Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-23
2004-02-24
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06696246
ABSTRACT:
BACKGROUND OF THE INVENTION
a) Field of the Invention
This invention relates generally to methods of detecting nucleic acid sequences using oligonucleotide probes or arrays of probes that have a crosslinking moiety in assays where either the probe or the target molecule is bound to a solid support. The surface-bound DNA or RNA may be immobilized cellular or subcellular systems, or immobilized arrays of DNA or RNA preparations.
b) Description of Related Art
Numerous useful techniques in the biological sciences involve the immobilization of biological material on a solid support of some kind. Immobilized nucleic acid hybridization assays constitute an important class of these methods. Examples of nucleic acid-based assays where the sample being assayed is immobilized include in situ hybridization and blotting assays. Examples of assays where the sample is contacted with immobilized probes include gene chip technologies.
In situ hybridization techniques are a valuable method for identifying the presence of specific nucleic acid sequences within cellular or subcellular systems. Unlike in vitro techniques in which the nucleic acids of interest are retained in some manner while the remainder of the sample is degraded in order to perform the assay measurement, in situ techniques allow one to assay for the presence of specific sequences among substantially intact cellular or subcellular structures.
In blotting assays, DNA or RNA bound to a membrane (or filter paper), in many cases after having been migrated through a gel, is probed for the presence of a specific nucleic acid sequence. Immobilization techniques for DNA assays were first demonstrated by Southern,
J. Mol. Biol
., 1975, 98, 503. Since then many derivative procedures have been developed. These derivative procedures include Northern blot techniques in which RNA is immobilized and assayed via hybridization and dot blot techniques where a solution containing nucleic acid molecules is directly immobilized on the membrane or filter and assayed via hybridization.
In immobilized nucleic acid-based assays, oligonucleotide probes are contacted with the immobilized sample, or a sample is contacted with immobilized probes, and evidence that the probe has hybridized with its essentially complementary sequence is determined by development of a signal from a direct or an indirect reporter system.
Achieving a desirable signal-to-noise ratio is a major challenge of immobilized nucleic acid hybridization assays. In situ PCR is one method that has been attempted for improving the sensitivity of in situ assays. In this technique, cellular, subcellular, or tissue samples are prepared and primer pairs are introduced. The samples are then subjected to repeated thermal cycles in the same manner that PCR is typically carried out. It is expected that if the target sequence is present then copies of the PCR amplicons will be amplified. However, significant problems with the in situ PCR technique remain.
In addition to the problems encountered for solution-based PCR such as enzyme inhibition, false priming, and primer dimerization, there are other issues specific to the in situ technique that contribute to inconsistent assay results:
1. The extent of cell permeabilization. If the pore size is too small then polymerase enzymes may not be able to enter the cell. If the pore size is too large then the amplicon may freely leave the cell. These effects may be variable within one sample as well as from sample to sample.
2. Endogeneous inhibitors. Whereas for solution-based PCR assays a purpose of sample preparation procedures is to isolate the nucleic acids and remove enzyme inhibitors, in situ assays are necessarily performed within a cellular or subcellular environment and limit the possibilities for removing inhibitors.
3. Loss of amplicon. The permeabilized membranes may permit the amplicon to leave the cell during wash steps subsequent to the amplification reaction. It may also allow amplicons that have been washed out of adjacent cells to enter, leading to signal being observed in cells that did not originally contain the target.
4. Diffuse signals. The amplicons are not localized at the site of the target sequence, unlike probes that hybridize to the target. The amplicons freely move through the sample and may thus generate a diffuse signal that may be difficult to detect and not permit localization of the sequence being assayed. Together, the above factors account for some of the reasons for inconsistent results obtained by in situ PCR assays.
There is a recognized need to improve the detection sensitivity of probe-based hybridization assays for detection of immobilized nucleic acids. For example, methods to improve the rates of hybridization through the use of volume exclusion agents (U.S. Pat. No. 4,886,741) or increased probe concentrations (U.S. Pat. No. 5,707,801) have been disclosed. In U.S. Pat. No. 5,521,061, Bresser et al. describe the use of permeation enhancers and signal enhancers as a means to increase the sensitivity.
Previous disclosures indicate the need for improved methods for assays of immobilized nucleic acids, particularly in in situ hydridization assays.
The use of long probes (200 to 500,000 nucleotides in length) in these assays requires long hybridization times and contributes heavily to high background signals, because of countless opportunities for undesired, non-specific binding interactions provided by the additional sequence. However, long probes provide the advantage of being able to contain many reporter groups and thus provide stronger signals. Shorter probes (less than 200 nucleotides) offer the advantage of reduced hybridization times, but shorter probes are more susceptible to being washed away, thereby reducing the signal. Shorter probes also have the advantage of being prepared by automated synthesis procedures, but their use is limited in many types of assays because of the loss of hybridized probes during the critical washing steps.
Crosslinker-containing probes have been previously used in in vitro hybridization techniques (for instance, see U.S. Pat. No. 4,599,303, Yabusaki et al.). However, whether such probes are applicable to in situ hybridization or blotting techniques was not known. In in vitro techniques, the various biologic components have normally been chemically degraded by sample preparation steps, typical among which are boiling in alkaline solution, proteinase K treatment, and the like. In any event, the in vitro assays are typically designed to retain only the nucleic acid material on some solid support temporarily while removing the other components through removal of the supernatant solution in a series of wash steps. The hybridization step itself is usually performed in solution. If there should be nonspecific interaction between a probe and non-nucleic acid and biological components, this would not contribute to a false signal because these components are not retained in the assay.
However, in techniques in which the target DNA or RNA is immobilized during the hybridization step, especially in the presence of other biological components, non-specific interactions between the crosslinker-containing probes and any of these materials, including the solid support on which the target DNA or RNA is immobilized, would be disastrous to the outcome of the assay. Non-specific interactions between the crosslinker-containing probes and solid support material on which the biological sample is immobilized are of particular concern. For instance, positively-charged groups on a solid support material are helpful in the original immobilization of the target nucleic acid molecule, but they will also attract binding of the nucleic acid probe. Alternatively, exposed hydroxyl groups on the surface of a solid support material may form hydrogen bonds with the nucleic acid probe used. Crosslinker-containing probes are particularly problematic for non-specific binding, because they necessarily contain a highly reactive functional moiety. Although the intention is to use the crosslinking moiety to covalently att
Albagli David
Cheng Peter C.
Huan Bingfang
Van Atta Reuel B.
Wood Michael L.
Lilore Ralph T.
Naxcor, Inc.
Siew Jeffrey
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