Capillary electrophoresis system

Details Capillary electrophoresis system Capillary electrophoresis system

2000-10-26

2003-10-21

Bell, Mark L. (Department: 1755)

Chemistry: electrical and wave energy

Apparatus

Electrophoretic or electro-osmotic apparatus

C204S600000

Reexamination Certificate

active

06635164

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention pertains to the field of analysis and separation of DNA, RNA and proteins. In particular, the present invention refers to an apparatus that allows for the separation and analysis of DNA, RNA and proteins.
2. Description of the Related Art
This invention concerns an apparatus for separating and analyzing DNA, RNA and proteins. Particularly, it relates to a capillary electrophoresis system effective to sequencing of DNA and RNA or, measurement of polymorphism, based on the versatility of individuals.
Analyzing technology for DNA and RNA has become important more and more in medical and biological fields concerning DNA analysis and DNA diagnosis. DNA analyzers at high speed and high throughput have been developed in relation with genome projects.
Capillary electrophoresis is a method of high speed and high resolution since higher electric fields can be applied due to high electric resistance and high efficiency of heat dissipation compared with slab gel electrophoresis. (Anal. Chem. 62, 900 (1999) (Prior art 1)).
As shown in
FIG. 16
, an electrophoresis system using a single capillary is known (Prior art 2: WO95/21378). The prior art 2 includes the following descriptions regarding FIG.
1
. Nucleic acid fragments are electrophoretically separated in a capillary
134
filled with a flowing polymer solution as an electrophoretic separation medium. The temperature of the capillary during electrophoresis is an important parameter that gives an effect on both the extent of denaturation of samples and the separation speed of fragments. An elevated temperature can be used for lowering the viscosity of the separation medium during introduction of the separation medium into the capillary, to shorten the time of filling. A temperature control unit
130
is used for keeping the capillary
134
filled with the electrophoretic separation medium to a predetermined temperature. Since the resolution and the electrophoresis time are determined partially based on the length of the capillary, the temperature control unit contains a portion of the capillary for the length from 30 to 35 cm. The temperature of the capillary is controlled by using an insulated pressure plate and bringing the capillary in contact with a thermally controlled surface.
A sample is introduced into the capillary
134
filled with the separation medium. A sample injection end
142
of the capillary
134
and a cathode
150
are immersed in a sample
158
contained in a sample container
126
disposed to an auto-sampler
166
, a sample elution end
138
of the capillary
134
and an anode
146
are immersed in an electrophoretic buffer
154
contained in an electrode vessel
118
and the sample is introduced into the capillary by application of electric field. An injection voltage is applied to the capillary by a power supply unit
122
connected to the anode
146
and the cathode
150
. The injection voltage and injection time are controlled by a computer
174
. After the injection of the sample into the capillary, electrophoretic separation is conducted.
The auto-sampler
176
changes the position for the sample injection end
142
of the capillary
134
and the cathode
150
from the container
126
containing the sample
158
to a container
170
containing an electrophoretic buffer
182
. A voltage is applied to the capillary
124
by the power supply unit
122
connected to the anode
146
and the cathode
150
, and sample ingredients pass the capillary by electrophoresis depending on the size thereof
In order to avoid abnormality in fragment phoresis by siphoning of the electrophoresis medium, it is important to keep the liquid levels of the buffer identical between the container
170
and the container
118
during electrophoretic separation.
The fragment is detected after the separation by a detection unit
162
. The detector used herein can include (1) a unit for spectrally separating an emission light (such as a grating or a prism), (2) an array of a plurality of detection elements sensitive to irradiation of light (e.g., diode array, CCD, photomultiplier), (3) an excited light source (e.g., incandescent lamp, arc lamp, laser, laser diode) and (4) a spectral array fluorescence detector using an optical system enabling directionation and conditioning for both of excited light and emitted light.
Before electrophoretic separation of the next sample, the electrophoresis medium in the capillary is replaced with a new one. There are described two methods of replacing the inside of the capillary with the new electrophoresis medium. In one method, the electrophoresis medium is replaced by the following procedures. A positive pressure from a pump
104
is applied to a container
108
containing a new electrophoresis medium and the medium is pumped out from the vessel
108
to a tee
111
. Since a valve
112
is closed at this instance, the new electrophoresis medium flows mainly to a sample injection end
142
. After the capillary is filled with the new electrophoresis medium between the sample injection end
142
and the tee
111
, the valve
112
is opened and a new electrophoresis medium flows from the container
108
to a container
118
for containing a buffer. As a result, the capillary is completely filled with the electrophoresis medium between the sample injection end
142
and the sampling elution end
138
. Since the length of the capillary between the sample elution end
138
and the tee
111
is shorter than the length of the capillary between the sample injection end
142
and the tee
111
, when the valve
112
is opened to apply a pressure to the container
108
, the new electrophoresis medium flows mainly to the sample elution end
138
. After the capillary is filled with the new electrophoresis medium between the sample injection end
142
and the sample elution end
138
, the valve
112
is opened so as to connect the sample injection end
142
and the sample elution end
138
to form a current path between electrodes
146
and
150
. The foregoing provides an are explanation for
FIG. 1
of the prior art 2.
An electrophoresis system for DNA using a single capillary is supplied as a commercial product from Perkin Elmer Co. (name of product: ABI Prism 310). A high throughput system capable of analyzing a plurality of samples simultaneously, by arranging 96 capillaries into an array is supported as a commercial product from Perkin Elmer Co. (name of product: ABI Prism 3700).
In genome analysis, DNA fragments formed by finely fragmenting large size DNA, at random are read and an original DNA is read by joining the result of reading. As the DNA read length capable of being read by electrophoresis at one time is increased, the efficiency and the speed of the entire analysis also increase.
Accordingly, in the capillary electrophoresis system, a capillary of increased effective length is used so that long DNA can be read. For example, in ABI Prism 310 or ABI Prim 3700, 600 base lengths can be read in about two hours, by electrophoresis under standard conditions (200 V/cm, 50(C), by setting the effective separation length to 50 cm and using a polymer solution POP 6 supplied from Perkin Elmer Co.
As the speed, the throughput and the read length have been increased for the capillary electrophoresis systems, it is expected that the entire base sequencing of the human genome will be completed substantially in 2001. After the base sequences for the entire genome are found, the necessity for reading long DNA will be reduced. It will only be necessary to read specific regions on the genome.
For example, it has now been advanced in a large scale national project to investigate single nucleotide polymorphisms present on the genome, on every group of persons and examine the relationship thereof with diseases or reactivity to chemicals. In the featured medical treatment and new industries, it will be important to relate not only the single nucleotide polymorphism but also specific sequences with diseases or various phenotypes. It is also possible t

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