In situ hybridization method

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

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435 5, C12Q 168, C12Q 170

Patent

active

057190232

DESCRIPTION:

BRIEF SUMMARY
1. FIELD OF THE INVENTION

The present invention relates to a diagnostic method for performing in situ hybridization with double-stranded DNA targets.


2. REFERENCES



3. BACKGROUND OF THE INVENTION

In situ hybridization employs direct hybridization of a DNA probe with DNA or RNA in biological structures, typically permeabilized cells, subcellular fractions, or fixed chromosome preparations. Because the method can yield morphological information about the localization of specific-sequence target nucleic acid(s) in fixed biological structures, it is applicable to many areas of biomedical research, such as developmental biology, cell biology, genetics and particularly gone mapping, pathology and gone diagnostics.
In most applications, in situ hybridization is directed toward a target sequence in a double-stranded duplex nucleic acid, typically a DNA duplex associated with a pathogen or with a selected sequence in viral or cell chromosomal DNA. In this method, as it has been practiced heretofore, a single-stranded labeled probe is added to the permeabilized structure, which has been heated to a temperature sufficient to denature the target duplex nucleic acid, and the probe and denatured nucleic acid are allowed to react under suitable hybridization, or reannealing conditions. After removal of unbound (non-hybridized) probe, the structure is processed for examination for the presence of reporter label, allowing the site(s) of probe binding to target duplex nucleic acid to be localized in the biological structure, i.e., in the context of cell or subcellular morphology.
The method has been widely applied to chromosomal DNA, for mapping the location of specific gene sequences, and distances between known gene sequences (Lichter, Meyne, Shen), for studying chromosomal distribution of satellite or repeated DNA (Weier, Narayanswami, Meyne, Moyzis, Joseph, Alexandrov), for examining nuclear organization (Lawrence, Disteche, Trask), for analyzing chromosomal aberrations (Lucas), for localizing DNA damage in single cells or tissue (Baan) and for determining chromosome content by flow cytometric analysis (Trask). Several studies have reported on the localization of viral sequences integrated into host-cell chromosomes (e.g., Harders, Lawrence, Lichter, Korba, Simon). The method has also been used to study the position of chromosomes, by three-dimensional reconstruction of sectioned nuclei (van Dekken), and by double in situ hybridization with mercurated and biotinylated probes, using digital image analysis to study interphase chromosome topography (Emmerich).
Another general application of the in situ hybridization method is for detecting the presence of virus in host cells, as a diagnostic tool (Unger, Haase, Noonan, Niedobitek, Blum). In certain cases where the number of virus particles in the infected cell is very low, it may be necessary to first amplify viral sequences by in situ adopted polymerase chain reaction (PCR) methods (Haase, 1990, Buchbinder).
The in situ hybridization method described above has a number of limitations. The most serious limitation is the requirement for denaturing the duplex target DNA, to form the necessary single-stranded form of the target. Denaturation typically is performed by heating the sample or treating with chemicals and heat. The heat treatment can produce spurious and unwanted changes in the nucleic acid being examined, related to structural changes and nucleic acid reassociation with repeated sequences within the DNA. The repeated DNA sequences can randomly reassociate with one another. The step also adds to the time and effort required in the method.
Secondly, where the target sequence of interest is present in very low copy number, the method is limited, by renaturation kinetics, to long renaturation times. Even then, the method may be incapable of producing probe/target renaturation events at low target concentration. This limitation may be partly overcome, as indicated above, by first amplifying the target duplex in situ by modified PCR methods. However, the PCR appro

REFERENCES:
patent: 4683202 (1987-07-01), Mullis
patent: 4888274 (1989-12-01), Radding et al.
Di Capua, E., et al., "Characterization of Complexes between recA Protein and Duplex DNA by Electron Microscopy," M. Mol. Biol. 157: 87-103 (1982).
Fan, Y.-S., et al., "Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes," Proc. Natl. Acad. Sci. USA 87: 6223-6227 (1990).
Honigberg, S.M., et al., "Ability of RecA protein in promote a search for rare sequences in duplex DNA," Proc. Natl. Acad. Sci. USA 83: 9586-9590 (1986).
Infantolino, D., et al., "HBV-DNA by in situ hybridization. A method to improve sensitivity on formalin-fixed, paraffin-embedded liver biopsies," Liver 9: 360-366 (1989).
Saiki, R.K., et al., "Enzymatic Amplification of .beta.-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia," Science 230: 1350-1354 (1985).
Griffith et al., "Intercalating Drugs Markedly Affect the Ability of the E. coli RecA Protein to Insert Small Primers Into Homologous Duplex DNA," J. Cell Biochem. (Suppl. 13E, 287) (1989).
Koch et al., "Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ," Chromosoma 98:259-265 (1989).
Haase et al., "Amplification and detection of lentiviral DNA inside cells," PNAS 87:4981-4975 (1990).
Weier et al., "Two-color hybridization with high complexity . . . ," Chromosome 100:371-376 (1991).
Van Dekken, H., et al., "Three-dimensional analysis of the organization of human chromosomes domains in human and human-hamster hybrid interphase nuclei," Journal of Cell Science 94:299-306 (1989).

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