TPA includes the protection of target protein sequences and detection using special probes.

Cutter Probe (CP) and Cutter Probe Analysis (CPA)

Cleavage probes can be used in known assays that include nucleic acid cleavage novel methods and compositions and require cleavage of nucleic acids. Preferred methods include oligonucleotide probe sequences complementary to the target sequence to be cleaved, wherein reactive groups, such as halogenated nucleotide derivatives, in the target sequence are incorporated into the probe at positions parallel to the position to be cleaved at the target. For example, substituted bromouracil (BU) is incorporated at the thymidine position in the probe where the complementary adenine is a cleavage site in the target nucleic acid (opposite strand). Cleavage probes cleave only DNA or RNA at defined sites. When BU is substituted with thymidine, the other thymidine present in the cleavage probe along with the other three bases forms the basis for the specificity of placement and cleavage by the base cleavage probe. Cleavage probes have sequences that are parallel to the target backbone. Such novel cleavage probes can be used for cleavage probe analysis (CPA) for detection of target nucleic acids or any other assay that desires cleavage of nucleic acids. Other uses of cleavage probes include disruption of specific regions of target nucleic acids and detection of known point mutations. BU, a reactive group in CP, is achieved by appropriate forms of specific energy, light energy or chemical agents and the target sequence is cleaved.

CP can be used for novel analysis of the present invention. In addition, once the target sequence is bound by the cleavage probe, the target / probe complex resists specific nuclease degradation and is protected from PNAS, TPA protected target nucleic acid structures (PNAS) and partial double helix target probe complex (PDTP) in RFTA. ).

Cleaver probes are described in US Provisional Patent Application No. filed February 24, 1998, which is incorporated herein by reference in its entirety. 60 / 075,812, and No. 5, filed March 5, 1998. 60 / 076,872.

Restriction Fragment Target Analysis (RFTA)

Restriction fragment target analysis (RFTA) is disclosed in US Provisional Patent Application No. filed November 12, 1997, which is incorporated herein by reference in its entirety. 60 / 065,378, and No. Filed February 24, 1998. 60 / 075,812.

In general, RFTA can be used for the detection of single- or double-stranded RNA or DNA targets. Preferably, the RFTA method and composition comprise at least one primary probe and at least one secondary probe. The probe is preferably a Japanese chain. The primary probe may have at least two zones. At least one zone of the primary probe is complementary to the target and at least one other zone of the primary probe is complementary to the secondary probe. The secondary probe is not complementary to the target but only to the region of the primary probe that is complementary to the secondary probe. Additional zones can be added to the probe.

The RFTA method of the present invention for both DNA and RNA applications has several advantages over conventional DNA or RNA blotting procedures that utilize membrane hybridization after migration of target nucleic acids after electrophoretic separation. RFTA is considerably faster and more convenient to perform than membrane hybridization and requires less technical skills and special equipment such as electro- or vacuum-delivery systems, UV crosslinkers or vacuum ovens, and hybridization ovens and water baths. RFTA is also difficult to perform than standard RFLP analysis in that it uses a relatively large amount of hybridization and therefore uses much less probes to hybridize in small quantities prior to electrophoresis, as opposed to proportionally slower hybridization kinetics. It is quite inexpensive. No special electrophoretic system is required for RFTA, and all of the available detection systems work as well as on membranes, even on fixed or dried gels, although not better. In addition, RFTA can be more sensitive than membrane hybridization because it does not require nucleic acid delivery or membrane crosslinking, which can result in damage or loss of specific signals due to inefficient delivery. Finally, RFTA is designed for eliminating the need for performing a replication separation step and / or for subsequent re-detection, such as several gels being multiplexed, in that several probes can be tested simultaneously in a single sample on a given electrophoretic gel. There are advantages, such as the removal of hybridized membranes (both steps are expensive and time-consuming).

An example of a preferred RFTA method of the present invention uses the following steps: 1) isolation and purification of sample nucleic acid using a target nucleic acid; 2) cleavage of the target nucleic acid by cleavage of the sample nucleic acid; 3) ds nucleic acid sample into the Japanese chain. Denaturation; 4) Binding of the purified nucleic acid sample with a labeled target specificity primary probe complementary to at least a portion of the target sequence of interest. Add the probe in molar excess under conditions that allow spontaneous hybridization of the probe to the target nucleic acid; 5) isolation of the target nucleic acid-probe complex; And 6) detection of the target nucleic acid-probe complex.

Targeted Protection Assay (TPA)

Methods and compositions comprising a TPA embodiment can be used for the detection of 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 biology techniques. In addition, TPA embodiments can be used with novel nucleic acid cleavage such as CP.

Preferred embodiments of TPA, RNA-TPA in the in vitro construction of a double stranded hairpin DNA probe of various lengths, preferably having a sequence rich in polypurine or polypyrimidine, fold centrally and form a double helix DNA structure.

TFO hairpin capture probe from mRNA TPA

Regions should be identical in polypyrimidine-rich mRNA. This is necessary due to the requirement for pyrimidine purine pyrimidine motifs for triple helix formation. The dsDNA hairpin capture probe is characterized by having two zones: the 3 'end is a polypurine rich area, the 5' end is a polypyrimidine rich area, and the two areas are centered by the extension forming the loop of the hairpin. Connected. The hairpin capture probe again folds itself to form a hairpin. Also, the 3 'end must be conjugated with a biochemical hook (digoxigenin or DIG) that is not in close proximity at 3'.

1.Isolation of mRNA by methods known to those skilled in the art and binding of specific mRNA molecules by dsDNA hairpin probe of step 1. The probe / target sequence is currently protected nucleic acid sequence (PNAS). The unique RNA target site is located between the mRNA and the 3 'poly A and 5' mRNA ends (closer to 5 '). This is the first level of specificity.

2. Removal of the double-stranded structure, such as by the addition of nucleases such as Exo III, which will degrade dsDNA probe molecules that do not become triple-stranded PNAS due to the presence of target mRNA molecules.

3. Then separate the PNAS. In a preferred method, PNAS is attached to a solid substrate using methods known to those skilled in the art. Such methods include, but are not limited to, attachment of probe / target complexes and magnetic beads by using biochemical hooks or specific binding pairs such as DIG (digoxigenin) and BIOTIN and anti-DIG, and streptavidin coated magnetic beads. ). The length of the target can vary and is preferably in the range from 8 to 25 nucleotide bases to kilobase length fragments.

4. The presence of the target is identified by a reporter probe molecule, preferably poly (dt) oligo, of approximately 25 nucleotides, which will hybridize with the 3 ′ adenine mRNA molecule. Each reporter probe molecule has at least one member of a binding group associated with the reporter probe molecule.

The ability to accurately determine the presence of low copy number RNA targets would aid therapeutic applications and allow for the determination of the presence of a single DNA by an indirect approach.

Compositions of the invention comprising aspects of CP, RFTA, and TPA include compositions that are essential for practicing the methods taught herein. For example, the composition used in the method for RFTA includes a primary probe having one or more regions of nucleotides complementary to the target sequence and one or more regions of nucleotides complementary to the secondary probe sequence, and a labeled secondary probe can do. Compositions or kits comprising selected primary and secondary probes, together with nucleases and buffers, are included in the present invention. It is understood that individual molecules, probes and components may also be provided separately.

It is therefore an object of the present invention to provide methods and compositions for detecting specific gene sequences in humans, plants and animals, preferably with varying levels of specificity.

It is another object of the present invention to provide novel specific nucleic acid cleavage methods and compositions comprising cleavage probes.

It is another object of the present invention to provide a method and composition for detecting DNA sequences comprising specifically designed probes, including primary and secondary probes.

It is an object of the present invention to provide methods and compositions for detecting RNA sequences comprising specifically designed probes, including primary and secondary probes.

It is an object of the present invention to provide a method and composition for detecting a nucleic acid sequence comprising a triple helix structure.

It is an object of the present invention to provide methods and compositions for detecting specific gene sequences in humans, plants and animals with varying levels of specificity.

It is an object of the present invention to provide a method and composition for detecting nucleic acid sequences for the actual detection of microorganisms or pathogens in humans, plants and animals.

It is an object of the present invention to provide methods and compositions for the detection of nucleic acid sequences for the determination of genetic relationships such as paternity or species identification, or for determination of potential donors of organs or tissues.

It is an object of the present invention to provide methods and compositions for the detection of nucleic acid sequences for forensic decision or for blood supply protection.

It is an object of the present invention to provide methods and compositions for the detection of nucleic acid sequences for the analysis of genetic diseases in humans, plants and animals.

These and other objects, features, and advantages of the present invention will become apparent after review of the detailed description of the described embodiments and the claims.

Cross Reference to Related Applications

This application is directed to US Provisional Patent Application No. 1, filed December 12,1997. 60 / 065,378, and US Provisional Patent Application No., filed February 24, 1998. 60 / 075,812, and US Provisional Patent Application No., filed March 5, 1998. Claim priority on 60 / 076,872.

Technical Field

The present invention includes methods and compositions for detecting nucleic acid sequences. More specifically, the present invention encompasses methods and compositions for detecting specific gene sequences using different nucleic acid target detection and recovery strategies. The present invention also encompasses novel nucleic acid cleavage methods.

1 is an embodiment of a cleavage probe used for cleavage of single stranded nucleic acid.

2 is an embodiment of a cleaver probe used to cleave a target nucleic acid from a sample.

3 is an embodiment of a cleavage probe used in an assay to recover cleaved target nucleic acid from a sample.

4 is an embodiment of a single chain cleavage probe (triple-forming oligonucleotide, TFO) used for cleavage of a double stranded target.

5 is an embodiment of a single stranded truncated probe (TFO) used to disrupt a double stranded gene target.

6 is an embodiment of a restriction fragment target assay (RFTA) in which the target / probe complex is separated by size separation.

7 is an embodiment of RFTA in which the target / probe complex is separated by in vitro format.

8 is an alternative embodiment of an RFTA primary probe for use with target / probe separation by size separation. 8I and 8II are primary probes with hairpin loops.

9 is an alternative embodiment of an RFTA primary probe for use with target / probe separation by in vitro format. 8II, 8III and 8iv are primary probes with hairpin loops.

10 is an embodiment of RNA / RFTA analysis for gel basal separation that is characteristic of cleavage probes.

11 is an aspect of RFTA / RNA application.

12 is an embodiment of a TPA / RNA capture assay.

13 is an embodiment of signal amplification for RNA / TPA tube analysis.

14 is an embodiment of TPA / RNA.

15a and b show aspects of TPR / RNA by signal amplification.

16 is an embodiment of a triple helix lock.

The present invention includes methods and compositions for detecting target nucleic acid sequences in a sample that are believed to contain the target. Methods and compositions include compositions and methods necessary for the direct detection of both RNA and DNA sequences. The methods and compositions can detect target sequences in a range of nucleic acid amounts, preferably in the range of nanograms to milligrams. Steps to enhance the specificity of the assay are described in the order in which they are performed and named by the level of specificity. The methods and compositions of the present invention include diagnostics and treatments, such as detection of microorganisms such as viruses and other microorganisms, and pathogens of humans, animals and plants; To be used for diagnosis and treatment such as diagnosis of infectious diseases in humans, animals and plants, analysis of blood products, and for genetic analysis for use in fields such as tumors, forensics, paternity, tissue or organ transplantation and genetic disease determination Can be. These assays can also be used to detect contamination of food, soil, water and blood products and to check air quality.

Specificity is a concept that is currently evolving in DNA diagnostics. As used herein, specificity levels are recognized as a single event that must occur to visualize the expected end result. In nucleic acid diagnostics, such events may include:

Specific nucleic acid restriction reaction;

Specific nucleic acid probe hybridization reactions;

Specific binding of PNAS with a fixed substrate; or

Signal visualization reaction.

For the purposes of turning and improving DNA diagnostic techniques, additional levels of specificity are added. Additional levels of specificity include effectively increasing the size of the target by screening the target again in size as a result of probe hybridization. This level is found in the preferred embodiment of RFTA using the gel format. Methods and compositions of the present invention that include non-enzymatic cleavage of CP site specificity of a target nucleic acid have the advantage of providing two levels of specificity for analysis.

This application is fully incorporated by reference in US Provisional Patent Application No. 60 / 065,378, filed Nov. 12, 1997, and US Provisional Patent Application No. 60 / 075,812, filed February 24, 1998, and 1998. Priority is claimed to US Provisional Patent Application No. 60 / 076,872, filed March 5, 2011.

The present invention uses methods and compositions that are well known molecular biology techniques. The present invention contemplates nucleases, probes, hybridization strategies, methods for catching elution, various methods of size detection or a combination of elution by detection. For example, isolation of nucleic acids as used herein can be performed by techniques known to those skilled in the art. Separation and purification methods are described in Sambrook et al., Molecular Cloning; A well known laboratory manual, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, New York (1989), incorporated herein by reference.

Other well known techniques include visualization of such nucleic acids for labeling and detection of nucleic acids. Such labels are called reporter molecules, and some probes containing labels are called reporter probes. The label used may be various ones currently available and may be used directly, such as radioactive, or fluorescent molecules; Indirect like biotin / avidin or digoxigenin; Enzymes such as alkaline phosphatase or peroxidase coupled with colorimetric and / or fluorescent substrates; Bioluminescent molecules, or combinations of one or more systems. This example is not intended to be limited to the only method of detection. Methods of labeling nucleic acids or probes can be used in the present invention. The detection method will depend on the labeling system chosen for the design of each individual test.

For example, labeled nucleic acids can utilize fluorescence and can be achieved by placing multiple FITC molecules (fluorescein isothiocyanate) on the nucleic acid, or other fluorescent molecules known to those skilled in the art. In addition, where signal amplification is required for increased specificity of detection, chemiluminescence, bioluminescence, or chemifluorescence (all enzyme mediated), radioactivity or other labels known to those skilled in the art can be used. Gel visualization can use radioactivity or chemifluorescence for radiographic visualization or others known to those skilled in the art. One method involves placing the gel in an ATTOPHOS® solution (Boehringer Mannheim) after running it. Alkaline phosphatase, a label on nucleic acid, reacts with ATTOPHOS® to generate fluorescence. Another method of direct detection of specific sequences is by Afquorin Bioluminescence (SeaLite Sciences, Inc.). The present invention contemplates a technique for charging as many as 100 signal amplification molecule loading capacities to a single target known by those skilled in the art.

In addition, target sequences can be determined by separation of the target from the analysis. Two well known techniques include the removal of a target by size separation techniques or binding to capture molecules. These two techniques are referred to herein in gel format and in vitro format, respectively.

Gel formats involve separation by size separation and do not use biochemical hooks and solid substrates, such as in vitro format capture. Instead, the target / probe complexes are separated by size selection of the target / probe complexes in other possible ways. Preferred methods are gel electrophoresis, but other sized sorting techniques such as chromatography or different centrifugation are also contemplated by the present invention.

In general, target / probe complexes are typically separated by size from non-hybridized probes by, but not limited to, polyacrylamide or agarose gel electrophoresis, capillary gel electrophoresis. Specific target / probe molecules are identical according to the label placed on the specific nucleic acid probe.

The expected fragment size of the target / probe complex, and thus the position moving in the gel, can be calculated. Descriptions of calculations can be found in a number of standard documents, such as Sambrook et al. After a period of time, the expected travel distance of the known sized fragments can be calculated taking into account the type of gel and the amount of current used to run the gel. The shift reflects the delay coefficient (Rf) of the desired label residue. Rf is defined as the distance from the loading point to the moving point on the gel of the target complex divided by the movement of the smallest molecule (usually the dye front) that is not delayed by the gel. In other words, Rf of the probe / target complex is defined as the travel distance of the probe / target complex divided by the travel distance of the dye front. Due to the fact that the dye fronts move identically under the same conditions, Rf is generally a constant number, so the Rf of the complex is proportional to the travel distance of the target / probe complex.

Transfer to the intrinsic (normative) gel position adds one additional level of specificity due to the presence of the label, not just the size of the probe / target complex. Target sequences with labeling probes can be detected at specific sizes without interference in detection by other non-labeled non-target nucleic acid sequences that are of similar size and run at the same location on the gel. Target / probe complexes are visualized in the gel by methods known in the art.

In vitro formats use specific binding pairs such as DIG or biotin / streptavidin, enzymes / substrates, or antibodies / antigens. Such binding pairs are also referred to as biochemical hooks. One of the binding pairs is bound to a probe, referred to as a capture probe, or a portion of a probe that binds to another probe or target sequence. The other binding pair member is attached to the surface of the solid support or to a surface that can be used for separation of trapped molecules from solution, such as magnetic beads. Solid supports include plastic plates, siliconized plates, and plastic beads from which the target can be cut or eluted for further analysis. This allows for the analysis of one or more targets in a single assay.

Two preferred embodiments of the invention include restriction fragment target assays (RFTA) and target protection assays (TPA). It is also contemplated that the invention may be used with novel methods and compositions for cleaving nucleic acids with cleavage probes (CP) having reactive groups such as halogenated nucleotides. Methods and compositions of CP can be used in assays where cleavage of nucleic acids is desired.

CP method and composition for cleavage of nucleic acid sequences

As used herein, CP is a nucleic acid probe with reactive groups that, when activated, form free radicals. Preferably, the reactive group is a halogen-substituted nucleotide derivative, preferably bromine or chlorine, most preferably a halogen-substituted pyrimidine derivative, but the nucleotide may be halogen-substituted. However, it is contemplated to include by the term CP those functionally equivalent molecules, ie those that are characterized by high efficiency and after energy injection of two free radicals that cleave the sugar-phosphate backbone of the nucleic acid backbone. The present invention also encompasses molecules that produce sufficient free radicals to cleave the backbone sugar backbone of nucleotides in multiple stranded complexes. The term BU (bromouracil) is used herein for ease of understanding and is not limited to compounds that may be used in the probe molecule. Other possible reactive groups contemplated by the term BU or CP include chloruracil, bromocytosine and chlorocytosine, which are known to produce free radicals upon activation.

One example of the use of such a probe includes injecting energy into a 5'bromouracil (BU) containing probe. Uracil free radicals are formed that will limit the nucleic acid backbone placed parallel to the BU at high frequency.

Bromine free radicals will limit the nucleic acid backbone with a low frequency. In this method, the addition of the internal addition dye produces bromouracil activation, producing bromine free radicals that will cleave the high frequency of the opposite strand.

The methods and compositions of the present invention include cleavage of the target nucleic acid using a cleavage probe. The target nucleic acid may be single stranded (ss) or double stranded (ds). Preferably CP is a Japanese chain, but other forms or associated structures are also contemplated in the present invention. New cleavage probes can be used for cleavage probe analysis (CPA) to detect the presence of target nucleic acids. Other uses of cleavage probes include disruption of specific regions of target nucleic acids and detection of known point mutations.

Restriction of nucleic acid targets has been achieved with restriction endonucleases that are specific for the enzyme used and that cleave the nucleic acid in the sequence only if the restriction site is located throughout the genome. Restriction of nucleic acids in this manner is limited because restriction sequences may not always be found at the desired position in the nucleic acid target region where they are conserved. Moreover, many restriction enzymes have inherent requirements, such as the requirement for methylation of bases for cleavage. The method of the present invention does not have this restriction.

The present invention includes the use of novel nucleotides that are denatured to contain reactive groups that cause specific destruction in the nucleic acid backbone, for example, when activated by action by specific energy or chemical agents. The present invention contemplates the use of UV energy or other frequencies that can cause such double strand breaks. In addition, the present invention includes, but is not limited to, bromine and chlorine, which are capable of incorporation or conjugation into nucleic acid sequences and cause destruction in one or both strands of the nucleic acid upon exposure to light or other stimuli. Consider the use of photoreactive compounds.

The methods and compositions of CP use probe sequences that are complementary to the target sequence to be cleaved. In the cleavage probe, reactive groups, such as halogenated nucleotide derivatives, are incorporated at certain positions in the probe that are arranged parallel to the position to be cleaved at the target. For example, bromouridine is incorporated in place of thymidine in CP where complementary adenine is cleaved at the target. Cleavage probes cleave DNA or RNA only at desired locations. When BU is substituted with a thymidine moiety, the complementary sequence of the probe molecule forms the basis for specificity of placement on the target and specific site cleavage by the cleavage probe.

Preferred embodiments use bromouridine. The activity of CP with BU is based on photosensitization of bromouracil-substituted DNA. The BU substituted DNA can be cleaved by light rays in the range of high wavelength visible light ranging from short wavelength UV 254 nm to long wavelength UV 313 nm and even 313 nm or more, and x-rays and gamma rays. Although not wishing to be bound by a particular theory, it is believed that this type of energy converts halogenated pyrimidines (BU) into two free radicals, namely bromine free radicals and uracilyl free radicals. The urasilyl radicals destroy the sugar phosphate backbone on the backbone into which the BU is incorporated, and the two adjacent bases upstream and downstream and the bromine free radicals destroy the sugar phosphate backbone on the target backbone. The double strand break effect at the bromouridine base is significant at 254 nm. In vivo, however, thymidine dimers are liable to form as an adjuvant reaction. In the in vivo method of the present invention, when thymidine dimer is not desired, the preferred embodiment uses a 313 nm UV wavelength. In in vitro systems, approximately 254 nm UV exposure is preferred if thymidine dimer is harmless to the application.

The advantage of using a base cleavage probe is to limit the target at the target site on a DNA or RNA molecule. This allows for cleavage of the sample nucleic acid that is believed to contain the target at the exact size of the target and measurement of the target's margin. This cleavage results in a smooth terminal target with no 5 'or 3' overhang.

A method and composition aspect of CP is shown in FIG. 1. Single chain target 50, DNA or RNA, is cleaved by hybridization with at least one ss cleavage probe 54,56. BU will displace thymine in probes 54 and 56 and will be placed in parallel with the adenine to be cleaved at the target. Upon activation, the BU interacts with adenine at the target 50. After the BU-containing probes 54,56 hybridize with specific regions on the target nucleic acid backbone 50, the target / indicator probe complex is exposed to UV. Light exposure causes chain break at both the BU insertion site on probes 54 and 56 and parallel placement sites on opposite target nucleic acid backbone 50.

In probes, BU molecules are located adjacent to each other or scattered throughout the probe at arbitrary locations. Using this technique, nucleic acids can be selectively cleaved at any position. Cleavage is not limited to sites recognized by restriction endonucleases that cleave only specific sequences. By this novel method, non-enzymatic, but sequence-specific nucleic acid cleavage is possible for both ends of the target. Alternatively, the CP may be complementary to the target and the activity of the CP cleaves the ds target / probe complex. The present invention is not used for only one or two BU sites, and complete cleavage may include two or more neighboring BU molecules.

Restriction of Nucleic Acids and Protection of Kb Length Fragments

Another aspect of the invention is that double stranded DNA sample 64, which is believed to contain the target nucleic acid sequence 50, as can be seen in Figure 2, can be used with CP 54,56. Sample sequence 64 is isolated by denaturation followed by hybridization with CP (54,56). The activity of CP limits the entire target at restriction site 1 and restriction site 2. In step D, the CP is on either end of the target to produce a truncated ss target.

Preferred Embodiments for CP of ssDNA with ssRNA Target

Cleavage probe analysis (CPA) can be automated and miniaturized and provides results of high specificity and high sensitivity, since the minimum of these three specificities can be identified in this format. This technique can still ensure very high sensitivity while processing a wide range of nucleic acids. Also, if desired, target / probe complexes made by CPA can be used with other DNA analysis techniques such as PCR, other amplification techniques described above, and DNA-chip analysis.

Preferred CPA three-step method

1) sample ss nucleic acid isolation;

2) hybridization and restriction with cleavage probes to form probe / target complexes; And

3) target separation and detection. Target separation can be accomplished by known techniques, preferably in vitro format or size separation.

CPA using an in vitro format for target / probe complex isolation is shown in FIG. 3 and can be used for RNA or DNA. One or more sequence specificity CPs can be used for analysis. In FIG. 3A, the sample nucleic acid 64 to be tested is isolated and purified. In the example shown in FIG. 3, the sample is DNA 64. This sample may be RNA, usually ss.

In step B of FIG. 3, CP oligos 54, 56 are designed and generated based on the target sequence and the desired cleavage site or sites. CP-anchor 56 has a biochemical hook bonded thereto at any position. CP-reporter 54 has a reporter molecule conjugated to it in a position that aids in identifying the target / probe complex.

In step C, the sample nucleic acid believed to contain the target is denatured if ds.

In step D, the following components are mixed under conditions that allow hybridization: denatured sample nucleic acid; Cleaver probe pairs; And magnetic beads 72 coated with an immobilized substrate for binding to CP anchor / target complexes, such as anti-DIG 88. The label on the CP reporter 54 is indicated by (*). In vitro VU lines limit target 50 at approximately 313 nm.

Step E of FIG. 3 shows the process of the complex settling into the solid substrate 72. Once bound, the restricted target / probe complexes are easily separated from high molecular weight genomic DNA, ssDNA (denatured) or RNA targets. For example, magnetic beads 8 with target / probe complexes attached by anchor capture molecules 88 are extensively washed. Target 50 with two probes 54, 56 is PNAS 90.

In step F the signal from the CP-reporter 54 is amplified.

In a preferred embodiment, each assay comprises two cutter probes referred to as a cutter probe-anchor pair (CP-anchor pair, CP1) and two referred to as a cutter probe-reporter pair (CP-reporter pair, CP2). Use a reporter probe.

Cleavage probe-anchor pairs are characterized by being able to bind only a portion of the 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 with another base at the probe. The anchor capture molecule 88 is anti-DIG.

The cleavage probe-reporter pair 54 is characterized by being able to bind with the rest of the ssDNA target, wherein the CP-anchor pair cannot bind to this sequence.

CP pairs bind to the degenerate strand or one RNA target of the complementary ssDNA target or double stranded DNA or RNA target.

The specific elements shown are used as examples and it is to be understood that the invention is not limited to these specific examples. The probe may vary in length. In addition, as shown in FIGS. 1B and 1C, if additional levels of specificity are required for CPA analysis, more cleavage probes 58, 60, and 62 may be added, each probe being added to an additional level of specificity. Contribute.

CPA has multiple levels of specificity, including:

1.Combination of BUCP-anchor pairs (BUCP1) 56

2. Combination of BUCP-Reporter Pair (BUCP2) 54

3. capture of (target-limited and protected) target / probe complexes; DIG / anti-DIG magnetic bead interaction.

In addition, the fact that the BUCP-anchor probe 56 and the BUCP-reporter probe 54 are non-complementary and that the target sequence 50 itself is a nucleic acid fragment linked to these two probes provides a high specificity analysis with a low background.

Sensitivity of single-stranded DNA (denatured-ssDNA) or RNA target detection is determined by the use of enzymes such as S1 or Mung Bean nucleases, or other similar functions, to degrade ssDNA and ssRNA from the 3 'to 5' It can be improved by treating new double-stranded target / probe complexes and other ssDNA / genomic DNA with enzymes. Once the probe binds to the target, the target / probe complex becomes a protected nucleic acid structure (PNAS), which is resistant to degradation by these enzymes. Such nuclease treatment is optional, but the present invention contemplates its use if necessary to increase the sensitivity of the assay, depending on the assay being performed.

CPA using size separation

The gel assay format of this preferred embodiment differs from the in vitro format in step F of FIG. 3. Gel assay formats consist of the following levels of specificity.

1. In the binding of CP-1, no anchor molecule, DIG, etc. are required, but the probe 56 will protect and limit the target 50.

2. In the binding of CP-2, the reporter molecule is not needed, but the probe 54 will protect and limit the target 50.

3. In gel learning, the restricted target 50 will travel to an expected distance, Rf, using the size separation technique described above. If additional levels of specificity are needed, they can be introduced by fragmenting two CPs with three probes that hybridize in series to protect and limit the target. 1 illustrates this probe fragmentation 58, 60, 62.

Aspects of CPA with Triple Helix Formation-TFO (Figure 4)

In this embodiment, target sequence 50 is double stranded and probe 68 is ss. When the probe 68 hybridizes to the target sequence, the probe 68 forms a triple helix structure. Such probes are called triple helix forming oligonucleotides (TFOs) and can be used to cleave specific double stranded gene fragments. The probe can bind (noncovalently) with either strand of the ds-target. This embodiment can be used for cleavage of genes in vivo and in vitro.

Cleavage of specific double-stranded gene sequences (dsDNAs) is of increasing interest due to the emergence of gene therapy and biotechnology. In these methods non-enzymatic restriction of DNA is new and advantageous over the use of endonuclease restriction of DNA. Endonuclease restriction requires 4 to 8 base restriction / recognition sites by restriction endonucleases (RE) that cannot be placed where desired to cleave the sequence. Large electronegative molecules such as bleomycin, EDTA, and others provide a method of generating weakly-specific cleavers by attaching to oligonucleotides that attach to nucleic acids and hybridize with DNA (ssDNA). The approach leads to separation of the DNA strands.

Problems due to the electronegative form of DNA cleavage are complicated by the need to denature, cleave and regenerate dsDNA to cleave gene fragments and the uncontrollable reactivity of highly electronegative cleavage molecules. These molecules can react with non-specific DNA sites themselves before probe hybridization with specific sites.

For example, the novel methods and compositions of the present invention for in vivo and in vitro gene cleavage are achieved by the use of TFO, CP forms. In vitro, the TFO specific for the unique dsDNA fragment is added in an amount several times greater for hybridization with DNA. TFOs exist in the major group of the helix, where they are bound by Hoogstein bonds (weak hydrogen bonds). In one aspect of the invention, the TFO is an oligonucleotide having two terminal thymidine bases substituted with BU called BU-TFO.

Treatment of BU-TFO protected target / probe complexes with UV at approximately 313 nm or any wavelength that excites bromine atoms to produce free radicals functions to cleave dsDNA at both ends of the TFO for short dsDNA cleavage.

In the second embodiment described in FIG. 4B, larger dsDNA fragments can be cleaved using two BU-TFOs. When flanking regions of large regions of DNA hybridize to CP or TFO, the entire DNA region is protected from nucleic acid degradation.

In vivo application of the BU-TFO cleavage probe requires transfection of cells with BU-TFO consisting of modified bases that are not recognized by cell membrane bound DNA nucleases. In vivo, the binding of two BU-TFO forms with the chromosomal site is followed by a lower amount of x-rays that appear to be involved in more invasive forms of energy, such as BU sensitizing effects.

These restriction methods and cleavage probe compositions are used for gene disruption in vivo and in vitro, and the binding of the TFO form is similar to that in the gene cleavage process. This aspect is shown in FIG. 5. Many closely spaced reactive groups, such as BU, are present throughout the TFO, resulting in deletion of unwanted nucleic acid sequences and normal cell destruction after ultraviolet irradiation.

Another aspect of the invention is the targeting of cellular mRNA or other RNA using BU substitution molecules. This results in the binding of the specific RNA molecule with the BU-cleavage probe for site selective cleavage. The production of bromine and other free radicals destroys mRNA transcriptional functions and cells will continue to survive without the specific mRNA population destroyed. BU-CP has an advantage over antisense DNA, where RNA is removed using the BU-CP probe, while with antisense DNA, the RNA is still present and only binds to the DNA. Binding conditions may change and RNA may be present for translation or other functions.

Point mutation detection using TFO

Another aspect of the compositions and methods of cleavage probes is the detection of documented point mutations. Examples of the use of CPs for point mutation detection are shown in Table 1. One aspect of this method involves denaturation and restriction of sample DNA that is believed to contain point mutations to obtain a target sequence. This restriction is achieved by separating small fragments of DNA that are known to contain point mutation sites using known restriction methods or more preferably two CPs. The third CP is used to hybridize to the target sequence. As can be seen in FIG. 1, if a point mutation is present, irradiation of the third CP causes cleavage in the target sequence and the CP complex. If no point mutation is present, cleavage does not occur. The sample can be run visually on the gel and see visually whether the complex is cleaved. One way to identify the presence or absence of point mutations is to label CPs that bind to the target sequence.

Table 1: Detection of single base mutations using a documented / characterized BU cleavage probe (BUCP) (the location of the BU is at the cleavage point).

+ * →-** BUCP action

DNA G-A Watson-Chain Cut

(Watson Sprint) T-A Watsons Cut Saw

C-A Watson-cut cutting

A-G Watson Not Printed

A-T Watson Uncut

A-C Watson Not Printed

C-T Watson Not Printed

C-G Watson Not Printed

T-C Watson Not Printed

T-G Watson Not Printed

G-T Watson Not Printed

G-C Watson Not Printed

DNA A-T Watson Chain Cleavage

(Creek Cut) C-T Watson Cut Cut

G-T Watson-cut cutting

A-G Watson Not Printed

A-C Watson Not Printed

C-G Watson Not Printed

C-A Watson Not Printed

G-A Watson Not Printed

G-C Watson Not Printed

T-A Watson Not Printed

T-C Watson Not Printed

T-G Watson Not Printed

RNA U-A RNA strand cutting

C-A RNA strand cutting

G-A RNA strand cutting

A-U RNA strand not cleaved

A-C RNA strand not cleaved

A-G RNA strand not cleaved

C-G RNA strand not cleaved

C-U RNA strand not cleaved

G-C RNA backbone not cleaved

G-U RNA strand not cleaved

U-C RNA strand not cleaved

U-G RNA strand not cleaved

+ * Wild type-** mutant

Restriction Fragment Target Analysis (RFTA)

Restriction fragment target analysis (RFTA) of the present invention directly detects both RNA or DNA targets. The target may be single stranded or double stranded. The RFTA method uses at least one primary probe and at least one secondary probe. All probes are Japanese. The primary probe has at least two zones. At least one zone of the primary probe is complementary to the target and at least one zone of the primary probe is complementary to the secondary probe. The secondary probe is not complementary to the target, but only complementary to the primary probe zone, which is designed to be complementary to the secondary probe. Additional zones can be added to the primary probe. The additional zone may be complementary to the target or secondary probe.

Once the probe / target complex is formed, it can be separated by linkage with a solid support in size separation or biochemical hook and test tube formats. Two separation methods are described above.

Techniques such as TPA and RFTA can analyze nucleic acids from milligrams to nanograms, picograms, and even less (pentamol) targets. Embodiments of the invention identify low copy number nucleic acids or other targets in very few samples. The processing power of such large amounts of nucleic acid is fundamentally starting from existing microgram analysis techniques, which significantly improves DNA diagnostics.

Apply DNA RFTA

RFTA can be used for a variety of DNA types, from simple detection of DNA sequences for identification of infectious agents to more complex applications where fine sequence analysis is desired, such as genotyping, viral strain, or genetic disease testing for histocompatibility antigens. It can be used in diagnostics. Small DNA sequence differences can also be determined by using sequence specificity probes or by selecting a restriction endonuclease used in the initial cleavage step and a combination of both, the latter method alters the size of the hybridized restriction fragment. . RFTA can be used to modify the variable number of serial iterations (VNTR), and the restriction fragment length polymorphism (RFLP) process to see differences in small serial iteration (STR) markers between samples. The RFLP process currently uses membranes for hybridization to perform forensic and paternity tests and to evaluate the success of bone marrow transplantation. The following detailed description relates to the application of methods and compositions for DNA (RNA is also contemplated), and modifications for RNA application are given below.

Examples of DNA RFTA embodiments are shown in FIGS. 6 and 7. 6 shows steps A-G; Step A, isolation and purification of sample DNA; Step B, release of the target sequence 50 from the sample DNA, step C, modification of the double stranded nucleic acid sample sequence; Step D, hybridization of the labeled primary probe 74 with the target sequence 50; Step E, hybridization with secondary probe 76; Step F, hybridization with another secondary probe 78; And step G, separation by size separation. The result is preferably applied to the assay gel using gel electrophoresis and the Rf of the labeled band is calculated.

Specificity levels described in the RFTA were determined when the target / probe complexes were separated by gel electrophoresis; 1) 5 'target cleavage mechanism, 2) 3' target cleavage mechanism (additional level of specificity different from the first target cleavage mechanism), 3) primary probe using label 74, 4) secondary probe 76, 5) secondary probe 78, and 6) predictable travel (Rf of the composite). Additional probes can be fragmented and added to further increase the level of specificity in a particular sequence.

Steps and Considerations of DNA RFTA Explained in FIG. 6:

Step A: sample DNA source; Isolation and Purification of DNA

Restriction fragment target analysis (RFTA) of the present invention detects target DNA sequences present in a sample. The method utilizes the purification of a DNA sample from a source that is believed to contain a target. Raw materials include, but are not limited to, cells, tissues, organelles, serum, water, and other fluids. The host or sample DNA may be in the form of high molecular weight genomic DNA. Purification may use amplified DNA that is one of a variety of methods known to those skilled in the art or intended for this purpose.

Step B: Cleavage of Target Nucleic Acid of DNA RFTA

Purified sample DNA is cut to cleave the target nucleic acid sequence present in the sample. Cleavage of the target nucleic acid is the first level of specificity in the RFTA method.

Cleavage can be accomplished in a number of different ways. One method involves cleavage with one or more sequence-specific restriction endonucleases, whose restriction sites are known to contact the target nucleic acid. Additional levels of specificity can be added using a second endonuclease that cleaves the target at a different site than the first restriction endonuclease.

Other types of nucleases can be used, such as non-specific endonucleases. For example, the nucleic acid can be cleaved using ribozymes, methods using ribonucleases, or other methods of cleaving nucleic acids. The choice of one or more endonucleases or other types of nucleases depends on the target sequence and the individual test design performed.

Other methods may be used instead of nuclease cleavage of DNA. The novel approach of the present invention is the above-described cleavage probe (CP) for selectively cleaving DNA and RNA at any site. Cleavage is not limited to sites recognized by endonucleases that cleave only specific sequences.

Step C: Modification in DNA RFTA

When the sample nucleic acid undergoes cleavage of the target nucleic acid, the whole sample DNA is denatured into individual complementary strands using known procedures such as heat, ionic or salt conditions, hydroxides, or pH conditions.

Step D: Hybridization of Primary Probe (s) in DNA RFTA

As can be seen in FIG. 6, after denaturation, the sample DNA is bound to the probe under conditions that allow hybridization. In a preferred embodiment, three probes, one primary probe and two secondary probes are used in the RFTA method. The primary probe is specially designed to bind only to the target so that only if the target is present in the sample the primary probe will bind. The present invention contemplates that the target and probe size may vary.

a) Primary probe design: In designing the primary probe (s) 74, consideration should be given to one or two sample strands of sample strands (known as Watson or Creek chains, abbreviated W or C in the figures). Can be used as the target backbone 50 in the present invention. The use of two strands results in double amplification of the signal. The latter embodiment requires the use of a strand specificity primary probe 74 specific for the particular target nucleic acid strand 50.

Primary probe 74 may have one or more zones. In a preferred embodiment, the primary probe has three zones. The first zone 80 may bind to the secondary probe 76 which may not be labeled by sequence homology or other means. The first zone 80 of the primary probe 74 cannot bind to the target nucleic acid sequence 50 by sequence homology or other means and will bind to the initial secondary probe 74. In general, the second zone 82 proximate to the middle of the primary probe 74 may bind to the target sequence 50 by sequence homology or other means. In this embodiment, the third zone 84 of the primary probe 74 may bind to another secondary probe 78 which may or may not be labeled by sequence homology or other means. The secondary probe cannot bind to the target sequence.

In certain formats, either or both of the first 80 and third 84 regions of the primary probe 74 have an attached reporter molecule, such as a label. The reporter can be lengthened or shortened to improve signal amplification.

For assays in which more than one sequence identification is desired, multiple primary probes are used in the (multiple) reaction mixture simultaneously with different sequences that bind to different target regions. These probes are not complementary to each other. The number of probes used is limited by capture-detection, separation and detection methods known to those skilled in the art.

Primary probes are the third level of specificity in the RFTA method.

b) Probe Labeling: The probe can be labeled with a detection molecule using radioactivity, fluorescence or other detection methods known to those skilled in the art. In one aspect, the primary probe is labeled along the entire length, regardless of how many different zones comprise the primary probe.

c) Hybridization of the primary probe (s): The primary probe 74 is placed in molar excess of sample DNA to ensure spontaneous reaction. Single-stranded sample DNA and the probe hybridize. Hybridization can be accomplished by known processes such as slow cooling, ion control, or pH neutralization. This is the third level of specificity of the assay.

Step E: Hybridization of the Primary Secondary Probe (s) in DNA RFTA

As can be seen in step E of FIG. 6, the first secondary probe is hybridized to the primary probe. Secondary probe 76 is a reporter probe used for a label to record a target. Secondary probes usually consist of oligonucleotide (ssDNA) sequences of, but not limited to, 10-25 mer probes. Secondary probes in gel format can be extensively labeled. Each secondary probe 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 probe 76 and the primary probe can be added simultaneously to hybridize with the sample DNA modified in molar excess. Alternatively, the secondary probe can be added after the primary probe has hybridized. Hybridization can be carried out by known procedures. This is the fourth level of specificity of the assay.

Step F: Hybridization of Another Secondary Probe in DNA RFTA

As can be seen in FIG. 6, if another secondary probe 78 is used, it is labeled and hybridized to the primary probe. The type of label can be determined by the selected detection method. All primary and secondary probes 76,78 can hybridize to cumulative levels of specificity at the same time. The addition of a second secondary probe is the fifth level of specificity.

Step G: Isolation and Detection in DNA RFTA

When the target 50 is present in the sample, the attached secondary probe or probes and the complex of the target sequence and the primary probe are double stranded structures (shown in step F of FIG. 6). The complex is separated from the remaining sample DNA and detected. The separation of the two aspects is (a) the size separation shown in FIG. 6 and (b) the capture probe shown in FIG. After gel electrophoresis, detection of the labeled band at the expected gel position (Rf) indicates the presence of a nucleic acid target with a sixth level of specificity.

In some cases, the target may be present in numbers lower than the primary and secondary probe concentrations. In this case, the primary and secondary probes can hybridize together without binding to the target and provide false-positive on the gel. To detect probes that do not hybridize with the target, a cleavage probe, such as a BU base cleaver with sequence specificity for the second portion of the primary probe (region complementary to the target), may be used to hybridize primary and secondary probes. Post-oxidation may be hybridized to the target / probe complex. The hybridized sample complex is then treated with UV (approximately 313 nm) to cut the probe / target complex into two smaller complexes. The two small complexes will be easily distinguished by calculating the Rf on the selection gel by size from the untargeted probe complex.

If the binding of the primary probe is nonspecific, the labeled band will appear at a different size position than the calculated Rf of the target / probe complex (which usually provides a small band) or the DNA band observed on the gel or membrane will not have a label. . Thus, some nonspecific combination of primary probes will not confuse diagnostic interpretation results in these RFTA tests.

The 3 'end of the target / probe complex removes the 3' end to cap free deoxyribose hydroxyl groups (i.e., hydroxyls that do not bind another nucleotide) at the 3 'end and design the target and secondary probe sequences So that gaps do not exist between them, thereby making them strong in nucleases. Likewise, capping at the 5 'end is achieved by removing free 5' hydroxyl or phosphate. Resistance to nucleases, although unnecessary in RFTA, can contribute to increasing the sensitivity of the assay for specific applications.

Another embodiment of the RFTA invention in which the target / probe complex is separated by size separation is shown in FIG. 7. Note that the probe / target complex binds to one or both of the nucleic acid strands. The present invention is contemplating signal amplification for detecting target sequences using such probe sets.

8-I shows an embodiment of an RFTA in which three different probes are used. The longer probe, the primary probe 74, binds with at least a portion of the target sequence 50, leaving a single chain region that is not bound at both ends 74. This is the end of the primary probe where the secondary probes 76 and 78 combine to form a complex separated by gel electrophoresis. If desired, there are ends that are not bound to the complex that can be processed by the exonuclease. One or more probes may be labeled. Watson or creek backbones can be used as target sequences in the use of probes with corresponding binding capacities. This structure provides 5 levels of specificity.

8-II illustrate another embodiment of binding the target sequence 50 internally to the hairpin using a probe 74 forming a hairpin loop at both ends. This probe / target complex is resistant to exonuclease cleavage. After formation of such probe / target complexes, exonuclease can be added to the sample, and all unbound targets and probes, and other nucleic acids, are cleaved away. The hairpin probe can be labeled by the method described above. This structure provides four levels of specificity.

8-III illustrate a preferred embodiment using two primary probes 74 and 86, each of which can form one hairpin loop on one end and the other end with a portion of the target sequence 50. Can be combined. In a preferred embodiment, the probes 74 and 86 are designed such that there is no significant gap between the ends of the two probes 74 and 86 allowing nuclease cleavage. The hairpin loops can be of any size and form any shape with a hairpin effect, a structure that binds to the internal sequence of the same strand at the same end. Such structures include hairpins, cloverleaf, side chain structures, and other structures known to those skilled in the art. The use of two different probes, each capable of binding to different portions of the target sequence, each forming a hairpin loop, creates a target / probe complex that is resistant to exonuclease cleavage. Thus, unbound or incompletely coupled probes 74 and 86 or target 50 are cleaved by exonuclease treatment.

In the embodiment of Figs. 8-III, the two probes 74,86 do not bind to each other and when the target 50 binds to the two probes 74,86, the background signal can be reduced to a significant extent and only observed in the gel. . In addition, unwanted complexes do not migrate to the expected Rf, which can be distinguished from the desired target. Thus, complexes without the desired target do not produce bands on the gel with the expected Rf.

Capture probe / tube format and detection

Another method contemplated in the present invention is to separate probe / target complexes using tube analysis and capture probes 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 (which may vary) using a capture probe for isolation of the target / probe complex from the sample nucleic acid. The sample believed to have the target sequence is hybridized with the three probes. Two of these probes are modified in the probes described in the RFTA method using size separation to separate probe / target complexes.

Probe structure

1) In one aspect of the in vitro format, the primary probe can have approximately 55 nucleotide bases, wherein approximately 15 nucleotide base regions are complementary to the target sequence. This zone is the center of the primary probe, with another zone of approximately 15 nucleotide bases on one end that binds the secondary probe and a third zone of approximately 25 nucleotide bases on the other end that binds another secondary probe or Can be placed nearby.

2) The first secondary probe is at least 15 nucleotide bases and is complementary to the first region of the primary probe but not complementary to the target sequence.

3) The second secondary probe is complementary to the end of the primary probe that is at least 25 nucleotide bases and does not bind to the first secondary probe but is not complementary to the target sequence.

In the separation of probe / target complexes by gel application described above, the secondary probe need not be labeled; Only the primary probe needs to be labeled. However, in these test tube or tube-based applications, only one secondary probe is labeled with any label known in the art.

The in vitro format of RFTA uses one secondary probe as a capture probe (conjugated with DIG or other biochemical `` hooks '' known in the art) to attach the target / primary probe complex to a solid support.

7 shows a preferred embodiment of this method: isolation of sample DNA, and restriction nuclease isolation of target sequences in the long sample DNA backbone. The target sequence 1 is denatured and hybridized with the primary probe, probe 2. The secondary probe 4, the capture probe, has a capture molecule attached thereto. In FIG. 7, the capture molecule is shown as digoxigenin (DIG) 3. However, it can be any other molecule with a similar function. Magnetic beads 5 are added to the test tube with antibodies to DIG to capture the target / probe complex. Step G of FIG. 7 preferably shows an embodiment in which a labeled third probe, reporter probe 6 is added to the target / probe complex. By combining these labeled probes, the entire structure is easily detected (visualized) in the in vitro container. The capture and reporter probes (both secondary probes) can be added in any order.

Target and probe size may vary, and reporter probes may be lengthened or shortened to produce an optimal signal. Secondary reporter probes are the only ones labeled in vitro analysis, which can be labeled using any method known to those skilled in the art; However, another label may be used if necessary. In in vitro applications, reporter probe signals are important for detection. For example, in tube format, the reporter signal should only be attached to the solid substrate when the target is present to bridge the gap between capture and the reporter probe.

9 shows tube application and modification in a target probe complex (PDTP-Partial Duplex Target / Probe Complex).

Tube-based assays can be used as preprocessing pools for DNA chips, PCR, and other nucleic acid or signal amplification techniques, for example to enrich all targets in milligrams of nucleic acid to mycogram amounts. Signal amplification can be any method known in the art.

The presence of the target / primary probe / capture probe / reporter probe complex directly depends on the presence of the target sequence for attaching all probes together. The absence of the target sequence should basically ensure that no nonspecific signal (background) is present.

Many other aspects of target / probe (PDTP) constructs used in the RFTA in vitro assay format are shown in FIG. 9. The use of either or both of the nucleic acid, Watson or creek strand is contemplated in these diagrams. The hairpin structure is shown for example only in FIG. 9, which is not intended to limit the type of structure formed.

9-I shows the target sequence 1 bound to the primary probe 2 with the capture molecule 3 attached directly to the capture probe 4. Labeled reporter probes 5 are added for structure detection. This embodiment has five levels of specificity and is not resistant to exonucleases unless the ends are capped by the addition of hinge / loop regions or other methods.

9-II illustrate an embodiment in which the secondary probe forms a hairpin or other similar structure. The target sequence 1 binds to the primary probe 2. The reporter probe 5 is labeled using methods known to those skilled in the art. The capture probe 4 with attached capture molecule 3 is also bound to the target / probe structure. This embodiment has five levels of specificity and is resistant to exonucleases.

9-III illustrate the use of two probes to form a hairpin loop, capture primary probe 2 and reporter probe 4. Each probe portion is bound to the target sequence (1). The primary probe 2 has a capture molecule 3 attached thereto to provide capture of the target / probe complex. This embodiment has four levels of specificity and is resistant to exonuclease cleavage.

9-IV illustrate the use of the reporter probe 4 to form a hairpin loop. The target sequence 1 binds to the primary probe 2. The primary probe 2 is coupled to the reporter probe 4 to form a hairpin structure. The primary probe 2 is also coupled to the capture probe 5 with the capture molecule 3 attached. This embodiment has five levels of specificity and is only partially resistant to exonuclease cleavage unless the ends are capped.

This aspect of RFTA can allow for a signal to be generated without a target sequence. In this case, care must be taken not to hook the reporter and the capture area. This can be accomplished in a number of ways:

i) hybridization step addition, after primary and secondary hybridization. At this point, the cleavage probe homologous to a portion of the target region and complementary to the primary probe hybridizes to the probe complex; This hybridization following UV treatment (approximately 313 nm) will separate the capture and reporter probes (in the absence of the target area).

ii) The primary probe can be fragmented into two probes, each binding to a different region of the target.

iii) other methods that do not hook the reporter and capture region using the nuclease resistance of the target stabilized target / probe complex.

9-V illustrate the use of a primary probe 2 bound to a target sequence 1. The reporter probe 5 and the capture probe 4 bind to the primary probe 2 together with the capture molecule 3. This embodiment has five levels of specificity and can be cleaved by exonucleases unless capping is used.

Additional levels of specificity can be achieved by adding multiple labels to the probe or by making one or two reporter probes. Table 2 features a plurality of probes in tube-based analysis.

Additional probes can be fragmented and added to specific sequences to further increase the level of specificity. For example, another embodiment of the target / probe construct includes the use of hairpin loops to reduce the number of probes from three to two.

Strengthening a sequence to nucleases can be used to reduce background or increase sensitivity at specific sequence locations, although unnecessary. Probes are designed so that no gap is formed between the probe and the target sequence to create a protected target sequence, or protected target nucleic acid sequence (PNAS) resistant to nucleases such as Exo III. In addition, the 3 ′ probe ends need to be capped, or dehydroxylated or modified by some methods known to those skilled in the art to be protected from nucleases. Other known modification means include, but are not limited to, the presence of a DIG, biotin, avidin, or hydroxylase reaction. Such a modification can be used with one of the above structures.

The use of elongated hairpin reporter probes allows the addition of more reporter molecules and enhances the visualization of the target.

RNA detection variant

For the detection of RNA target sequences, in addition to RNA sequence modifications, methods and compositions similar to the DNA methods and compositions described above can be used.

For DNA samples, the initial step is the purification of sample RNA by various methods known to those skilled in the art. In its simplest form, detection and characterization of specific RNA sequences is accomplished using hybridization of specific labeled nucleic acid probes followed by size separation and detection of specific targets described above.

In general, target cleavage from sample RNA is unnecessary because many RNA target sequences are found as discrete RNA molecules of different size, such as tRNA or mRNA. However, if desired, the target RNA can be cleaved in the present invention. For example, RNA molecules can be cleaved to the desired size to separate target sequences by binding complementary probes, DNA or RNA of the correct size and removing single stranded regions using single stranded nucleases. Other known enzyme and RNA manipulation methods can be used to size-sort RNA molecules such as ribozymes and CP cleavage probes.

Detection of specific RNA target sequences can be performed by gene expression analysis, detection of RNA virus (HCV, hepatitis C virus), or activity vs. activity. Useful in applications such as identifying latent virus (ie CMV, cytomegalovirus) infections. The invention can also be designed to be quantified for these applications.

For RNA application, in order to increase the pluripotency of RFTA, a reverse transcription step can be performed initially to produce single- or double-stranded cDNAs. This makes it possible to use restriction fragment analysis coupled with RFTA in substantially the same way as described for DNA applications. This type of analysis is applicable to the detection of tolerant or immune evasive similar species that may be generated due to RNA virus genotyping (HCV) and high mutation rates in some pathogens (eg HIV and HCV). Additional levels of sequence specificity can be added to RFTA by synthesizing cDNAs using sequence specific primers, such as random hexamers or oligo-dT primers, instead of relatively nonspecific primers, thereby allowing a defined size of RFTA to be analyzed. Generate a runoff reverse transcript. Two levels of these specificities, sequence specificity cDNA synthesis coupled with sequence specificity RFTA, reduce the likelihood of false-positive signals, increasing overall test specificity.

RFTA Direct Analysis of RNA Targets:

Most RNA targets such as mRNA, viral RNA, tRNA and others can be detected directly by RFTA in both gel and in vitro formats. Thus, there is no need to perform a reverse transcriptase reaction that converts RNA to cDNA for later detection.

Preferred embodiments of the present RNA RFTA invention are shown in FIG. 11 (mRNA RFTA gel analysis). Size separation can be performed by in vitro methods, using capture molecules or other suitable molecules on the probe. In addition, the hairpin structure shown in the case of DNA RFTA can be used where appropriate.

Step I: Isolate mRNA.

Step II: Design the primary probe by selecting target sites on mRNA (polypyrimidine).

Step III: Hybridization of Primary Probe to mRNA Target.

Step IV: Treatment with Exoribonuclease (S1 and Ming Bean Nuclease).

Step V: Generation of Target Probe Complex (PDTP Complex).

Step VI: Secondary reporter probe poly dT (biotinylated) is added to the poly A 3 ′ end of the triple helix for hybridization to initiate signal amplification.

Step VII: Add the avidin-enzyme complex to the complex with conjugated biotin molecules.

Step VIII: Electrophoresis with predictable Rf.

Step IX: Gel Placement in Pigment Substrate Solution

This assay provides 4 levels of specificity.

Embodiments of mRNA RFTA Gel basic assay using BU cleavage probe include the following steps:

Phase I: mRNA Isolation

Step II: Hybridization with BU Cleaver Probe

Step III: UV light irradiation to complete the restriction

Phase IV: Hybridization with Primary Probe

Step V: Hybridization with Secondary Reporter Probes

Step VI: Running the complex on gel with predictable Rf.

It has 4 levels of specificity.

An example of this method is shown in FIG. Total mRNA isolated (some of which contain the target sequence) is present in the sample. The probe 2 containing the BU cleavage site is bound to the target sequence 1. The target is cut using UV. A labeled probe 3 is added that binds to the single chain region of the target sequence.

In a further embodiment, a third probe is added that binds to the labeled probe 3. The complete double-stranded structure of the probe / target (PDTP) is run on a specific Rf on the gel.

RNA cleavage by hybridization of the target with the base cleavage probe 2, denoted herein as the primary probe fragment, is not necessary in all cases as described above.

If a base cleavage probe is not used, the target hybridizes with the primary probe (3) having two regions that hybridize to the RNA target and a third region with a 5 'end or 3' end that does not hybridize to the target. Can be. The total length of this probe is labeled as described above. One variable in the RNA embodiment is to enhance specificity by adding a second primary probe, which may or may not be fully labeled and acts as a base cleavage probe (2) that will bind in series close to the first primary probe.

The site of the nucleic acid probe to hybridize to the RNA target may be a fragment. As a result, for each additional probe fragment produced, a different level of specificity is added. The fragmented probe may be an oligonucleotide that is readily substituted with a base cleavage probe molecule, all described in the RFTA format described herein.

Hybridize with an unlabeled secondary probe that binds to the third site (4) of the primary probe.

S1 nuclease treatment or techniques known to those skilled in the art are optional steps in the presence of base cleavage probes and necessary steps in the absence of cleavage probes. Removal of non-hybridized mRNA will yield predictable R _f values for the expected target / probe complexes. If the ssRNA tail is not removed, the R _f absolute value of the tail / target / probe complex will be slightly differently modified but is a predictable R _f value. The size of the final complex is not critical. The ability to detect a target depends on the predictability and reproducibility of the R _f shift of the complex.

Other possible embodiments encompassed by the present invention for cleaving non-hybridized ssRNA tails include binding EDTA molecules or bleomycin to the ends of the probes adjacent to the ssRNA tails. Hybridization of RNA with probes having other similar molecules known to those of skill in the art will result in cleavage of the target RNA and removal of the ssRNA tail (s).

Another approach to cleaving non-hybridized ssRNA tails is to approach the probe to the mRNA tail with one or more thymine bases. Substituting these with BU and then exposing the hybridized complex to UV, preferably long wavelength, approximately 313 nm will separate the mRNA tail non-enzymatically. Other aspects of the CP described above may also be used.

When the target / probe complex learning in polyacrylamide or agarose gel, a double-stranded target / probe complex is visualized and R _f is calculated by comparing the R _f of the nucleic acid size standard. Obtaining the expected R _f value for the target / probe complex confirms the presence of the desired RNA target in the sample (with four specificities).

RFTA / RNA Gel Assay: Specificity Step (4)

1. Combination of Primary Probes

2. Exoribonuclease treatment that increases sensitivity / specificity by reducing background signals (non-specific RNA is destroyed)

3. Combination of Secondary Probes

4. Movement of the target / probe (R _f , delay coefficient) to the fixed point of the gel

Omitting excoribonuclease treatment changes the specificity step to three steps. However, substitution of the base cleavage probe for exoribonuclease increases the specificity of a total of five levels by adding two levels of specificity.

RFTA Direct Analysis of RNA Targets: in Vitro Format

Target complexes can also be detected by a gel format which involves attaching capture molecules to the probe.

Advantages of the Invention for DNA and RNA Detection

Advantages of DNA RFTA:

Multilevel Specificity

The capture and reporter probes do not bind to themselves but only to conventional targets.

PCR can be run on a DNA probe.

DNA-chip technology can also detect target / probe complexes.

(w) or (c) strands can be detected independently or together.

Benefits of RNA RFTA:

Multilevel Specificity

The capture or reporter probes do not bind themselves (but directly to each other), but only to conventional targets.

5 'poly A sites of approximately 200 to 250 mer can be used for signal amplification (using poly dT labeled probes).

RNA can be analyzed directly by performing a reverse transcription (RT) step.

The present invention, including RFTA aspects of both DNA and RNA applications, has a number of advantages over conventional DNA or RNA blotting procedures that employ membrane hybridization following migration of target nucleic acids following electrophoretic separation. RFTA is considerably faster and more convenient to implement than membrane hybridization, and requires less technology and specific equipment such as electro- or vacuum-transfer systems, UV crosslinkers or vacuum ovens, and hybridization ovens and baths. RFTA is also more substantial than standard RFLP analysis in that it uses proportionally slower rates than using membrane hybridization, and much less probes are used to hybridize to smaller volumes prior to electrophoresis compared to hybridization to relatively large volumes. As it runs cheaper.

No additional specific electrophoretic system is required for RFTA, and if not better, it performs well in the laboratory or on the original gel with analysis on gel running or fixed or dried gels as if the available detection systems were analyzed on the membrane. In addition, RFTA can be more sensitive than membrane hybridization because it does not require nucleic acid transfer or membrane crosslinking, which can result in loss of certain signals due to damage or insufficient migration. Since RFTA does not use DNA amplification, high rates of non-specific signals are not often seen in PCR-based tests. Finally, RFTA provides electrophoresis which eliminates the need for multiple probes to run on duplicate samples all at once, or to run duplicate gels that are both expensive and time-consuming and / or to strip already hybridized membranes for subsequent re-probes. It has the advantage that it can be used with multiple probes in the same sample on the lane or gel.

A major problem in the above-described DNA analysis, particularly in the field of diagnostics, was the inability of all diagnostic techniques to process 1 mg of nucleic acid in one test. This is necessary in all DNA analysis methods to diagnose the presence of minority targets during the early stages of infection. High sensitivity is not available in the above technique. The method of the present invention can be used in all DNA analysis and diagnostic techniques, whether amplified (PCR, etc.) or not, and on chip.

An embodiment of mRNA RFTA in vitro assay is shown.

Step 1: Isolation of mRNA

Step 2: The target site on the mRNA (polypyrimidine) is selected and the primary probe is defined.

Step 3: Hybridize Primary Probe to mRNA Target

Stage 4: Hybridization with Secondary Capture Probe

Step 5: Treatment with Exoribonuclease (S1 and Mung Bean nuclease)

Step 6: Create Resulting PDTP Complex

Step 7: Addition of Secondary Reporter Biotinylated Amplification Probe

Step 8: Add Conjugate

Step 9: Binding PDTP to Solid Substrate

Step 10: bind PDTP to solid substrate

Step 11: Wash

Step 12: Add the pigment source substrate and observe the color development

It has four levels of specificity.

Alternative Aspects of mRNA RFTA, Tube-Base Format

Step 1: mRNA isolation

Step 2: Select mRNA Target and Set Primary Probe (BUCP)

Step 3: Hybridize BUCP and mRNA Target Sites (BUCP-1, Capture)

Step 4: Hybridize with the Primary Probe

Step 5: Capture PDTP Complex

Step 6: wash

Step 7: Hybridize with secondary reporter probe and determine presence of label

It has three levels of specificity.

The use of the methods and compositions of the present invention does not limit the size of the nucleic acid sample that can be analyzed in a single tube, but rather enhances DNA chip and PCR techniques with improved sensitivity and specificity due to the fact that there will be a large amount of possible non-specific contaminants. Will effect.

Many methods of known DNA analysis techniques are unable to test sufficient amounts of nucleic acids to enhance their sensitivity. The methods of the present invention pre-treat the genome or other large amounts of DNA (mg of non-specific nucleic acid to the target-rich ug present in the sample) to enable known DNA analysis techniques in the presence of selected target nucleic acid sequences. Specifically and accurately.

Targeted Protection Assay (TPA)

Another aspect of the invention for detecting nucleic acid target sequences is called Target Protection Assay (TPA). TPA is described in U.S. Patent Application on October 26, 1996, which is incorporated herein. Patent application 08 / 739,069, to the present invention U.S. Patent, and U.S. application filed February 24, 1998. Provisional Patent Application 60 / 075,812 and 60 / 076,872, filed March 5, 1998.

TPA technology is a method for direct analysis of specific DNA and RNA targets and is sensitive enough to detect single DNA (gene) replication. The TPA method also detects both ds and ssDNA.

The methods and compositions of the present invention have the ability to handle a wide range of RNAs that impart the sensitivity needed for the development of applications that have been described previously and could not be achieved with prior art. TPA treatment enables diagnostic techniques during early infection. The importance of detecting RNA targets during the early stages of infection is due to the fact that both replicating RNA viruses, tumor development and infectious disease inventions require protein synthesis in the host, but more important is the generation of specific mRNA species.

One aspect encompassed by the present invention includes indirect single gene duplication detection. For each activated gene, 10/1000 of the mRNA molecule is generated. When a single gene is activated, 20,000 specific mRNA targets will be present in cells and tissues and can be identified.

Preferred embodiments of the present invention include the following steps. First, a sequence specific target site is selected on RNA. Second, RNA samples that are presumed to have the target sequence are separated by methods known to those skilled in the art to obtain denatured linear RNA. Sample RNA is purified using DNA amplified from or available for such purposes. Cleavage of the target from the sample nucleic acid is unnecessary because of the fact that the mRNA is already of the expected size.

In a third step, certain single-stranded RNA targets are protected by hybridizing to double-stranded DNA structures, which form partial triple helix molecules called protected target nucleic acid structures (PNAS). An aspect of triple helix structure formation is shown in FIG. 12. In this embodiment, the reporter probe is DNA, the double-stranded DNA capture molecules vary in length, and the biochemical hook is such a molecule known to those skilled in the art for this purpose, for example DIG. Such DNA capture molecules may be hairpin structures that form ds molecules that act like the ds structure of FIG. 12.

TFO hairpin capture probe from mRNA TPA

The site should be identified in polypyrimidine-rich mRNA. This is necessary because it is a requirement for the pyrimidine-purine-pyrimidine motif for the formation of the triple helix structure. The ds DNA hairpin capture probe is characterized by having two sites:

The 3 'end is a site of various lengths rich in polypurine.

The 5 'end is a site of various lengths rich in polypyrimidine.

Both sites are connected in the middle by six base stretches forming a loop of hairpins.

After all, the hairpin capture probe is a DNA molecule that twists itself to form a hairpin. Also, the 3 'end may be conjugated with a biochemical hook, but not close to the 3' end. The target is hybridized to the dsDNA hairpin probe with RNA strand origin.

Additional aspects of the invention include adding a poly dT labeled probe to the captured mRNA molecule. This enhances the reporter signal. 13 illustrates this embodiment. It is included in the present invention that repeating sequences such as the poly A sites shown in the mRNA examples can be used for this new signal amplification.

mRNA TPA gel-based assays comprise the following steps:

Step 1: Isolation of mRNA

Step 2: Selection of Target Site and Determination of DNA Capture Probe

Step 3: Hybridization of mRNA Target with dsDNA Probe

Step 4: Treatment with Exonucleases to Destroy the Non-mRNA-Coupled dsDNA Probes to Improve Assay Specificity

Step 5: Attach PNAS to Solid Support

Step 6: wash

Step 7: Signal amplification by hybridizing PNAS with poly dT reporter probe (25 mer biotinylated molecules)

Step 8: wash

Step 9: Add an avidin-enzyme conjugate to the target solution that binds to the biotin molecule on the conjugated poly dT amplification probe

Step 10: Wash

Step 11: Isolate PNAS from Magnetic Beads While Preserving PNAS Structural Strength

Step 12: Run soluble PNAS on an electrophoretic gel to predict R _f

Step 13: Place the gel in the pigment substrate solution and incubate to develop

It has five levels of specificity.

This aspect is not intended to be limiting, and other equivalent or similar variables may be used. Target site size and probe length can vary from short or long, but depend on the type of format expected for the target and analysis. Hybridization is accompanied by differently defined probes, identically to the RFTA method described above. The probe used herein may be a hairpin or other structure that forms a double stranded structure. Hairpin structure is a preferred embodiment.

Formation of PNAS is the first level of specificity of the methods and compositions of the present invention. This level of specificity is necessary to function at sites rich in polypurine or polypyrimidine in DNA double helix protective molecules, so that low and some non-specific RNA species can bind to dsDNA protective molecules. One way to solve this problem is the addition of the recA protein, which allows the use of a variety of target protective molecules (dsDNA) depending on the four general bases rather than the two bases of the poly-rich site.

An example of a preferred embodiment of the above having a target sequence with an 18 nucleotide base long sequence is mRNA TPA gel analysis.

An embodiment of the mRNA TPA gel-based assay is shown in FIG. 14.

An embodiment of mRNA TPA in vitro or capture assay is shown in FIG. 15.

Step 1: Isolation of mRNA

Step 2: Selection of Target Site and Determination of Capture Probe

Step 3: Hybridize Target and Probe

Step 4: Exo III Treatment to Remove dsDNA Capture Probe Not Protected by Target

Step 5: Attach PNAS to Solid Support

Step 6: wash

Step 7: Hybridization with Reporter Probe (Poly dT, Biotinylated and 25 mer)

Step 8: wash

Step 9: Add Avidin-Enzyme Conjugate

Step 10: Wash

Step 11: Isolate PNAS from Magnetic Beads without PNAS Structure Degradation

Step 12: Gel Electrophoresis with Specific R _f

Step 13: Place the gel on the pigment substrate and visualize the band on the gel (R _f calculation)

This assay has six levels of specificity. The DNA probe used herein may be a hairpin or other structure that forms a double stranded structure. The hairpin structure is a preferred embodiment.

RNA / TPA PNAS (target / probe complex) of the TPA process can be isolated and detected in both in vitro and gel assay formats. After hybridization of the TFO forming the PNAS, the subsequent step is determined by the detection method.

RNA / TPA in vitro applications provide very high specificity because the assay is initiated by at least five levels of specificity. The analysis allows for the direct analysis of large amounts of RNA.

Exo III treatment is required to remove non-specific DNA hybridized by the capture probe.

Examples of mRNA TPA probe basic assays using DNA hairpin structures are described.

Step 1: mRNA isolation

Step 2: Selection of Target Site and Determination of Hairpin Capture Probe

Step 3: Hybridization of Target and Hairpin Probe

Step 4: Exonuclease III treatment to remove dsDNA hairpin capture probe that is not protected by the target

Step 5: Attach PNAS to Solid Support

Step 6: wash

Step 7: Hybridization with Reporter Probe (Poly dT, Biotinylated and 25 mer)

Step 8: wash

Step 9: Addition of avidin-enzyme conjugate

Step 10: Wash

Step 11: Add Pigment Substrate for Color Development

It has five levels of specificity.

Aspects of mRNA CPA Gel Format Analysis

Step 1: mRNA isolation

Step 2: Hybridize the two BU cleavage probes and target restriction

Step 3: Settling PNAS on Fixed Substrate

Step 4: Run the gel and determine the R _f by band signal visualization

This assay has three levels of specificity.

18 shows mRNA CPA in vitro format analysis.

Step 1: mRNA isolation

Step 2: Hybridize two BU cleavage probes and target restriction

Step 3: Settling PNAS on Fixed Substrate

Step 4: wash

Step 5: PNAS Detected by Signal Depth

Another embodiment that may be used with triple helix structure formation is a triple lock. Under certain conditions, exposure of dsDNA fragments to Exo III (exonuclease) results in 3 ′ DNA on the W and C chains. Can decompose in 5 'direction. Triple Helix Structure Formation The double helix structure of the DNA of the oligonucleotide (TFO) prevents degradation by Exo III in a short time.

In order to achieve stability of the triple helix structure, an embodiment of the triple helix structure lock is shown in FIG. FIG. 16 illustrates mRNA molecules forming a triple helix structure with dsDNA capture probes defined to produce major grooves in which mRNA can be located. This hairpin also eliminates possible sites of Exo III attack on the 3 'DNA double helix structure ends.

There is also a need to protect the only 3 'probe ends remaining against Exo III degradation. This invention in the Triplex Lock is characterized by having a hairpin DNA capture probe that is stranded rich in polypurine (3 'terminus) and polypyrimidine (5' terminus), which are twisted together and connected to the proximal end by a string of bases (7) And poly dT was used herein.

Extension of the 3 'end of the three capture probes by 12-15 bases of DNA to hybridize via conventional hydrogen bonds with the existing mRNA target results in a 3' probe end that resists Exo III degradation by DNA-RNA hybrid production. Create The presence of Exo III for smooth and 3 'exposed ends achieves the goal of protecting the 3' end of the probe (ssmRNA 3 'end produces an overhang) and Exo III is the 3' probe end (15 bases) Extra stretches of A, T, C and G) will not be able to simultaneously cleave hydrogen) with RNAs that send specific ends that resist 3 'to 5' degradation.

Advantages of RNA / TPA

The main advantage of using RNA / TPA is that all signal amplification techniques and DNA on chip and PCR achieve target detection as sensitive as single gene replication detection by the use of the methods and compositions of the present invention. It also provides a reduction, protection, enrichment of a mg amount of nucleic acid to a single ug of nucleic acid in a patient's specimen, containing all targets in the sample of origin. This is shown in the figure.

The viral load problem in HIV treatment has been described so far as unsuccessful. While current PCR measures undetectable levels of viral load, AZT + cocktail therapy removes all signs of HIV virus from an individual, inherent limitations in PCR, poor performance of RT-PCR, and RT (reverse transcription). Both enzymes and PCR treatments are used together to attempt to identify results that have little or no success because of the complexity created.

The methods and compositions of the present invention are used for diagnosing viral load, by allowing the inventors to follow mRNA levels during HIV treatment control. For example, protease inhibitor therapy suffers from PCR ability to determine when treatment should be stopped and when viral loads decrease. PCR has the inherent drawbacks affecting its sensitivity and specificity, and most importantly, the identification of low copy number targets (DNA) in excess genomic DNA and the lack of the ability to directly analyze RNA targets. The present invention solves this problem by identifying and analyzing the targets that are not abundant in excess DNA / RNA directly by single gene or RNA target replication. Eventually, parameters that TPA can deliver for analysis of viral loads will include monitoring the number of the following nucleic acid types:

1. dsRNA of HIV virus sensitive to single gene replication

2. Sensitive to dsDNA (Integrated) -Single Gene Replication of HIV Proviruses

3. Sensitive to 10,000-20,000 mRNA molecules thought to be mRNA-single gene replication specific for viral proteins

Currently, signal amplification through bioluminescence is sensitive to single gene DNA or RNA targets by indirect evaluation of mRNA analysis. As bioluminescence and other signal amplification techniques improve, mRNA targets can be assessed directly at the level of single messenger replication in large quantities 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 analysis. Lymph nodes containing a single cancer cell, by themselves, target at least 10,000 to 20,000 mRNA targets specific for tumor cell surface markers, receptor proteins, which can be detected by isolation of all RNA in all lymph nodes (mg amount) and a single analysis thereof. Will exist.

Another use of the invention is the direct identification of hepatitis C RNA and mRNA. This direct detection enables the early detection of infectious viruses and helps to ensure the safety of blood and plasma supplies worldwide.

Another use of the present invention is that a number of treatment modalities can be monitored by mRNA target analysis specific to the condition to be treated. Diagnosis of hormonal abnormalities (hypo- and hyper-states) can be monitored by similar mRNA analysis. Growth factor treatment can also be monitored by direct mRNA irradiation in the case of gene restriction issues. In recent years, with myriad human medical applications, there is an equal or stronger list of agricultural and veterinary applications.

Compositions of the present invention include compositions of ingredients necessary to practice the methods taught herein. For example, a composition comprising a primary probe at a nucleotide site complementary to a specific target sequence and at least one secondary probe sequence, and a labeled secondary probe. Included in the present invention are compositions or kits, ribozymes and buffers comprising primary and secondary probes selected according to nucleases. It is understood that each molecule, probe and component may also be provided separately or in combination.

The invention is illustrated by the appended examples, which should not be construed as limiting the scope thereof in any way. On the contrary, a resort may have a number of other aspects, modifications, and equivalents, and after reading the description herein, it is suggested to those skilled in the art without departing from the spirit of the invention and / or the scope of the claims appended hereto. Can be.

Example 1

General format of target detection assay using dsDNA target sequence with PNAS initiated by triple helix structure: DNA TPA

Isolation of DNA

The following protocol is an exemplary procedure for rapidly separating DNA from large volumes of blood: 150 ml of blood (hairpin, ACD or EDTA) collected from venipuncture tubes, together with 500 ml centrifuge tubes, isoton II (Coulter Diagnostics) Dilute to 150 ml. Add 30 ml of 10% Triton X-100 and mix vigorously for 3 seconds. Cell nuclei are pelleted for 5 minutes at full speed (12,000 x g). After removing the supernatant, the pellet was 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 2.5 mg / ml protease K), 55 Incubate at 95 ° C. for 15 minutes at 95 ° C. for 10 minutes (until protease K is inactivated) and then slowly cool to room temperature. 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 by addition of 0.2 volume of 10 M ammonium acetate and 2 volumes of ethanol. 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. Gentle sonication or cutting may be necessary to achieve complete dissolution of the pellets. Approximately 1 mg of total genomic DNA will be recovered from 150 ml of total blood (approximately 150 million aggregated cells). RNA preliminary techniques can also be applied.

Formation of PNAS Mediated by Triple Helix Structure Formation

50 μl 10 × TFO buffer (0.25 M Tris-acetate, pH 7.0, 0.5 M NaCl, 100 mM MgCl ₂ , 50 mM-mercaptoethanol, 0.10 mg / ml BSA, and 40 mM spermine-) in 0.5 ml of DNA sample in water. HCl) is added followed by 10 nanomoles of specific TFO. Incubate for 10 minutes at a temperature and time sufficient for stable PNAS formation, for example 37 ° C., before proceeding to the next step.

zymolysis

Add 500 units of each restriction enzyme (50 μl in most cases) and 4,000 units of Exo III (40 μl of 100,000 u / ml stock). The reaction mixture is incubated at 37 ° C. for an additional 50 minutes and then the enzyme is inactivated by biochemical or biophysical methods. The sample is now ready for the next step.

Capture system

To the digested DNA mixture, 10 nanomoles of DIG labeled capture probe and 0.5 ml 2.5x hybridization buffer (5.0 M NaCl, 0.5 M NaOAc, pH 4.5) are added. The mixture is incubated for a time sufficient to form a stable hybridization complex at an optimal hybridization temperature, for example 1 hour, then 100 μl of anti-DIG coated magnetic beads, washed and resuspended in hybridization buffer. do. After an additional hour of incubation, the beads are separated using a magnetic particle concentrator and washed eight times with 0.5 ml hybridization buffer. The sample is now ready for the final step of the DNA triple helix structure TPA process.

FITC labeled reporter probes are used and detection is performed using anisotropic fluorescence. Initial anisotropy of a 1.0 ml solution containing 10 nanomoles of reporter probe in hybridization buffer is measured and then added to the washed magnetic beads. The mixture is incubated for 1 hour at 50 ° C. under gentle locking, then all the contents (including the beads) are transferred to an Abbott TDM sample vial. The anisotropy is then remeasured against the initial value for analysis. Fractional binding is expressed as f _b = ( ^r obs- ^r in) / ( ^r b- ^r in), where f _b is fractional binding, ^r in is initial anisotropy, robs is anisotropy observed after hybridization, and r _b Is the total binding (measured by titrating light probes with excess binder).

Example 2 HIV

Human immunodeficiency virus type 1 (HIV-1) is one of two pathogens of AIDS. In recent years, serum assays that detect the presence of anti-HIV antibodies are used to screen blood and blood products. While generally evident, these tests show false-positive results due to cross-reactive antibodies or negative-negative responses when the infection is in the early stages of infection prior to the onset of the immune response for which it is measured. In the latter case alternative methods such as TPA are particularly useful and large amounts of sample DNA can be processed and tested in a single assay tube. Direct analysis of viruses using co-cultures as susceptible cell lines exists, but this method is more laborious and requires several days to complete. The following example will illustrate a large amount of blood extraction in the worst case: from individuals who have been recently infected with low levels of infected CD4 positive cells.

1. DNA is extracted from 150 ml total blood (140 million leukocytes) as described in Example 1. The purified DNA is resuspended in 0.5 ml water.

2. Add 50 μl 10 × TFO buffer and 10 nanomolar TFO:

HIV-1 TFO:

5'-TTT TCT TTT CCC CCC T-3 '

3. Incubate at 37 ° C. for 10 minutes.

4. Add 500 units of Sau 3A and 4,000 units of Exo III.

5. Incubate at 37 ° C. for 50 minutes and then at 60 ° C. for 20 minutes.

6. Add 0.5 ml 2.5x hybridization buffer and 10 nanomoles of DIG labeled capture probe.

HIV-1 Capture Probe:

5'-ACT GCC ATT TGT ACT GCT GT-DIG-3 '

7. Incubate at 50 ° C for 1 hour.

8. Add 100 μl of washed DIG coated magnetic beads.

9. Incubate at 50 ° C. for 1 hour by locking.

10. Place the tube in the magnetic concentrator to remove the liquid.

11. Wash 8 times with 0.5 ml hybridization buffer.

12. Measure the anisotropic fluorescence in advance and resuspend the beads in 1.0 ml of hybridization buffer containing 10 nanomoles of reporter probe.

HIV-1 Reporter Probe:

5'-GAA TAG TAG ACA TAA TAG TA-FITC-3 '

13. Incubate at 50 ° C. for 1 hour.

14. Re-measure anisotropy and analyze the fraction of bound probe (fb) as given above.

As a rule, after step 13, the beads were redefined with a magnetic particle concentrator and washed eight times with hybridization buffer, placed in a fluorometer for direct fluorescence measurements (-exe = 490 nm, -em = 520 nm) or the beads were fluorescent microscope Can be placed on the slide for viewing.

Example 3

Single-chain M 13 mp 18 Bacteriophage DNA digested with BUCP

Replication of filamentous bacteriophages occurs together in E. coli cilia containing host bacteria and infected cells are released into the supernatant without degradation while producing hundreds of viral particles per cell per generation. The titer of bacteriophage in culture of infected bacteria can reach 10 ¹² pfu per ml. The extracellular infection strand is the M13 (+) strand, a single stranded DNA molecule (approximately 7.2 kb in length).

The system to be used to describe the BU cleavage probe DNA restriction utilizes the ability of the BU cleavage probe to hybridize to specific sequences on circular M13mp18 bacteriophages. The following is a sequence of 2500 to 2525 bases.

F = fluorescent molecule

2. The second half of E. coli grown in nutrient broth will be centrifuged at 12,000 x g for 10 minutes, and the resulting supernatant is poured into the tube, dropping two drops of chloroform to sterilize the bacteriophage stock.

Various concentrates of M13 DNA are hybridized to the BU cleavage probe sequence that was previously presented by culturing a 10 times excess cleavage probe relative to the number of M13 molecules to be limited, thereby ensuring complete hybridization (probe M13 Tm of the interaction is calculated at 44 ° C. by base content experiments). The hybridization temperature should be around 37 ° C. in the hybridization buffer 1 M salt.

High purity washes are added at 37 ° C. at low salt concentrations to remove BUCP probes that bind to non-specific sites.

Circular DNA along with the attached probe is then irradiated in Petri dishes with a long wavelength UV (313 nm) at a content rate of 14.6 W _m ⁻² . However, prior to irradiation, Hoechst Dye # 33258 must be added to ensure high frequency cutting by the bromine free radicals of the target (opposite back sugar-phosphate backbone of M13 mp 18 bacteriophage).

Restriction of bacteriophage DNA is visualized by performing agarose (1%) gel electrophoresis showing cleavage induced in the probe backbone and M13 DNA backbone. The BU cleavage probe has at least one similar fluorescent source molecule on both sides of the BU molecule. M13mp 18 bacteriophage DNA will have different fluorescent dyes and other fluorescent source dyes that cause different wavelengths to fluoresce than the first, and gel analysis will identify Rf of circular (non-limiting target) and linear target (target limiting).

These fluorescent dyes are close to infrared in the spectrum, minimizing the experimental background. Analysis of the gel in the Molecular Dynamic Fluorescence Analyzer will provide a profile.

If the target was not limited, M13 leaves an intact circular fragment of DNA that migrates in the gel with specific R _f . Restriction of the target will move the gel Rf to another location demonstrating the production of cut and linear bacteriophages. The reaction conditions may be conditions controlled by irradiation with another small band of denaturing gel (2) indicating cleavage probe removal (undamaged cleavage probe bands will be observed if the experimental conditions are not satisfactory).

Example 4

General format of DNA RFTA using ssDNA target sequences with specific target / probe complexes (PDTP)

The selected target is the sensitive strand (w) sequence of the toxin producing gene of Bacillus Anthracis. The presence of the target in the DNA sample indicates the presence of bacteria and infection.

Isolation of Bacterial Targets from Serum Specimens

1. Centrifuge 50-100 mL of serum sample at 4 ° C. at 12,000 × g.

2. Resuspend the pellet with 567 μL TE buffer by repeated pipetting. 30 μl of 10% SDS and 3 μl of proteinase K 20 mg / ml are added, mixed and incubated at 37 ° C. for 1 hour.

3. Add 100 μl 5 M NaCl and mix thoroughly. Add 80 μl of CTAB / NaCl solution, mix and incubate at 65 ° C. for 10 minutes.

4. Add isovolume chloroform / isoamyl alcohol, mix and microcentrifuge for 4-5 minutes. Transfer the supernatant to a new tube. If it is difficult to remove the supernatant, use a toothpick to remove the interface.

5. Add isovolume phenol / chloroform / isoamyl alcohol, mix and microcentrifuge for 5 minutes. Transfer the supernatant to a new tube.

Some bacterial backbones at the interface formed after chloroform extraction are not dense enough to allow easy removal of the supernatant. In this case, most of the interface can be removed with sterile toothpicks before removing the supernatant. The remaining CTAB precipitate is then removed by phenol-chloroform extraction.

6. Add 0.6 volume isopropanol and mix gently until DNA precipitates. Transfer the precipitate to 1 ml of 70% ethanol with a sealed Pasteur pipette and wash.

Alternatively, the precipitate can be briefly centrifuged and washed with 1 ml of 70% ethanol.

7. Discard supernatant for 5 minutes by centrifugation and dry briefly in lyophilizer. Resuspend in 100 μl TE buffer. Use 10-15 μl per restriction digest.

DNA RTFA

Identification of Toxin Genes in Bacillus anthracis

Once the DNA is isolated it must be restricted in one or two restriction endonucleases with positions adjacent to the selected target site (various lengths).

Enzymatic degradation

Add 500 units of each restriction enzyme (50 μl in most cases). The reaction mixture is incubated at 37 ° C. for an additional 50 minutes and then the enzyme is inactivated by biochemical or biophysical methods. The sample is now ready for the next step.

The following is the sequence for all probes and structures containing RFTA:

Base 581-660 B.anthracis Toxin Gene

3 '(541) actttgagtg gtccgtctt tatccccctt gtacagggg cgggcggtca tggtgatgta

(601) ggtatgcacg taaaagagaa agagaaaaat aaagatgaga ataagagaaaa agatgaagaa-5 '

Target sequence:

3'GTACAGGGGG, CGGGCGGTCA TGGTGATGTA 5 '(double helix at this point)

Primary probe:

5'CCAGT ACCACTACAT AGCTTGCTAC TCAGG3 '

Binding site reporter region of target

Secondary probe:

5'TAGCG TTACGACGCG CATCTCCCCC GCCCG3 '

Binding site of the capture site target

Take probe:

3'ATCGC AATGCTGCGC5 '

DIG

Reporter probe:

3'TCGAACGATG AGTCC5 'B = Biotin

B B B B B

Once the DNA is restricted, the DNA must be denatured to release the ssDNA target.

DNA denaturation

After limitation, DNA should be denatured by heating to 94 ° C. for at least 1 minute (at 94-C) or by alkali treatment (0.4 N No. OH plus 25 mM EDTA).

The primary probe is then hybridized to the site of the target sequence. If the hybridization conditions are similar, capture probes can be added simultaneously in a mixture (10 nanomoles of DIG labeled probe) and bind to other sites of the target site. The temperature of hybridization is typically 20 ° C. below the melting point of the ds nucleic acid. Conventional hybridization conditions require 10 times less each probe and incubate at 50 ° C. for 20-60 minutes, and add higher salt (5 × 55 PE) buffer in neutral OH.

Capture system

To the DNA mixture is added 10 nanomoles of DIG labeled capture probe and 0.5 ml 2.5x of hybridization buffer (5.0 M NaCl, 0.5 M NaOAc, pH 4.5). The mixture is incubated for a sufficient time, for example 1 hour, to form a stable hybridization complex at the optimal hybridization temperature, then 100 μl of anti-Dig coated magnetic beads are added, washed and resuspended in hybridization buffer. . After incubation for an additional hour, the beads are separated using a magnetic particle concentrator and washed eight times with minimal damage with 0.5 ml hybridization buffer (1 × SSPE) (lower salt). After washing the magnetic beads are prepared for hybridization of the reporter probe substituted with biotin every nth base. Reporter probes (25 mer) are incubated at 5 ° C. for 20-60 minutes at 50 ° C. and wash water is introduced to wash unbound probes at 45 ° C. with 1 × SSPE buffer. All tube washers containing magnetic beads are operated in a magnetic tube holder to prevent bead / target loss.

The next step is to initiate signal generation by adding the avidin-horse radish peroxidase conjugate. The conjugate is added in 10-fold excess in the tube containing the beads on which the target is trapped. Beads are similarly washed to remove unbound conjugates and contacted with peroxidase, which produces a soluble color by adding the pigment source substrate tetramethyl benzidine.

The intensity of the color is proportional to the number of targets present.

The descriptions of all patents and applications cited in this application are incorporated by reference to further describe the technology to which the invention relates.

Although the method of the present invention has been described with reference to specific aspects of the details, these details are not limited to the spirit of the invention except as included in the appended claims.

Claims (23)

Providing a cleavage probe having a reactive group that is complementary to the target nucleic acid sequence; The cleavage probe and the target nucleic acid sequence are combined under conditions that allow hybridization and target / probe complex formation; Wherein the activation comprises activating a reactive group that disrupts the target / probe complex at the position of the reactive group in the cleavage probe and at a complementary position in the target nucleic acid sequence. The method of claim 1 wherein the reactive group is a halogenated nucleotide. The method of claim 2, wherein the reactive group is a purine or pyrimidine homologue. The method of claim 3, wherein the reactive group is bromouracil and the activation method is light irradiation. The method of claim 1, wherein the cleavage probe further comprises a capture molecule. A composition for detecting a target nucleic acid sequence in a sample, comprising a cleavage probe having a reactive group bonded to the target nucleic acid sequence. 7. The composition of claim 6 further comprising a reporter molecule. a) obtaining an unlabeled nucleic acid sequence isolated from a sample presumed to contain the target nucleic acid sequence; b) cleaving the target nucleic acid sequence; c) denature the sample and the cleaved target nucleic acid sequence; d) contacting the target-protecting molecule, the portion of which specifically binds to the target nucleic acid sequence, with the unlabeled nucleic acid sequence of step a) under hybridization conditions sufficient to form a protected target nucleic acid sequence (PNAS); e) contacting at least one secondary probe with the non-binding site of the double-stranded target-protecting molecule; f) detecting the PNAS. The method of claim 8, wherein the target nucleic acid sequence is single chain. The method of claim 8, wherein the target is double stranded. The method of claim 8, wherein the target-protecting molecule and the secondary probe are single chain. The method of claim 8, further comprising a secondary probe having a target-protecting molecule or reporter molecule. A composition for detecting a target nucleic acid sequence in a sample, comprising a target-protecting molecule and a secondary probe bound to the target nucleic acid sequence. The composition of claim 13, further comprising a reporter molecule. a) obtaining an unlabeled nucleic acid sequence isolated from a sample presumed to contain the target nucleic acid sequence; b) cleaving the target nucleic acid sequence; c) contacting the unlabeled nucleic acid sequence of step a) under hybridization conditions sufficient to form a protected target nucleic acid sequence (PNAS), wherein the target-protecting molecule, wherein the portion specifically binds to the target nucleic acid sequence; d) contacting at least one secondary probe with the non-binding site of the target-protecting molecule; e) detecting the PNAS. The method of claim 15, wherein the target nucleic acid sequence is single chain. The method of claim 15, wherein the target-protecting molecule is double stranded. The method of claim 15, wherein the target-protecting molecule produces a hairpin structure. The method of claim 15, wherein the PNAS is triple helix. The method of claim 15, further comprising a secondary probe having a double-stranded target-protecting molecule or reporter molecule. The method of claim 15, wherein Exo III degrades the double stranded molecule after the secondary probe is bound. A composition for detecting a target nucleic acid sequence in a sample comprising a double-stranded target-protecting molecule and a secondary antibody coupled with a target nucleic acid sequence. The composition of claim 23 further comprising a reporter molecule.