Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-15
2001-08-21
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C530S388100, C536S023100
Reexamination Certificate
active
06277579
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the general field of detection of nucleic acid sequences, and specifically in the field of detection of specific RNA sequences.
BACKGROUND OF THE INVENTION
The RNA or DNA for many genes, including those associated with disease states, and microorganisms and viruses have been isolated and sequenced. Nucleic acid probes based on such sequences are currently available to identify a large number of genes and infections. Nucleic acid probes are detectable nucleic acid sequences that hybridize to complementary RNA or DNA sequences in a test sample. Detection of the probe indicates the presence of a particular nucleic acid sequence in the test sample for which the probe is specific. In addition to aiding scientific research, DNA or RNA probes can be used to detect the presence of viruses and microorganisms such as bacteria, yeast and protozoa as well as genetic mutations linked to specific disorders in patient samples.
Grunstein, et al.,
Proc. Natl. Acad. Sci. USA
72:3961 (1975) and Southern,
J Mol. Biol.
98:503 (1975) describe hybridization techniques using radiolabelled nucleic acid probes. Nucleic acid hybridization probes have the advantages of high sensitivity and specificity over other detection methods and do not require a viable organism. Hybridization probes are often labelled with a radioactive substance that can be easily detected.
Probes have been indirectly labelled in an attempt to avoid the problems associated with direct radioactive labelling. The most common method of indirect labelling is to attach biotin, a small vitamin, to the nucleic acid probe using a chemical or enzyme technique. Following hybridization to the specific RNA, the biotin is detected by reaction with streptavidin, a protein which binds biotin tightly and has been labelled with an enzyme or fluorochrome. Bound biotin-streptavidin complex can be detected by reaction with color-producing substrates and the fluorochrome can be seen when reacted with incident light of appropriate wavelength. However, indirect labelling of hybridization probes with biotin or other haptens often increases the “hydrophobicity” of the probe. The probe tends to interact non-specifically with materials other than the complementary nucleic acid target, leading to high background. High background reduces sensitivity and increases the likelihood of a false-positive result. Indirect labelling is also less sensitive than direct labelling because the labelling density is limited; only a small fraction of the bases are labelled giving a limiting number of sites for signal generation. An increase in the labelling density of a probe leads to increased non-specific binding, higher background, and ultimately, failure of the probe to hybridize with its target due to the interference of the hapten with base pairing. Indirectly labelled probes are therefore not well suited to clinical diagnosis.
Hybridization of a probe to the specific RNA sequences has been detected with the use of an intercalating agent such as acridine orange or ethidium bromide as described in U.S. Pat. No. 4,563,417 to Albarella et al. The intercalating agent becomes inserted between hybridized base pairs of probe and sample nucleic acids and causes the tertiary structure of the helix to unwind. An antibody specific for the newly formed antigenic determinant created by the intercalating agent and the unwound helix is detected by conventional means. This method lacks selectivity for the target hybrids because intercalating agents fail to recognize specific sequences. Furthermore, the antibodies recognize only the intercalating agent
ucleic acid complex, but do not detect a specific sequence. Therefore, additional selection or purification steps are required to prevent non-specific signal, making this approach poorly suited for clinical diagnosis.
Hybridization of the probe to the specific RNA sequences can also be detected with the aid of an antibody specific for a labelled probe as described in U.S. Pat. No. 4,743,535 to Carrico. The probe is labelled with a detectable substance such as flavin adenine dinucleotide (FAD) or a fluorescent agent. An antibody specific for the labelled probe, after it has hybridized to the specific RNA sequence, is detected by a biochemical reaction. This method of detection also creates non-specific binding and the likelihood of false-positive results and is not well suited for clinical screening.
Monoclonal antibodies to DNA-RNA hybrids are now available. U.S. Pat. No. 4,732,847 to Stuart et al. and the publication of Stuart et al.,
Proc. Natl. Acad. Sci. USA
78:3751 (1981) describe a method of hybridization detection of specific nucleic acid sequences on a solid surface involving a monoclonal antibody specific for a poly(A)-poly(dT) duplex. In Stuart, DNA/RNA hybrids are formed by annealing DNA or RNA sequences complementary to the sequence of interest.
A monoclonal antibody specific for DNA-RNA hybrids secreted by hybridoma HB 8730, is disclosed in U.S. Pat. No. 4,833,084 to Carrico et al. In Carrico, DNA/RNA hybrids are formed by annealing of RNA and DNA polynucleotides, or by transcription of DNA.
The isolation of anti-DNA-RNA hybridomas has improved the development of assays for genetic mutations linked to specific defects and the detection of bacterial and viral infections. However, assays utilizing these anti-hybrid monoclonal antibodies secreted from the hybridomas often employ large probes leading to a high level of non-specific binding causing false positive results. Boguslawski et al.,
J Immunol. Methods
89:123-130 (1986) developed a hybridization assay using anti-hybrid coated polystyrene beads isolated on filter paper in an attempt to reduce non-specific binding and avoid complicated washing procedures.
Therefore, it would be useful to have a method to detect RNA sequences that is easy to use, highly specific, accurate and sensitive enough for clinical screenings.
Accordingly, it is an object of the present invention to provide a method of detecting specific RNA molecules in a sample with a relatively large signal based on specific hybridization of a relatively small oligonucleotide primer.
It is further an object of the present invention to provide an assay having minimal false positives.
It is further an object of the invention to provide an assay to detect RNA indicative of an infection, a disease state, or predisposition to a disease.
It is further an object of the invention to provide an assay for detecting reverse transcriptase activity and testing for reverse transcriptase inhibitors.
BRIEF SUMMARY OF THE INVENTION
Disclosed is a method of detecting RNA molecules of interest in which reverse transcription primers unique to the RNA molecule of interest are used for reverse transcribing the RNA with a reverse transcriptase lacking RNAse H function and the resulting RNA/DNA hybrid is detected with an antibody specific for RNA/DNA hybrids. The primers are immobilized on a solid support in order to associate the RNA/DNA hybrid with the solid support. This allows easy separation of hybrids form sample solution and specific detection of RNA molecules based on the position of the hybrid on the solid support. This method can be used to detect the presence of one or many specific RNA molecules which may be present in a sample, including RNA from different organisms (such as viruses, bacteria, fungi, plants, and animals), or RNA indicative of an infection, a disease state, or predisposition to a disease in an animal. The specificity of detection is increased relative to current detection methods involving probe hybridization since the reverse transcription primers are shorter and less subject to non-specific hybridization. Specificity of the disclosed method can also be increased by using a thermostable reverse transcriptase and performing reverse transcription at a high temperature.
The disclosed method can be used to detect the presence of one or many specific RNA molecules which may be present in a sample. The method can be used to detect, for example, continuous a
Collier Clayton D.
De La Rosa Abel
Lazar James G.
Brusca John S.
Digene Corporation
Holland & Knight LLP
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