Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-06
2004-02-03
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C530S388210, C536S024300
Reexamination Certificate
active
06686151
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the general field of detection of biological molecules, including DNA, RNA, protein and the like, and specifically in the field of detection of RNA:DNA hybrids on a solid phase, as further described herein, using a hybridization assay.
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, nucleic acid probes may 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 radiolabeled 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 labeled with a radioactive substance that may be easily detected.
The existing hybridization techniques that utilize radioisotopes to label probes introduce additional expenses caused by the high costs of disposal of radioactive waste products and the need for monitoring personnel and the workplace for contamination. In addition, the short half-life of radioactive compounds such as
32
p requires that radioactive probes be produced frequently. Radioactive nucleic acid hybridization is therefore discouraged in commercial areas such as clinical diagnosis.
Probes have been indirectly labeled in an attempt to avoid the problems associated with direct radioactive labeling. One common method of indirect labeling is to attach biotin, a small vitamin, to the nucleic acid probe using a chemical or enzyme technique. Following hybridization to the specific nucleic acid, the biotin is detected by reaction with streptavidin, a protein that binds biotin tightly and has been labeled with an enzyme or fluorochrome. Bound biotin-streptavidin complex may be detected by reaction with color-producing substrates and the fluorochrome may be seen when reacted with incident light of appropriate wavelength. However, indirect labeling 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. The biotin label increases non-specific binding, which leads to high background, thereby reducing sensitivity and increasing the likelihood of a false-positive result. Indirect labeling is also less sensitive than direct labeling because the labeling density is limited; only a small fraction of the bases are labeled giving a limiting number of sites for signal generation. An increase in the labeling 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 labeled probes are therefore not well suited to clinical diagnosis because of its inaccuracy and false positive results.
Hybridization of a probe to the specific nucleic acid 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 time consuming and labor intensive approach poorly suited for clinical diagnosis.
Hybridization of the probe to the specific nucleic acid sequences may also be detected with the aid of an antibody specific for a labeled probe as described in U.S. Pat. No. 4,743,535 to Carrico. The probe is labeled with a detectable substance such as flavin adenine dinucleotide (FAD) or a fluorescent agent. An antibody specific for the labeled probe, after it has hybridized to the specific nucleic acid 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.
Attempts have been made to increase the sensitivity of nucleic acid assays by target amplification. Methods of amplifying nucleic acid sequences are commercially available. These methods include the polymerase chain reaction (PCR), the ligation amplification reaction (LCR), and the transcription based amplification reaction (TMA). PCR technology is described in
PCR Protocols A Guide to Methods and Applications
by Michael A. Innis, David H. Gelfand, John J. Sninsky and Thomas J. White, pp. 39-45 and 337-385 (Academic Press, Inc., Harcourt Brace Jovanovich, Publishers, 1990). PCR technology is also described by Marx, J. L.,
Science
140:1408-1410 (1988) and in U.S. Pat. Nos. 4,683,195 and 4,683,202, to Mullis. Ligation amplification reaction is described by Wu, D. Y and Wallace, R. B,
Genomics
4:560-569 (1989) and Barringer, K. J., et al.,
Gene
89:117-122 (1990). Transcription based amplification reaction is described by Kwoh, D. Y., et al.,
Proc. Natl. Acad. Sci. USA
86:1173-1177 (1989). These methods have the advantages of high sensitivity, but the disadvantages of having a lengthy, tedious, and expensive sample preparation, being prone to false-positive results from reaction product contamination, and having the inability to accurately quantify the initial amount of target nucleic acids. Amplification reaction products are most often detected by a hybridization assay.
The degree of sensitivity achieved in assays for the detection of nucleic acid molecules, either RNA or DNA, in a sample is generally lower for RNA than DNA because RNA is subject to degradation by endogenous RNAses in the sample, resulting in less RNA available for detection. In addition, background interference caused by contaminants in the sample is difficult to eliminate without causing further degradation of the target nucleic acid, such as RNA.
Hybridization assays for the detection of nucleic acid molecules, i.e. RNA, have been developed. For example, a hybridization protection assay for RNA is commercially available from Gen-Probe Inc. (San Diego, Calif.). The hybridization protection assay employs a single-stranded nucleic acid probe linked to an acridinium ester, as described by Engleberg, N. C.,
ASM News
57:183-186 (1991), Arnold et al.
Clin. Chem
. 35:1588-1594 (1989) and U.S. Pat. No. 4,851,330. Hybridization of the probe to a target RNA molecule protects the acridinium ester bond from heat hydrolysis so that the detected chemiluminescent signal is proportional to the amount of target RNA in the sample. The sensitivity of this protection assay is limited by background luminescence caused by non-hybridized probe.
Polyclonal and monoclonal antibodies and other similar entities are commonly used for detection purposes. Specifically, polyclonal antibodies recognize a plurality of epitopes, while monoclonal antibodies only recognize one s
De La Rosa Abel
Lazar James G.
Lorincz Attila T.
Strange Christina M.
Williams Inna R.
Brusca John S.
Digene Corporation
Morgan & Finnegan L.L.P.
Moroz, Esq. Eugene
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