In-situ hybridization method using RecA protein and RecA protein

Details In-situ hybridization method using RecA protein and RecA protein In-situ hybridization method using RecA protein and RecA protein

1995-10-30

1999-10-12

Zitomer, Stephanie W.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 71, 435 405, 4351723, 435810, 530350, 5303871, 536 243, 536 2431, 536 2432, 935 77, 935 78, C12Q 168, C12Q 108, C07K 1400, C07H 2104

Patent

active

059653612

DESCRIPTION:

BRIEF SUMMARY
DESCRIPTION

1. Technical Field
The present invention relates to an in situ hybridization method utilizing RecA protein useful in the detection of a double-stranded target nucleic acid sequence existing in cells or cell structures, a kit and RecA protein including a label or ligand used to practice the method.
2. Background Art
In situ hybridization is a method for hybridizing a nucleic acid probe directly to a target nucleic acid sequence such as DNA and RNA in a cell. This method is applied to fixed permeabilized cells or cell structures (for example, organelles such as nuclei and mitochondria, and parasites such as bacteria and virus) and biological materials such as a fixed chromosome specimen, with a nucleic acid sequence contained therein utilized as a target. Thus, the presence of the target nucleic acid sequence can be detected in a place where it is present, i.e., in situ, and information about the localization of the target nucleic acid sequence can be obtained. In this manner, this method is applicable to a wide range of areas of biomedical research such as developmental biology, cell biology, genetics (in particular, gene mapping), pathology and gene diagnostics.
In isitu hybridization, a target is generally a double-stranded nucleic acid; typically a specific sequence of pathogen or virus and a specific gene of chromosomal DNA. Conventional in situ hybridization adopts a single-stranded labeled nucleic acid probe, which is added to permeabilized cells and heated to a temperature sufficiently high for denaturing a double-stranded target nucleic acid sequence. The probe and the denatured target nucleic acid sequence are then allowed to be hybridized to each other under suitable conditions. Then, probe which has not been bound to the target nucleic acid sequence is removed, and the labelled probe bound to the target nucleic acid sequence in the cells is detected.
This in situ hybridization is widely applied to areas of chromosomal DNA research such as: mapping of the location of a specific gene sequence, and distance from a known gene sequence on a chromosomal DNA (Fan, Y. S., et al., Proc. Natl. Acad. Sci. USA, 87, 6223, 1990, etc.); study of the distribution of satellite DNA or other repeated sequences on a chromosome (Kenji Sugimoto, et al., Clinical Genetics and Molecular Medicine, 1, 348, 1993, etc.); analysis of chromosomal aberrations (Hopman, A.H.N., et al., Histochemistry, 89, 307, 1988, etc.); analysis of a DNA damage site (Baan, R. A., et al., Prog. Clin. Biol. Res., 340A, 101, 1990, etc.); analysis of a chromosome content with a flow cytometer (Trask, B., et al., Hum. Genet., 78, 251, 1988, etc.); and analysis of gene copy number (Kallioniemi, O. P., et al., Proc. Natl. Acad. Sci. USA., 89, 5321, 1992; Kallioniemi, A., et al., Science, 258, 818, 1992, etc.). Furthermore, the method is utilized in research for localization of viral nucleic acid sequences integrated into a host-cell chromosome (Lawrence, J. B., et al., Proc. Natl. Acad, Sci. USA., 87, 5420, 1990, etc.). It is also applied to study of the location of a chromosome by three-dimensional reconstruction of a sectioned nuclei.
Another general application of the in situ hybridization method is as a diagnostic tool for the detection of the presence of virus in a host cell (Han, K. H., et al., J. Virol. Methods, 37, 89, 1992, etc.). In the case where the number of virus particles contained in an infected cell is very small, viral sequences are previously amplified by in situ polymerase chain reaction (in situ PCR) methods (Hasse, A. T., et al., Proc. Natl. Acad. Sci. USA, 87, 4971, 1990).
The in situ hybridization method described above has a number of limitations. For example, it is necessary to denature a double-stranded target nucleic acid sequence. Denaturation is generally conducted by heat treatment of the target nucleic acid sequence, or a sample including the nucleic acid sequence, in the presence or the absence of a specific drug. This heat treatment frequently causes an undesired change in the nucleic acid in the sample

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