Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1992-06-22
2001-07-31
Zitomer, Stephanie (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S259000, C435S287200, C435S288300, C435S288400, C435S288700, C436S501000, C436S508000, C536S023100, C536S024200, C536S024300, C536S024320
Reexamination Certificate
active
06268128
ABSTRACT:
BACKGROUND
Biological samples generally contain nucleic acid sequences which encode information unique to its biological source. For example, all species of acteria which belong to a certain genus (e.g., Campylobacter or Enterobacter) share certain physical characteristics which are encoded by the same nucleotide sequences present in most or all of the species within the genus. Thus, an assay which is specific for the genus can be based upon these common nucleotide sequences.
Detection of nucleotide sequences in a sample can be carried out using nucleotide probes specific for these target sequences. For example, Nagata et al.,
FEBS
, 183:379-382 (1985), describe the use of UV irradiation to bind heterologous high molecular weight DNA to polystyrene microtiter wells in order to detect specific sequences within the immobilized DNA by way of specific DNA probes.
Zouali and Stollar,
J. Immuno. Methods
, 90:105-110 (1986), describe a technique for the attachment of high molecular weight nucleic acids to polystyrene microtiter wells using pre-treatment of the support with UV irradiation.
Polsky-Cynkin et al.,
Clinical Chemistry
, 31:1438-1443 (1985), describe the use of immobilized capture probes in clinical assays.
Kremsky et al.,
Nucleic Acids Research
, 15:2891-2909 (1987) and Wolf et al.,
Nucleic Acids Research
, 15:2911-2926 (1987) describe a technique for the covalent attachment of oligonucleotides to latex coated polystyrene beads.
Stabinsky, U.S. Pat. No. 4,751,177, describes a single-step target capture that utilizes a hybridization of a tailed capture probe in solution followed by a solid phase capture with oligo-(dT)-controlled pore glass.
Soderlund, UK Patent Application GB 2169403A (1985), describes several affinity-based capture hybridization methods which use two probes, detector probe and a capture probe that contains one member of an affinity pair.
Collins, European Patent Application Number 265 244, describes a nonisotopic reversible target capture protocol which makes use of dA-tailed oligonucleotide probes and oligo(dT)-magnetic particles and poly(dT) filters.
Presently available nonisotopic assay methods are either lacking in sensitivity for certain applications, or are too complex or too slow to be clinically useful. They also require a sample to be split in order to perform multiple assays thereon, resulting in decreased sensitivity. Most of the prior art methods also employ solid phases that are not easily separated from viscous clinical samples, such as stool. It would be helpful to have a rapid, nonisotopic assay useful for assaying complex or unpurified samples that is highly specific, simple to use useful with RNA as well as DNA targets and applicable to clinical and food samples with no prior purification of the nucleic acids of the samples.
SUMMARY OF THE INVENTION
The present invention pertains to a method of determining (detecting and/or quantitating) target nucleic acid sequences in a sample, which is simple and rapid and does not require the use of radioactive materials. In the method of the present invention, oligonucleotide probes (capture probes), which are specific for nucleic acids to be detected (target nucleic acids), and bound to an appropriate support, are contacted with a sample to be analyzed for the target nucleic acids, under conditions appropriate for hybridization of complementary nucleic acid sequences to occur. In general, the sample has been previously treated in such a manner that the molecular structure of the cells is disrupted (i.e., the cell structure, such as the chromosomes and membranes are broken, and the cellular cytoplasm is dispersed).
In the method of the present invention, a sample is treated to release the nucleic acids of cells contained in the sample, and is combined with a capture probe, which is reversibly attached or preimmobilized on a support, such as polystyrene, by means of a homopolymer tail whose sequence is complementary to a sequence present on the support surface. Hybridization of complementary nucleic acid sequences results in capture of target nucleic acids from the sample. Capture of the target on the solid supports also serves to separate the target nucleic acids from sample impurities prior to nonradioisotopic or radioisotopic detection. Target nucleic acids can be detected and/or quantified by hybridizing the captured target with a labeled probe, for example.
The present method has numerous advantages over presently-available methods. For example, the present method makes it possible to: (1) analyze many samples (e.g., 20 or more) nonisotopically in a short time; (2) carry out analyses without sample filtration or cell culture or prior purification of nucleic acids; (3) run a panel of tests on a sample simultaneously without crosstalk; (4) run multiple tests on a small volume sample; and (5) use capture probes without prior purification. In addition, the method can be efficiently performed using a single labeled probe, since one generic probe can be constructed (e.g., by cloning) to hybridize to all target nucleic acids that make up a screen or a panel. The present assay method allows the non-radioactive detection of as little as one hundred attomoles of target nucleic acid in any type of cell extract (bacterial, mammalian, yeast, plant), in food and clinical samples and other impure biological specimens in about two hours.
In addition to its use in detecting and/or quantitating the level of target present in a sample, the present invention can also be used, without the customary phenol extraction, to isolate nucleic acid targets from crude specimens for cloning (or subcloning) and/or amplification. Substantial purification of the target prior to either cloning or target amplification (such as the PCR method, Mullis, U.S. Pat. No.4,683,202) reduces the level of background (and thus improves the specificity) of these procedures as well as removing numerous interfering substances present in crude specimens. In addition, if necessary, the sensitivity of the detection of targets by the present method can be substantially improved by inserting an optional target amplification method between target capture and detection or by using reversible target capture methods as disclosed herein.
The method of the present invention is particularly useful for precious or small volume samples because it is not necessary to divide the sample into smaller samples for each test to be carried out. Another advantage of the present invention is that a nonspecific or generic reporter probe can be used because the possible loss of signal due to the presence of interfering substances or high levels of competitor organisms is avoided by capturing target nucleic acid sequences from the sample being tested prior to labeling.
The present invention also includes kits for rapid analysis of samples by the method of the present invention. A kit can contain, for example, suitable solid supports, such as dipsticks or wells, which contain a substratum, which is discussed in greater detail hereinbelow, and a specific capture probe prehybridized to the substratum, and an agent, such as a lysis solution, for disrupting cells to free cellular nucleic acids for detection. Alternatively, the kit can contain capture probes which are not bound to the solid phase. This requires the user to perform the prehybridization step. Such a system allows the user more flexibility since the user would prepare the appropriate capture probe-solid phase adducts whenever desired. The kit can, optionally, contain a labeled probe and a means for detecting the labeled probes, positive and/or negative control samples, elution buffers for carrying out reversible target capture and amplification or cloning reagents.
REFERENCES:
patent: 4358535 (1982-11-01), Falkow et al.
patent: 4751177 (1988-06-01), Stabinsky
patent: 4797355 (1989-01-01), Stabinsky
patent: 5288609 (1994-02-01), Engelhardt et al.
patent: 5457025 (1995-10-01), Collins et al.
patent: 0 139 489 (1985-06-01), None
patent: 0200057 (1986-05-01), None
patent: 0198413 (1986-10-01), None
patent: 023
Collins Mark L.
Morrissey David V.
Farrell Kevin M.
Vysis, Inc.
Zitomer Stephanie
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