The present invention comprises methods and compositions for detecting nucleic acid sequences. More particularly, the present invention comprises methods and compositions for detection of specific genetic sequences using nucleic acid target protection strategies. The methods and compositions of the present invention can be used in the detection of microorganisms, for diagnosis of infectious diseases in humans, animals and plants; assays of blood products, and for genetic analysis for use in such areas as early detection of tumors, forensics, paternity determinations, transplantation of tissues or organs and genetic disease determinations.
Many target and signal amplification methods have been described in the literature, but none are believed to offer the combination of high specificity, simplicity, and speed. General reviews of these methods have been prepared by Landegren, U., et al., Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990). These methods include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridization, Qxcex2 bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (NASBA) and in situ hybridization. Some of these various techniques are described below.
Polymerase Chain Reaction (PCR)
PCR is the nucleic acid amplification method described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis. PCR consists of repeated cycles of DNA polymerase generated primer extension reactions. The target DNA is heat denatured and two oligonucleotides, which bracket the target sequence on opposite strands of the DNA to be amplified, are hybridized. These oligonucleotides become primers for use with DNA polymerase. The DNA is copied by primer extension to make a second copy of both strands. By repeating the cycle of heat denaturation, primer hybridization and extension, the target DNA can be amplified a million fold or more in about two to four hours. PCR is a molecular biology tool which must be used in conjunction with a detection technique to determine the results of amplification. The advantage of PCR is that it may increase sensitivity by amplifying the amount of target DNA by 1 million to 1 billion fold in approximately 4 hours. The disadvantage is that contamination may cause false positive results, or reduced specificity.
Transcription-based Amplification System (TAS)
TAS utilizes RNA transcription to amplify a DNA or RNA target and is described by Kwoh et al. (1989) Proc. Natl. Acad. Sci., USA 86:1173. TAS uses two phases of amplification. In phase 1, a duplex cDNA is formed containing an overhanging, single-stranded T7 transcription promoter by hybridizing a polynucleotide to the target. The DNA is copied by reverse transcriptase into a duplex form. The duplex is heat denatured and a primer is hybridized to the strand opposite that containing the T7 region. Using this primer, reverse transcriptase is again added to create a double stranded cDNA, which now has a double stranded (active) T7 polymerase binding site. T7 RNA polymerase transcribes the duplex to create a large quantity of single-stranded RNA.
In phase 2, the primer is hybridized to the new RNA and again converted to duplex cDNA. The duplex is heat denatured and the cycle is continued as before. The advantage of TAS over PCR, in which two copies of the target are generated during each cycle, is that between 10 and 100 copies of each target molecule are produced with each cycle. This means that 106 fold amplification can be achieved in only 4 to 6 cycles. However, this number of amplification cycles requires approximately three to four hours for completion. The major disadvantage of TAS is that it requires numerous steps involving the addition of enzymes and heat denaturation.
Transcriptions Amplification (3SR)
In a modification of TAS, known as 3SR, enzymatic degradation of the RNA of the RNA/DNA heteroduplex is used instead of heat denaturation, as described by Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874. RNAse H and all other enzymes are added to the reaction and all steps occur at the same temperature and without further reagent additions. Following this process, amplifications of 106 to 109 have been achieved in one hour at 42xc2x0 C.
Ligation Amplification (LAR/LAS)
Ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and Wallace, R. B. (1989) Genomics 4:560. The oligonucleotides hybridize to adjacent sequences on the target DNA and are joined by the ligase. The reaction is heat denatured and the cycle repeated. LAR suffers from the fact that the ligases can join the oligonucleotides even when they are not hybridized to the target DNA. This results in a high background. In addition, LAR is not an efficient reaction and therefore requires approximately five hours for each cycle. Thus, the amplification requires several days for completion.
Qxcex2 Replicase
In this technique, RNA replicase for the bacteriophage Qxcex2, which replicates single-stranded RNA, is used to amplify the target DNA, as described by Lizardi et al. (1988) Bio/Technology 6:1197. First, the target DNA is hybridized to a primer including a T7 promoter and a Qxcex2 5xe2x80x2 sequence region. Using this primer, reverse transcriptase generates a cDNA connecting the primer to its 5xe2x80x2 end in the process. These two steps are similar to the TAS protocol. The resulting heteroduplex is heat denatured. Next, a second primer containing a Qxcex23xe2x80x2 sequence region is used to initiate a second round of cDNA synthesis. This results in a double stranded DNA containing both 5xe2x80x2 and 3xe2x80x2 ends of the Qxcex2 bacteriophage as well as an active T7 RNA polymerase binding site. T7 RNA polymerase then transcribes the double-stranded DNA into new RNA, which mimics the Qxcex2. After extensive washing to remove any unhybridized probe, the new RNA is eluted from the target and replicated by Qxcex2 replicase. The latter reaction creates 107 fold amplification in approximately 20 minutes. Significant background may be formed due to minute amounts of probe RNA that is non-specifically retained during the reaction.
Chiron Signal Amplification
The Chiron system, as described by Urdea et al. (1987) Gene 61:253, is extremely complex. It utilizes 12 capture oligonucleotide probes, 36 labeled oligonucleotides, 20 biotinylated immobilization probes that are crosslinked to 20 more enzyme-labeled probes. This massive conglomerate is built-up in a stepwise fashion requiring numerous washing and reagent addition steps. Amplification is limited because there is no cycle. The probes simply form a large network.
ImClone Signal Amplification
The ImClone technique utilizes a network concept similar to Chiron, but the approach is completely different. The ImClone technique is described in Kohlbert et al. (1989) Mol. and Cell Probes 3:59. ImClone first binds a single-stranded M13 phage DNA containing targeted probe. To this bound circular DNA is then hybridized about five additional DNA fragments that only bind to one end and the other end hangs freely out in the solution. Another probe set is then hybridized to the hanging portion of the previous set of probes. The latter set is either labeled directly with an enzyme or it is biotinylated. If it is biotinylated, then detection is via a streptavidin enzyme complex. In either case, detection is through an enzyme color reaction. Like the Chiron method, the ImClone method relies on build-up of a large network. Because there is no repeated cycle, the reaction is not geometrically expanded, resulting in limited amplification.
While the nucleic acid amplification methods described above allow for the detection of relatively small quantities of target nucleic acid molecules, there is a need for the ability to detect target nucleic acid molecules in a shorter amount of time with less background interference. Problems inherent in PCR and other amplification techniques involve sample contamination during the collection techniques and the presence of amplicons (amplified target DNA). There are problems with non-specific target amplification mediated by closely related sequences and the production of primer dimers. There is also poor control of specificity, resulting in false positive reactions, and poor control of sensitivity, resulting in false negative reactions. PCR results must often be confirmed and validated by other techniques such as probe hybridization, Southern blotting or in situ hybridization.
Additionally, PCR and amplification techniques can only be used with very small amounts of starting sample DNA, in the range of a maximum of 1 microgram. This negates use of PCR techniques for the detection of low copy number nucleic acid targets. For example, early detection of HIV infection, soon after the initial viral infection, would be almost impossible to detect using PCR.
Thus, compositions, methods and kits are needed that are capable of detecting specific nucleic acid sequences and isolating them. Especially needed are methods and kits that would allow for the detection of low copy number nucleic acid target sequences. Additionally, there is need for methods and kits that provide the flexibility that would allow for isolation of nucleic acid sequences using a desired level of specificity.
What is also needed are methods that do not use amplification techniques, but do allow for the isolation of a specific target sequence from any amount of starting nucleic acid, especially large amounts, and have the flexibility to accomplish the isolation at several levels of specificity, depending on the level of specificity desired.
In accordance with the present invention, methods and compositions are provided for the detection of specific nucleic acid sequences from cellular or tissue sources. More particularly, the present invention includes methods and compositions for the detection of nucleic acid sequences using a protection molecule that forms a protected nucleic acid sequence (PNAS) such as a triplex or duplex nucleic acid structure that includes the target nucleic acid sequence. The targent nucleic acid sequence is the specific sequence being detected. An assay using the methods of the present invention may include one, two or three levels of specificity to minimize false positive signals. An assay using the methods or compositions of the present invention can be performed on large amounts of purified DNA in a single test, with high levels of sensitivity, thus eliminating the need for in vitro DNA amplification procedures.
When the target nucleic acid sequence is double-stranded, the structure formed with the protection molecule is a triplex. When the target nucleic acid sequence is single-stranded, the structure formed with the protection molecule is a duplex. In this disclosure, where triplex structures are discussed, one can also substitute duplex structures or structures using PNA (peptide-nucleic acid) and the appropriate nucleases. Assays using the methods of the present invention may be referred to as TPA, Target Protection Assays.
The initial level of specificity utilizes protection molecules such as oligonucleotides or peptide-nucleic acids (PNA) to bind to specific target sequences of interest. Such binding may be accomplished by formation of Hoogstein-type hydrogen bonds. The protection molecule, bound to the target nucleic acid sequence, forms the protected nucleic acid sequence (PNAS). Once these PNAS structures are formed and stabilized in solution, the non-specific DNA is digested. For example, this digestion can be accomplished with a combination of endonucleases and a double-strand-dependent exonuclease, such as DNA Exonuclease III (Exo III). The endonucleases used in this example are designed to cut on both sides of the PNAS, leaving approximately 20 base pairs of DNA on each side of the sequence. Exo III, an exonuclease which progressively cleaves one strand of the DNA from the 3xe2x80x2 end, is inhibited by the triple helix structure. Using a combination of nucleases, the unprotected DNA sequences are digested completely. A method of the present invention involving a lower level of specificity would employ an affinity molecule for capture and a reporter molecule for labeling in conjunction with the protection probe.
However, if a higher level of specificity is required, 5xe2x80x2 flanking regions can be generated on either or both sides of the PNAS to allow for assays employing two further levels of specificity. The structure formed, a PNAS with flanking regions is termed PNAS/tail. Following the selected digestion around the PNAS, a capture probe, such as an oligonucleotide complementary to one of the single stranded flanking regions, is added. The capture probe is allowed to hybridize to a single-stranded region. For example, the capture probe could be an oligonucleotide that would bind to a single-stranded region and have an affinity molecule attached. For example, the affinity molecule could be didoxigenein or biotin. The capture probe comprises an affinity molecule and is capable of associating with the PNAS.
A capturing system is used to isolate the PNAS with the capture probe attached. Any capturing system that is capable of binding to the capture probe and separating the PNAS/tails with affinity molecule from the mixture is contemplated. In the example used above, such a capture system may comprise using magnetic beads coated with anti-didoxigenein antibodies for binding to the didoxigenein-capture probe portion or, streptavidin for binding to the biotin-capture probe portion. The PNAS/tails with affinity molecule, now attached to the magnetic beads, are separated from non-specific complexes and washed to remove any non-specific nucleic acid sequences. Such washing may use any washing technique known in the art. For example, a magnetic particle holder could be used. Again, should this be the level of specificity required, the present invention comprises assays that also have a reporter molecule associated with the protection molecule or the capture probe.
A third level of specific detection involves the addition of a labeled reporter probe. The reporter probe comprises a detectable label and is capable of associating with the PNAS. For example, the reporter probed may comprise an oligonucleotide complementary to the 5xe2x80x2 single-stranded tail that is part of the PNAS/tail. This 5xe2x80x2 region may or may not be on the opposite flanking tail to which the capture probe binds. The reporter probe may be labelled with any labels known in the art such as radioactivity or non-radioactive labels such as labeled with biotin or didoxigenein for indirect detection, or directly with a fluorescent reporter molecule, e.g., fluorescein, or chemiluminescent or bioluminescent labels. An excess of reporter probe is added to the washed magnetic bead-triplex complex and allowed to hybridize. Detection of the bound labelled reporter probe can be accomplished after washing by using detection devices specific for the type of label used. For example, if a fluorescent labeled reporter probe is used, the labeled sequences can be detected using a fluorometer or viewing the beads through a fluorescent microscope. Alternatively, the amount of bound probe can be directly assessed by fluorescent anisotropy with an analyzer such as the Abbott TDM analyzer.
Compositions of the present invention include compositions comprising the components to practice the methods taught herein. For example, a composition comprising a labeled protection molecule with an affinity molecule could be used in an assay with a first level of specificity. A composition comprising a labeled protection molecule and a capture probe could be used in a level two specificity assay. A composition comprising a protection molecule, a capture probe and a reporter probe could be used in a level three assay. It is to be understood that the individual molecules, probes and components can also be provided individually.
The present invention is especially useful for detecting specific genetic sequences. The present invention comprises methods such as the Target Protection Assay (TPA) in all its formats, which have the advantage of allowing the processing of very large amounts of purified nucleic acids, thus eliminating the need for artificial amplification procedures such as PCR, while enabling the detection of a specific target sequence. In addition, the three levels of specificityxe2x80x94PNAS formation, capture probe binding, and reporter probe bindingxe2x80x94reduce technical problems such as those associated with false positive signals from non-specific amplification and/or hybridization.
The present invention comprises a method for detecting a target nucleic acid sequence, comprising obtaining isolated nucleic acid sequences from a sample suspected of containing a target nucleic acid sequence; contacting a protection molecule with the nucleic acid sequences under hydridizing conditions sufficient to form a PNAS; and detecting the PNAS. The methods may further comprise the steps of digesting the isolated nucleic acids containing one or more PNAS with nucleolytic enzymes to form a PNAS/tail; and hybridizing a capture molecule to the PNAS/tail; prior to the step of detecting the PNAS. Additionally, the methods may further comprise the step of hybridizing of a reporter molecule to the PNAS/tail; prior to the step of detecting the PNAS. A method for detecting specific nucleic acid sequences, comprising obtaining isolated nucleic acid sequences from a sample suspected of containing a target nucleic acid sequence; contacting a protection molecule with the nucleic acid sequences under hydridizing conditions sufficient to form a PNAS; digesting the isolated nucleic acids containing one or more PNAS with nucleolytic enzymes to form a PNAS/tail; hybridizing a capture molecule to the PNAS/tail; hybridizing of a reporter molecule to the PNAS/tail; and detecting the PNAS.
The present invention comprises compositions for detecting specific nucleic acid sequences, comprising a protection molecule capable of binding with a specific nucleic acid sequence. A composition of the present invention may further comprise a capture molecule. Additionally, a composition of the present invention may further comprise a reporter molecule.
The methods and compositions of the present invention should be ideal for the detection of viruses and other microorganisms such as pathogens of humans, animals and plants, as well as genetic analysis of polymorphic gene sequences such as HLA typing. The methods of the present invention can be used in forensics, paternity determinations, or transplantation or organs or tissues, or genetic disease analysis.
Accordingly, it is an object of the present invention to provide methods to detect specific genetic sequences.
It is yet another object of the present invention to provide methods for detecting specific DNA sequences involving triplex nucleotide structures.
It is another object of the present invention to provide methods for detecting specific RNA sequences involving triplex nucleotide structures.
It is yet another object of the present invention to provide methods for detecting specific DNA sequences involving duplex nucleotide structures.
It is another object of the present invention to provide methods for detecting specific RNA sequences involving duplex nucleotide structures.
It is another object of the present invention to provide methods for detecting specific RNA sequences involving PNA structures.
It is another object of the present invention to provide methods for detecting specific DNA sequences involving PNA structures.
It is yet another object of the present invention to provide methods for detecting specific DNA sequences involving antibodies.
It is yet another object of the present invention to provide methods for detecting specific RNA sequences involving antibodies.
Another object of the present invention is to provide a method of detecting nucleic acid sequences involving radioactive labeled nucleic acids.
It is another object of the present invention to provide a method of detecting nucleic acid sequences involving non-radioactive labeled nucleic acids.
It is yet another object of the present invention to provide a method of detection of specific genetic sequences with variable levels of specificity.
Another object of the present invention is to provide a method of detecting nucleic acid sequences for the determination of the identity of microorganisms.
It is another object of the present invention to provide a method of detecting nucleic acid sequences for the determination of the identity of human pathogens.
It is yet another object of the present invention to provide a method of detecting nucleic acid sequences for the determination of the identity of animal pathogens.
It is yet another object of the present invention to provide a method of detecting nucleic acid sequences for the determination of the identity of plant pathogens.
It is another object of the present invention to provide a method of detecting nucleic acid sequences for the determination of the genetic relationship, such as paternity or species identification, of a sample.
It is yet another object of the present invention to provide a method of detecting nucleic acid sequences for the determination of potential donors of organs or tissues for transplantation purposes or for protecting the blood supply.
It is another object of the present invention to provide a method of detecting nucleic acid sequences for use in forensic determinations.
It is yet another object of the present invention to provide a method of detecting nucleic acid sequences for the analysis of genetic diseases.
It is another object of the present invention to provide methods for testing body or tissue fluids to detect microorganisms or other pathogens.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.