Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-08-07
2001-09-25
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007920, C536S024300, C359S368000
Reexamination Certificate
active
06294331
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods of detecting genetic and phenotypic markers in biological samples using spectral imaging and brightfield microscopy to detect the presence of chromogenic dyes.
2. Background Art
In cytopathological diagnostic laboratories, cytological specimens are routinely stained with permanent dyes such as hematoxylin and eosin and for decades, pathologists have based their diagnosis of disease on cyto- and histological features as seen under a light microscope. Unlike fluorescent dyes, permanent dyes do not fade or bleach so that second opinion diagnosis, re-examination of archived material and even retrospective studies, can be performed. Thus, for routine cytopathological diagnostic purposes, fluorescence microscopy is not preferred for these reasons as well as because of high auto-fluorescence inherent to the tissue type or which might be induced by fixation.
Immunohistochemical and in situ hybridization methods have become increasingly important for research and diagnosis of disease. Also, multi-parameter cytochemical analysis is required when rare or unique material is to be studied. Many fluorescent markers with emission spectra ranging from blue to infra-red have become available for multi-color detection due to advances made in conjugation chemistry. Thus, although fluorescence microscopy could be used for these multi-parameter applications, for the above-mentioned reasons, it is often not possible to use methods employing fluorescence.
The present invention overcomes previous shortcomings in the art by providing methods for analyzing both genetic and phenotypic markers in a single biological sample through the use of bright field spectral imaging of chromogenic dyes. Such analyses are valuable in a variety of clinical applications, such as, for example, the diagnosis and characterization of cancer and the analysis of chromosomal aberrations in pre- and post-natal diagnostics.
An important aspect of the present invention that overcomes a severe limitation in the art is that by using the methods provided herein, multiple probes, both to genetic and/or phenotypic markers, and therefore multiple chromogenic dyes can be used in the same sample and the individual dyes can be distinguished using spectral imaging, even where the sample has been previously stained with a cytological stain which otherwise would obscure the signal from the genetic or phenotypic probes. Using these methods, a pathologist for example, can stain a tissue sample to observe a general morphological aspect of cells in the sample, and a geneticist can subsequently use that stained sample to diagnose cells in the sample for the presence of a genetic or phenotypic marker, such as a chromosomal aberration associated with cervical cancer, with much more clarity, accuracy, ease, and efficiency than using previously available methods.
SUMMARY OF THE INVENTION
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to the present invention provides an improved method for detecting a genetic marker in a biological sample comprising contacting the biological sample with a nucleic acid probe linked to a detectable moiety, whereby the detectable moiety can be detected by the presence of a chromogenic dye associated with the detectable moiety, obtaining a spectral image of the biological sample using brightfield microscopy, and detecting the presence of the chromogenic dye, thereby detecting the genetic marker in the biological sample.
The present invention also provides an improved method for detecting a phenotypic marker in a biological sample comprising contacting the biological sample with a compound comprising a detectable moiety, whereby the compound associates with the phenotypic marker and whereby the detectable moiety can be detected by the presence of a chromogenic dye associated with the detectable moiety, obtaining a spectral image of the biological sample using brightfield microscopy, and detecting the presence of the chromogenic dye, thereby detecting the phenotypic marker in the biological sample.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.
REFERENCES:
patent: WO 97/21979 (1997-06-01), None
patent: WO 97/22848 (1997-06-01), None
Malik et al. Fourier transform multipixel spectroscopy for quantitative cytology. J. Microscopy vol. 182 pp. 133-140, 1995.*
Schröck, et al., “Multicolor Spectral Karyotyping of Human Chromosomes”,Science, vol. 273, pp. 494-497, Jul., 1996.
Speel, et al., “Cytochemical detection systems for in situ hybridization, and the combination with immunocytochemistry. ‘Who is still afraid of Red, Green and blue?’”,Histochemical Journal, 27:833-858, 1995.
Garini, et al., “Spectral karyotyping”,Bioimaging, 4:65-72, 1996.
Hopman Anton H. N.
MacVille Merryn V. E.
Ried Thomas
Brusca John S.
Needle & Rosenberg
The United States of America as represented by the Department of
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