Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1999-02-03
2002-09-24
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C422S105000, C436S525000, C435S286400, C435S286500, C435S287700, C435S287800, C435S309100
Reexamination Certificate
active
06455325
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a liquid processing method making use of a pipette device and an apparatus for the same, with which works including for quantifying, separating, taking out, pipetting, clarifying, condensing, diluting a liquid or a target high molecular substances included in a liquid such as useful substances such as antibiotic substance, genetic substances such as DNA, and immunological substances such as antibodies, and/or works for extracting, recovering and isolating the target high molecular substance can automatically and accurately be executed by means of absorbing and discharging the liquid through a liquid absorbing/discharging line in the pipette device.
BACKGROUND ART
In recent years, research activities for DNA or the like are very active in many fields including engineering, medical science, agriculture, physical science, pharmacology, and the purpose includes genome sequencing, clinical diagnosis, improvement of agricultural products, bacteriological inspection of foods, drug preparing systems or the like.
As described above, when various types of immunoassay applicable in a very wide range with high expected possibility in its application or structural analysis of molecular level organisms, microorganisms, or substances such as cells, DNA, RNA, mRNA, plasmid, virus, or bacteria (described simply as a target high molecular substance in the present specification) is performed, it is necessary to carry out with high precision works such as those for quantifying, separating, taking out, pipetting, clarifying, condensing, diluting a sample or a target high molecular substance included in the sample or works for extracting, recovering, or isolating a target high-molecular substance as a preprocessing.
To explain structural analysis of a gene such as DNA diagnosis as an example, at first it is necessary to extract, recover, and isolate a DNA region including a target gene. The technology for extracting, recovering, and isolating genes has already been established as the gene cloning technology or genome sequencing technology, and at present it is believed that, by spending enough time and expenses, any gene can be separated and obtained. For this reason, if a target gene DNA has been extracted, recovered, and separated, any type of gene analysis is possible as a principle by making use of the separated gene DNA.
However, in a case of man, for instance, a particular target gene DNA is one millionth or below of all genome DNA, and for this reason actually a quantity of DNA obtainable for testing is quite small, while a quantity of DNA and RNA not necessary for a particular experiment is quite large, which makes it difficult to execute analysis smoothly.
For the reasons as described above, to execute structural analysis of a gene such as DNA diagnosis, it is important to extract, recover, and isolate a DNA area including a target gene. Description is made hereinafter for a basic method of extracting, recovering, and isolating a DNA.
A DNA exist in a nucleus as a complex with a protein in a cell. In the basic sequence for extracting a DNA, a cell or a cell nucleus is processed with SDS (surfactant dodecil sodium sulfate) to make the DNA soluble, and proteins contained therein are removed with a proteolytic enzyme or phenol.
In other words, when a DNA is separated from the tissue, at first the tissue taken out is put in ice and kept therein for a certain period of time under a low temperature, then this cooled tissue is divided to small pieces each with the weight of around 0.1 g, which are washed with a ice-cooled buffer solution A (0.01 M Tris HCl, pH 7.8, 0.1 M NaCl, 2 mM MgCl
2
). This tissue is put in the above-described buffer solution A having a volume 20 times larger as compared to that of the tissue and is homogenized 5 or 10 times with a Potter type homogenizer. Then the tissue is put in a centrifugal tube together with the buffer solution and is subjected to centrifugation (2,000 rpm, for 5 minutes). The cell nucleus or the cell precipitates, so that the supernatant is aborted. When extracting a DNA from cultured cells, the cells are well suspended in an ice-cooled buffer solution B (0.01 M Tris HCl, pH 7.8, 0.1 M NaCl, 2 mM EDTA) and is subjected to centrifugation. The precipitated nuclei or cells are again well suspended in the buffer solution B having a volume 100 times larger than that of the specimen.
After the cells or cell nuclei are well suspended until a block of cells disappears, a 10% SDS solution is added by one twentieth thereof to lyse the cells. Then proteinase K (10 mg/ml) is added by one fiftieth thereof to the solution and reacted for 4 hours under a temperature of 50° C. so that the protein is lysed. During this reaction, the solution is sometimes agitated because the viscosity is high. Then phenol extraction is executed 3 times. In this step, the extracting work should be performed carefully so that no physical power is not loaded thereto.
Then the specimen is dialyzed for around 18 hours with a buffer solution C (10 mM Tris HCl, pH 7.8/0.1 mM EDTA) having a volume 100 times larger as compared to that of the specimen, and is kept under a temperature of 4° C.
Through the steps as described above, about 0.2 mg of DNA can be obtained from 0.1 g of tissue. What is described above is a process of extracting DNA from tissue or cells, and in addition there have been known a method in which plasmid DNA is obtained by way of the alkali method (the small quantity adjusting method), a method of recovering DNA by way of the boiling method, and a method of recovering closed-cycle bromide DNA by way of the large quantity adjusting method.
As described above, it is possible to extract, recover, and isolate DNA for structural analysis of a gene in, for instance, DNA diagnosis according to any of the known methods as described above, but a work for isolating DNA from the tissue or cells as described above is, as clearly understood from the sequence for extracting DNA from the tissue or cells as described above, extremely complicated, and a long period of time is required, which is disadvantageous.
In addition, any of various types of method including the centrifuging method, high speed liquid chromatography method, gel electrophoresis method, dispo-column method, dialysis method, glass powder method, magnetic particle cleaning nozzle method has been employed for structural analysis of DNA or the like extracted by the above-described means, and each of the methods has respective advantages and disadvantages, and at present a high precision and stable method for structural analysis has not been developed yet.
Namely, in a case of centrifugation, automation of processes for loading and taking out vessels is very difficult, and also it is very difficult to mechanically separate supernatant from precipitates after centrifugation, and for this reason its applicability for various purpose is disadvantageously poor.
In a case of high speed liquid chromatography, a separation column is basically consumable, injection for a sample to the column or time management for separation can not be mechanized, and also different samples pass through the column, which disadvantageously makes it impossible to completely prevent contamination of the column.
Furthermore, in a case of gel electrophoresis, adjustment of gel can not be mechanized, and this method has generally been used as a basic technique for separation of DNA, but the separated pieces must be taken out manually, which is disadvantageous.
The dispo-column method is one of technic which can be embodied as a kit for separation of a particular DNA piece, but the cost is very expensive, and its applicability is narrow. In addition, controls over pipetting and liquid passing through the column are difficult, and there are many problems in mechanization of this method.
In the dialysis method, a long period of time is required for dialysis, and also it is hard to apply this method when a quantity of sample is small, so that this method has not been used widely.
The glass powder method is an
Chin Christopher L.
Do Pensee
Precision System Science Co., Ltd.
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