Sample loading sheet

Details Sample loading sheet Sample loading sheet

2000-02-08

2002-04-23

Sisson, Bradley L. (Department: 1655)

Chemistry: molecular biology and microbiology

Apparatus

Including measuring or testing

C435S287200, C435S006120, C436S094000, C536S023100, C536S024300, C204S456000, C422S068100

Reexamination Certificate

active

06376231

ABSTRACT:

BACKGROUND OF INVENTION
This invention relates to a sample loading sheet for loading an assay sample in specified lane positions in a gel electrolyte layer in an electrophoresis plate to be used in a gel electrophoretic apparatus. More particularly, the invention relates to a sample loading sheet for loading an assay sample in specified lane positions in a gel electrolyte layer in an electrophoresis plate to be used in a gel electrophoretic apparatus such as a DNA base sequencer capable of determining the base sequences of DNA by fluorescence labeling in an efficient and rapid manner.
Gel electrophoresis is practiced extensively as a technique for determining the base sequences of DNA and other proteins.
Conventionally, the sample to be subjected to electrophoresis is labelled with a radioisotope for analysis but this method has had the problem of being painstaking and time-consuming. Furthermore, the use of radioactive substances always calls for utmost safety and management and analysis cannot be performed in areas other than facilities that clear certain regulations. Under the circumstances, a method that uses fluorophores to label the sample and which detects fluorescences as emitted upon irradiation with light is being reviewed.
In this method, fluorophore-labelled DNA fragments are caused to migrate through a gel and a light excitation portion and a photodetector are provided for each electrophoresis track in an area 10-70 cm below the start point of electrophoresis. The DNA fragments are assayed as they pass through the line connecting the light excitation portion and the photodetector. A typical procedure of the method is described below. First, using as a template the DNA chain to be determined for its base sequence, DNAs of various lengths with known terminal base species are replicated by a method involving an enzymatic reaction (the dideoxy method). Then, the replicated DNAs are labelled with a fluorophore. Stated more specifically, there are prepared a group of adenine (A) fragments, a group of cytosine (C) fragments, a group of guanine (G) fragments and a group of thymine (T) fragments, all being labelled with a fluorophore. A mixture of these fragment groups is injected into separate lane grooves in an electrophoretic gel and, thereafter, a voltage is applied at opposite ends of the gel. Since DNA is a chained polymer with negative charges, it will move across the gel at a rate in inverse proportion to its molecular weight. The shorter the DNA chain (the smaller its molecular weight), the faster will it move and vice versa; this is the principle behind the fractionation of DNA by molecular weight.
Japanese Laid-Open Patent Application (kokai) No. 21556/1988 teaches a DNA base sequencer that is adapted in such a way that a line on the gel in an apparatus for electrophoresis at which laser light is applied and the direction in which photodiodes are arranged are both perpendicular to the direction in which DNA fragments migrate in the apparatus.
The setup of this apparatus is shown schematically in FIG.
14
. In the apparatus shown in
FIG. 14
, a laser beam emitted from a light source
70
is reflected by a mirror
72
and launched horizontally from one side of an electrophoresis plate
74
at a predetermined point on the gel. As the fluorescence-labelled DNA fragments migrating through the gel pass through the irradiated region, they will fluoresce successively. The horizontal position of fluorescence emission tells the species of a particular terminal base, the time difference from the start of migration tells the length of a particular fragment, and the emission wavelength identifies the sample under assay. The fluorescence from each electrophoresis track is condensed by a lens
78
to focus at a light-receiving area
82
in an image intensifier
80
. The received signal is amplified and converted to an electric signal in a photodiode array
84
for the purpose of various measurements. The results of measurements are processed with a computer so that the sequences of the individual DNA fragments are calculated to determine the base sequence of the DNA at issue.
As shown in
FIG. 15
, the electrophoresis plate
74
comprises a pair of glass plates
86
and
88
between which is held a gel electrolyte layer
90
made of an electrophoresing gel (e.g. polyacrylamide gel). To regulate the thickness of the gel electrolyte layer
90
, a spacer
92
is provided between the two glass plates along both vertical edges. The top edge of the glass plate
88
is cut away in a specified depth across the entire width except both lateral ends. The resulting cutout
94
provides access for a buffer solution to make contact with the top edge of gel electrolyte layer
90
. The electrophoresis plate
74
has an overall thickness of about 10 mm but the thickness of the gel electrolyte layer itself is only about 0.3 mm. The upper edge of the gel electrolyte layer is comb-shaped (i.e., has indentations) and located substantially flush with the bottom
96
of the cutout
94
. Fluorophore-labelled DNA fragments to be assayed are injected into grooves
75
between the teeth of the comb.
Each of the grooves
75
into which the DNA fragments are to be injected has a width of about 1.5 mm and a depth of no more than about 5 mm. Two grooves are spaced apart by a distance of about 2 mm. Such small dimensions require that a fine glass tube, such as a capillary, be used to inject the samples into the grooves
75
. However, due to the transparency of the glass plates and the gel electrolyte, identifying or determining the positions of the individual grooves
75
is extremely difficult and the failure to inject the samples into the right grooves has been frequent.
To support the injection of DNA samples, a sharktooth comb of the shape shown in
FIG. 16
has been developed and used. The sharktooth comb is described in U.S. Pat. No. 5,744,097 which was issued to Machida et al. on Apr. 28, 1998 and herein incorporated by reference. The sharktooth comb indicated by
110
in
FIG. 16
has a series of teeth
113
formed on one of its longer sides. The sharktooth comb
110
may be made of a water-swellable material such as paper. As shown in
FIG. 17
, the sharktooth comb
110
is inserted, usually from the top edge of the electrophoresis plate
74
, into the gap between the two glass plates. The tips of the teeth
113
of the sharktooth comb
110
are slightly urged into the gel electrolyte layer. When the sharktooth comb
110
is immersed in a buffer solution, they swell and close all gaps present between the two glass plates. As a result, adjacent teeth
113
form walls that isolate two adjacent sample loading zones that are defined by spaces
115
.
FIG. 18
is a section taken on line XVIII—XVIII of FIG.
17
. As shown, the top edge of the glass plate
88
which combines with the other glass plate
86
to form the electrophoresis plate is partly cut away in the longitudinal direction. Since the teeth
113
have a specified length, an opening
117
is formed between the root of each tooth and the bottom edge
96
of the cutout
94
in the glass plate
88
. In the actual sample injecting process, the operator
13
looking through the glass plate
86
inserts the tip of a micro-injecting device
119
such as a capillary or plate chip into the sample loading space
115
via the opening
117
and injects a liquid sample
121
. This process involves extreme difficulty in checking the right injecting site through the glass plate and occasionally suffers from the problem of clogging of the injection device
119
. The problem of clogging can be avoided by substituting a micropipette but its use is not practically feasible since the openings
117
are very difficult to see and physically too small for the micropipette to be inserted.
To determine the base sequences of DNA, the four bases that compose the DNA, i.e., adenine (A), guanine (G), cytosine (C) and thymine (T), must be detected according to the correct order. A failure in sample injection is most likely to cause an error in the result of analysis. Hen

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