WO1996018099A1

WO1996018099A1 - Arrangement in a gel electrophoresis apparatus

Info

Publication number: WO1996018099A1
Application number: PCT/SE1995/001482
Authority: WO
Inventors: Bengt-Göran Andersson
Original assignee: Pharmacia Biotech Ab
Priority date: 1994-12-08
Filing date: 1995-12-08
Publication date: 1996-06-13
Also published as: SE9404274D0; EP0805979A1

Abstract

In a gel electrophoresis apparatus having a laser which is adapted to emit a laser beam of a predetermined wavelength to excite at least one substance (4) which is labelled with a fluorophore and which is caused to migrate along a gel (1), which is provided between two transparent plates (2, 3), a detector (5) which is located close to one (3) of the plates to detect the fluorescence emitted upon excitation of the fluorophore, and an optical filter (6) which is located between said one (3) of the plates and the detector and which is adapted to block light of said predetermined wavelength, the optical filter (6) is adapted to block also the intrinsic fluorescence of the plates (2, 3).

Description

ARRANGEMENT IN A GEL ELECTROPHORESIS APPARATUS

TECHNICAL FIELD

The present invention relates to an arrangement in a gel electrophoresis apparatus having a laser which is adapted to emit a laser beam of a predetermined wavelength to excite at least one substance which is labelled with a fluorophore, and which is caused to migrate along a gel which is provided between two transparent plates, a detector which is located close to one of the plates to detect the fluorescence emitted upon excitation of the fluorophore, and an optical filter which is located between said one of the plates and the detector, and which is adapted to block light of said predetermined wavelength. BACKGROUND OF THE INVENTION

In DNA sequencing, it is known to use, as fluorophore, i.a. a carbocyanine based dye marketed under the trademark Cy5 by Biological Detection Systems, Inc., Pittsburg, PA, U.S.A.. The dye Cy5 can be excited by means of a laser, e.g. a helium-neon laser, operating at a wavelength of 633 n , or a laser diode of a wavelength between 630 and 650 n .

The fluorescence emitted upon excitation of the dye Cy5 will have a wavelength of between 650 and 720 nm with a maximum at about 670 nm.

In order for light of the laser wavelength not to disturb the detection of the fluorescence emitted by the fluorophore upon excitation, it is known to provide an optical filter between the detector and one of the transparent plates between which the gel is provided, and which most often are made of glass, which filter exhibits a stop band for wavelengths below the wavelength of the fluorescence including the laser wavelength.

However, it has turned out that the transparent plates, most often of glass, as such constitute a major disturbing source in the detection of the fluorescence emitted by the fluorophore. BRIEF DESCRIPTION OF THE INVENTION

Therefore, the object of the invention is to bring about an arrangement which makes it possible to eliminate the effect of this disturbing source. This is attained according to the invention in that the optical filter is adapted to block also the intrinsic fluorescence of the plates.

BRIEF DESCRIPTION OF THE DRAWING The invention will be described more in detail below with reference to the appended drawing on which Fig. 1 is a schematic cross-sectional view of a portion of an embodiment of a gel electrophoresis apparatus with an arrangement according to the invention, and Fig. 2 is a schematic diagram of fluorescence and filter characteristics in the embodiment shown in Fig. 1.

PREFERRED EMBODIMENTS In Fig. 1, 1 denotes an electrophoresis gel provided between two plates 2 and 3. The plates 2 and 3 are transparent and are preferably made of glass but can also be made of plastic.

Along a path, not illustrated, in the gel 1, in the embodiment shown, at least one substance 4, labelled with a fluorophore, e.g. the carbocyanine based dye Cy5, mentioned in the introduction, is adapted to migrate in a manner not shown under the influence of an electric field.

At a predetermined location beside the gel 1, a laser, not shown, e.g. a helium-neon laser or a laser diode, is provided to supply into the gel 1, through one of the lateral edges of the gel 1, a laser beam of a predetermined wavelength to excite the fluorophore of the substance 4 migrating along the gel 1.

In Fig. 1, the substance 4 is schematically shown in the position where its fluorophore has been excited by the laser beam, not illustrated, going across the gel 1. To detect the fluorescence emitted upon the excitation of the fluorophore, a detector 5, e.g. a photo diode, is provided adjacent to the glass plate 3 in the embodiment shown. An optical filter 6 is provided between the detector 5 and the plate 3. According to the invention, the optical filter 6 is adapted to block not only light of the wavelength of the laser beam but also light originating from the intrinsic fluorescence of the glass plates 2 and 3 upon the excitation by the laser light.

In Fig. 2, reference numeral 7 designates laser wavelength, 8 is the characteristic of an optical filter according to the invention, 9 designates fluorescence from a fluorophore, and 10 is fluorescence from glass plates.

With reference to Fig. 2, in a preferred embodiment of the arrangement according to the invention using a laser wavelength λ of 633 nm and the carbocyanine based dye Cy5 as fluorophore, the fluorescence from the fluorophore detected by the detector 5 will exhibit a maximum at a wavelength of about 670 nm, while the maximum of the intrinsic fluorescence of the glass plates 2 and 3 will appear at a wavelength of about 770 nm.

According to the invention, the optical filter 6 is designed as a band pass filter to block light of the laser wavelength as well as light originating from the intrinsic fluorescence of the glass plates 2 and 3.

It is to be understood that in a practical embodiment of the gel electrophoresis apparatus schematically illustrated in Fig. 1, a number of detectors 5 and a number of optical filters 6 are provided to detect fluorophore labelled substances migrating along a corresponding number of parallel paths in the gel 1.

In case the carbocyanine based dye Cy5 is used as fluorophore, the optical filter 6 is preferably dimensioned in such a manner that it exhibits a pass band characteristic having a center wavelength of 680±10 nm, a half-width of 40±5 nm and an attenuation which is greater than OD5 between 400 and 640 nm, and between 720 and 800 nm, and greater than OD3 between 800 and 1000 nm, as schematically apparent from the filter characteristic illustrated in Fig. 2. Such filters can be manufactured by i.a. Omega Optical, Inc., Brattleboro, VT, U.S.A.. By using an optical filter in accordance with the invention, a considerably improved signal quality is attained at the same time as a greater freedom is obtained when selecting transparent plates between which the gel is provided.

Claims

1. An arrangement in a gel electrophoresis apparatus having - a laser which is adapted to emit a laser beam of a predetermined wavelength to excite at least one substance (4) which is labelled with a fluorophore, and which is caused to migrate along a gel (1) which is provided between two transparent plates (2, 3), - a detector (5) which is located close to one (3) of the plates to detect the fluorescence emitted upon excitation of the fluorophore, and

- an optical filter (6) which is located between said one (3) of the plates and the detector, and which is adapted to block light of said predetermined wavelength, characterized in that the optical filter (6) is adapted to block also the intrinsic fluorescence of the plates (2, 3).

2. The arrangement according to claim 1, characterized in that the optical filter (6) is a band pass filter.

3. The arrangement according to claim 2, in which the fluorophore is the carbocyanine based dye Cy5, characterized in that the optical filter (6) exhibits a band pass characteristic having a center wavelength of 680±10 nm, a half-width of 40±5 nm, and an attenuation which is greater than OD5 between 400 and 640 nm, and between 720 and 800 nm, and greater than 0D3 between 800 and 1000 nm.