Gel charger for capillary column

Details Gel charger for capillary column Gel charger for capillary column

1999-11-19

2002-08-06

Warden, Jill (Department: 1743)

Chemistry: electrical and wave energy

Apparatus

Electrophoretic or electro-osmotic apparatus

C204S451000, C204S453000, C204S455000, C204S604000, C204S605000

Reexamination Certificate

active

06428670

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a gel charger for charging a plurality of capillary columns used in a multi-capillary electrophoretic apparatus with a gel serving as a medium for electrophoresing samples.
2. Description of the Prior Art
An electrophoretic apparatus is used for separating and analyzing protein, peptide, sugar or the like, and plays an important part particularly for analysis of the base sequence of DNA.
A DNA sequencer having high sensitivity, a high speed and high throughput is necessary for sequence determination for DNA such as a human genome having long base sequence. As an example, a multi-capillary electrophoretic apparatus formed by arranging a plurality of capillary columns charged with a gel in place of a flat plate type slab gel is proposed. With such capillary columns, samples can not only be readily handled or injected but also electrophoresed at a high speed, and detected in high sensitivity as compared with the slab gel. If a high voltage is applied to the slab gel, a band is spread or a temperature gradient is caused due to influence by Joulean heat However, the capillary columns hardly cause such a problem and can perform detection in high sensitivity with small band spreading even if performing high-speed electrophoresis with application of a high voltage.
Capillary gel electrophoresis using a supporter (separation medium) having a molecular sieving effect in a glass capillary column having an inner diameter of 10 to 200 &mgr;m separates nucleic acid or protein by the molecular sieving effect of the separation medium. The separation medium is formed by cross-liked polyacrylamide prepared by polymerizing a cross-linking gel in the capillary column or a previously polymerized high polymer such as linear polyacrylamide or hydroxyethyl cellulose charged into the capillary column.
An initial capillary electrophoretic apparatus employs a single capillary column. In this case, an end of the capillary column is dipped in a gel stored in a container, which in turn is closed and pressurized for forcing and charging the gel into the capillary column. Alternatively, an end of the capillary column is dipped in the gel and another end of the capillary column is decompressed for sucking and charging the gel into the capillary column.
In a multi-capillary electrophoretic apparatus, a plurality of capillary columns are mounted on the multi-capillary electrophoretic apparatus in a state two-dimensionally arranged on a sample injection side and aligned with each other on a plane on a detection side. The plurality of capillary columns are preferably set in a capillary cassette in which the arrangement is fixed by a holder, in consideration of operability.
It is extremely troublesome and impossible in practice to charge the capillary columns one by one with a gel in the state of the capillary cassette. Therefore, it is desired to make it possible to readily charge all capillary columns included in a unit capillary cassette with the gel.
In relation to a separation medium polymerizing a gel in a capillary column, the state of the inner wall surface of the capillary column influences the polymerized state of the gel. If the state of the inner wall surface is inferior, the polymerized state of the gel is deteriorated to increase the frequency of generating bubbles in the capillary column during electrophoresis. Furthermore, if the state of the inner wall surface is inferior, frequency that the gel comes out from an end of the capillary column on a sample injection side is increased whether or not the gel is a cross-linked gel or a high polymer. Thus, reproducibility of an electrophoresis result is disadvantageously deteriorated.
While pretreatment of introducing an acidic solution into the capillary column or the like must be performed for solving this problem, it is extremely troublesome and impossible in practice to pretreat each capillary column contained in a capillary cassette.
SUMMARY OF THE INVENTION
Accordingly, an objective of the present invention is to provide a gel charger capable of readily pretreating and charging all capillary columns contained in a capillary cassette with a gel and improving reproducibility of an electrophoresis result in capillary electrophoresis.
The present invention is directed to an apparatus for charging a plurality of capillary columns of a capillary cassette, in which ends of the capillary columns, mounted on a multi-capillary electrophoretic apparatus, on a sample injection side are two-dimensionally arranged through a holding member of a holder and fixed with airtightness between the same and the holding member with a gel.
The capillary columns can be charged with the gel by either suction or pressurization. When charged with the gel, the capillary columns are not sealed one by one and subjected to suction or pressurization, but the holder airtightly fixing the capillary columns seals all capillary columns of the capillary cassette to charge all capillary columns with the gel simultaneously.
A number of silanol groups (—SiOH) are present on inner wall surfaces of glass capillary columns. The states of the silanol groups vary with the capillary columns. The states of the silanol groups remarkably influence a polymerized state of the gel, and hence the difference between the states of the silanol groups varying with the capillary columns vary the polymerized state of the gel.
Therefore, the apparatus according to the present invention introduces acid into the capillary columns before charging the same with the gel or a polymer for arranging the states of the silanol groups on the inner wall surfaces of the capillary columns (hereinafter referred to as acid treatment) and bringing the silanol groups into states suitable for gel polymerization. Consequently, the polymerized state of the gel in the capillary columns can be stabilized. On the other hand, acid inhibits gel polymerization and hence the capillary columns subjected to acid treatment are thereafter washed with pure water. If moisture remains in the capillary columns, gel polymerization is inhibited or the gel concentration is reduced. As a result, the capillary columns are dried with inert gas.
In a system performing gel charging by suction, a gel charger according to the present invention comprises a chamber airtightly fixing ends of capillary columns on a sample injection side, an inert gas supply mechanism supplying inert gas, decompression means decompressing the chamber, and an introduced substance selection mechanism for supplying ends of the capillary columns on a detection side opposite to the sample injection side with inert gas, an acidic solution, pure water or a gel. The chamber is provided on its upper surface with an opening, which in turn is provided with closure means receiving the ends of the capillary columns on the sample injection side therein and closing the opening with a holder, and a gas outlet is further provided. The decompression means is provided on the gas outlet of the chamber. The introduced substance selection mechanism comprises an inert gas supply port supplied with inert gas from the inert gas supply mechanism, an acidic solution container storing an acidic solution, a pure water container storing pure water and a gel container storing a gel, and comprises a mechanism moving the inert gas supply port, the acidic solution container, the pure water container and the gel container as well as ends of the capillary columns on a detection side opposite to the sample injection side so that the ends of the capillary columns on the detection side are inserted in any of the inert gas supply port, the acidic solution container, the pure water container and the gel container.
In operation, the gel charger closes the opening of the chamber with the closure means through the holder of the capillary cassette thereby fixing the detection side of the capillary cassette to the introduced substance selection mechanism. Then the gel charger arranges the acidic solution container, the pure water conta

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