Radiant energy – Luminophor irradiation
Reexamination Certificate
1998-03-06
2001-03-06
Hannaher, Constantine (Department: 2878)
Radiant energy
Luminophor irradiation
Reexamination Certificate
active
06198107
ABSTRACT:
BACKGROUND OF THE INVENTION
The separation of DNA fragments by polyacrylamide or agarose gel electrophoresis is a well-established and widely used tool in molecular biology (Sharp, P. A. et al., “Detection of two restriction endonucleases activities in
Haemophilus parainfluenzae
using analytical agarose-ethidium bromide electrophoresis,” (1973)
Biochemistry
12:3055). The standard technique for viewing the positions of the separated fragments in a gel involves the use of an ultra-violet (UV) transilluminator (Brunk, C. F. and Simpson, L., “Comparison of various ultraviolet sources forfluorescent detection of ethidium bromide-DNA complexes in polyacrylamide gels,” (1977)
Analytical Biochemistry
82:455). This procedure involves first staining the gel with a fluorescent dye such as ethidium bromide or SYBR® Green I. The DNA fragments, which bind the dye, are then visualized by placing the gel on a light-box equipped with a UV light-source. Typically the UV source, in combination with a built-in filter, provides light with an excitation maximum of around 254, 300 or 360 nm. The UV light causes the DNA-bound dye to fluoresce in the red (ethidium bromide) or green (SYBR Green I) regions of the visible light spectrum. The colored fluorescence allows visualization and localization of the DNA fragments in the gel. The visualization of DNA in a gel is used either to assess the success of a gene cloning reaction as judged by the size and number of DNA fragments present, or to identify a particular sized fragment which can be cut out from the gel and used in further reaction steps.
Transilluminators used in the art to visualize fluorophors are described in a number of patents, including U.S. Pat. Nos. 5,347,342, 5,387,801, 5,327,195, 4,657,655, and 4,071,883. Clinical examination of skin anomalies causing fluorescence have been described in U.S. Pat. No. 5,363,854 using visible light images as a control.
The use of UV light for viewing molecules in gels has two major disadvantages: (1) It is dangerous. The eyes are very sensitive to UV light and it is an absolute necessity that the viewer wear eye-protection, even for brief viewing periods, to prevent the possibility of serious damage. More prolonged exposure to UV light results in damage to the skin tissues (sunburn) and care must be taken to minimize skin exposure by wearing gloves, long-sleeved jackets and a full-face mask. (2) DNA samples are damaged by exposure to UV light. It has recently been documented by Epicentre Technologies that a 10-20 second exposure to 305 nm UV light on a transilluminator is sufficient to cause extensive damage to the DNA. This period of time is the absolute minimum required to excise a DNA band from a gel.
An alternative to UV transillumination involves the use of laser light sources. However, the use of laser light is not applicable to the simple and direct viewing of a DNA gel by the human eye. The extremely small cross-section of the laser light beam requires that a typical DNA gel be scanned by the laser, the fluorescence intensity at each point measured electronically and stored digitally before a composite picture of the DNA gel is assembled for viewing using computer software.
Visible light boxes for artists' uses are known to the art for visualizing non-fluorescing materials, e.g., as described in U.S. Pat. No. 3,802,102. The use of visible light to detect certain fluorescent dyes is suggested, e.g., in Lightools Research web page. However, no enabling disclosure for making such devices is provided. None of these references provides devices or systems for viewing fluorescence patterns using visible light.
Despite the recent development of dyes fluorescing in the visible spectrum (Haugland, R. [1996] “Handbook of Fluorescent Probes and Research Chemicals, Sixth Edition,” Molecular Probes, Inc., Eugene, OR, pp. 13-18, 25-29, 29-35), transilluminators and other devices to take advantage of the properties of such dyes have not been made available to the public. It is an object of this invention to provide devices and methods for directly and indirectly viewing and measuring patterns of fluorescence not involving the use of UV transillumination but rather being capable of using sources of visible light such as ordinary lamps, as opposed to lasers and the focused lights used in standard fluorometers.
All publications referred to herein are incorporated by reference.
SUMMARY
Avisible light system is provided for detection of patterns of fluorescence emitted by fluorophors capable of emitting light of an emitted wavelength range (emission spectrum) when excited by light of an excitation wavelength range (excitation spectrum). In one embodiment, the excitation wavelength range must be different from the emitted wavelength range, although these ranges may overlap, and at least a portion of the non-overlapping portion of the emitted wavelength range must be within the visible spectrum. Both the exciting and emitted wavelength ranges are within the visible spectrum.
In preferred embodiments, using color filters, light of the “excitation type” for the fluorophor is light within the excitation wavelength range for the fluorophor, and light of the “emitted type” is light within the emitted wavelength range for the fluorophor. The first filter preferably transmits at least about 70% of the light from the light source in the excitation wavelength range, and the second filter transmits at least about 95% of the light in the emitted wavelength range. The term “filter” as used herein includes combinations of filters.
In other embodiments using polarizing filters, the first filter transmits the light from the source in a narrow range of orientations, and the second filter is oriented to exclude light from the source, i.e., transmits only light orthogonal to that passed by the first filter, so that only light emitted by the fluorophor passes through the second filter.
This invention comprises a visible light system comprising:
a) a light source capable of producing visible light of the excitation type for the fluorophors;
b) a first optical filter placed between said light source and said fluorophors, which is capable of transmitting light from said light source of the excitation type for said fluorophors and of preventing transmission of at least a portion of the light from said light source of said emitted type; and
c) a second optical filter placed between said fluorophors and a light detector which second filter is capable of transmitting light of said emitted type and of preventing transmission of light from said light source of said excitation type, to form a viewable image of the pattern of fluorophors.
The fluorophors may be any fluorophors known or readily available to those skilled in the art, and are preferably used in the form of fluorophors bound to or in a biological sample. Fluorophors may be used to detect and quantify any desired substance to which they can be attached or into which they can be incorporated, e.g. organic molecules such as proteins, nucleic acids, carbohydrates, pigments, and dyes, inorganic molecules such as minerals, bacteria, eukaryotic cells, tissues and organisms. Fluorophors may also be an intrinsic part of an organism or substance to be detected, e.g., various dyes and pigments found in, for example, fungi, fish, bacteria and minerals.
The system of this invention may be incorporated into an integrated device such as a horizontal or vertical gel electrophoresis unit, scanner or other device in which detection of fluorescence is required.
The devices and methods of this invention are especially useful for viewing patterns, i.e., two-dimensional and three-dimensional spatial arrangements of fluorophors. Fluorescence detectors such as found in fluorometers are able to detect only the presence and intensity of fluorescence, and rather than generating an image generate a stream of data which must be interpreted by machine. The present invention allows direct viewing of two-dimensional (or three-dimensional) patterns of fluorophors by the human eye. Such patterns o
Clare Chemical Research, Inc.
Greenlee Winner and Sullivan P.C.
Hannaher Constantine
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