Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1994-03-15
2003-06-10
Jones, Dwayne C. (Department: 1614)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S452000, C204S455000, C204S456000, C204S461000, C204S601000, C204S603000, C204S605000, C204S606000, C204S612000
Reexamination Certificate
active
06576104
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an apparatus and a method for reading a gel electrophoresis pattern and, more particularly, to a gel electrophoresis pattern reading apparatus and a gel electrophoresis pattern reading method so adapted as to read a gel electrophoresis pattern with high sensitivity to detection of the electrophoresis pattern without requiring any expensive device configuration such as a special laser light source, the gel electrophoresis pattern being obtained by subjecting a sample containing nucleic acids or protein acids to electrophoresis in gel and separating the sample from the gel material.
2. Description of the Related Art
Heretofore, techniques for analysis by means of gel electrophoresis methods have been extensively utilized for the fragmentation and for the analysis of structures of protein acids and nucleic acids present as a polymer in the body of an animal or plant. The gel electrophoresis method has the advantage that a minute amount of a sample can be analyzed in an appropriate manner so that in many cases the gel electrophoresis method is utilized for experiments in which a sample can be procured in a very limited amount. Therefore, the analysis using the gel electrophoresis method requires a very high sensitivity to detection of the sample.
In conventional techniques, a sample to be analyzed is first labelled with a radioactive isotope, the labelled sample is injected into a gel, and the resulting gel material is subjected to electrophoresis. After the gel electrophoresis, the gel is attached to an X-ray film or the like for exposure to X rays to be emitted from the radioactive isotope labelled in the sample. After exposure of the gel material containing the labelled sample to the X-ray film or the like for a given period of time, the X-ray film is then developed, and the exposed pattern resulting from the radioactive isotope transcribed onto the X-ray film is read as a gel electrophoresis pattern in order to analyze the structures of the proteins or nucleic acids of the sample.
The radioactive isotopes, however, are so hazardous that they should be handled and managed with extreme care and with high security. Recently, techniques for handling laser light and technologies of laser light sources, optical sensors and signal processing have greatly developed leading to the development of fluorescence detecting methods for detecting an electrophoresis pattern without requiring the use of such hazardous radioactive isotopes. The fluorescence detecting methods comprise labelling a sample with a fluorescent substance, subjecting the sample to electrophoresis, exciting the labelled fluorescent substance directly with a laser light source, and detecting the resulting fluorescent pattern, thereby resulting in the detection of the electrophoresis pattern.
As an example of reading an electrophoresis pattern by the fluorescence detecting method, which has initially been developed, there is known a method for determining the sequence of a DNA as disclosed in Japanese Patent Unexamined Publication Kokai No. 61-173,158. An outline of a fluorescence detecting method utilized for the DNA sequence determining method will be described with reference to
FIGS. 4 and 5
, showing a schematic representation of the method for the detection of a gel electrophoresis pattern by the fluorescence detecting method, in which
FIG. 4
is a block diagram showing an outline of a gel electrophoresis apparatus and
FIG. 5
shows the details of a portion of a fluorescence detecting section of the gel electrophoresis apparatus of FIG.
4
.
First, a description will be made of the device structure of the gel electrophoresis apparatus with reference to FIG.
4
. The gel electrophoresis apparatus comprises a gel material
31
in which electrophoresis is to be conducted, a fine tube
32
for retaining the gel material
31
, an upper buffer solution container or tank
33
and a lower buffer solution container or tank
34
, between which an electrical field is applied to the gel material
31
held in the fine tube
32
, a first electrode
35
a,
a second electrode
35
b,
a light source
36
for exciting a fluorescent substance labelled in the electrophoresed sample, a detector
37
for detecting the fluorescence emitted from the sample, a data processing section
38
for processing signals transmitted from the detector
37
and converting the fluorescent signals into electrical signals, and an electric power source
39
for applying the electrophoresis electrical field between the first electrode
35
a
and the second electrode
35
b.
Next, an operation of the gel electrophoresis apparatus will be described by example where a DNA sample is electrophoresed as an object of electrophoresis and an electrophoresis pattern of the DNA sample is read with the gel electrophoresis apparatus. The DNA sample is first labelled with a fluorescent substance and the labelled sample is poured into the upper buffer solution container
33
from which the sample in turn is introduced into the fine tube
32
, and an electrophoresis voltage of from several kV to approximately 10 kV is applied from the electric power source
39
between the first and second electrodes
35
a
and
35
b.
As the DNA has negative charges, they migrate toward the positive electrode of the second electrode
35
b
upon application of such electrophoresis voltage and they eventually reach the position of the light source
36
. Thereafter, the fluorescent substance labelled in the DNA sample is excited in this position upon exposure to laser beams emitted from the light source
36
, thereby emitting fluorescence that in turn is detected and received by the detector
37
. The fluorescence received by the detector
37
is then converted into electrical signals and the detector
37
transmits the electrical signals to the data processing section
38
which in turn processes the electrical signals and determines the sequences of the DNA fragments separated by their molecular weights, thereby yielding an electrophoresis pattern.
The detector
37
is arranged such that the fluorescence emitted from the sample in the gel material
31
migrating within the fine tube
32
can be received in a manner as shown in
FIG. 5
, which is a partially transverse sectional view (looking down). As shown in
FIG. 5
, when the sample migrates downward through the gel material
31
and the sample reaches the position of the fine tube
32
which is irradiated with laser beams
40
emitted from the light source
36
, the fluorescent substance labelled in the DNAs of the sample is excited with the laser beams
40
, thereby resulting in the emission of fluorescence
41
that in turn is received by the detector
37
. The received fluorescence
41
is then transmitted to a photomultiplier of the detector
37
and the photomultiplier converts the fluorescence
41
into electrical signals and transmits the electrical signals to the data processing unit
38
. The data processing unit
38
is arranged such that the sequences of the DNA fragments in the sample are determined by the molecular weights on the basis of the peak positions of the intensity of the fluorescence
41
emitted from the DNA sample and received by the detector
37
.
When they are employed as a sample, DNA fragments are labelled with the fluorescent substance so as to have different fluorescent wavelengths corresponding to their ingredients, i.e. four bases comprising adenine, cytosine, guanine and thymine, and to determine the DNA sequences of the four bases simultaneously by causing the DNA sample to migrate down through only one fine tube. The fluorescent substance with which to label the DNA fragments, which can emit four different fluorescent wavelengths, may include, for example, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (EITC), tetramethylrhodamine isothiocyanate (TMRITC), and substituted rhodamine isothiocyanate (XRITC), respectively. Further, the gel electrophoresis apparatus of this type has a sensitivity to detect
Fujimiya Hitoshi
Nakamura Yoshitaka
Nasu Hisanori
Yamamoto Kenji
Delacroix-Muirheid C.
Hitachi Engineering Software Co., Ltd.
Jones Dwayne C.
Mattingly Stanger & Malur, P.C.
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