Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-30
2001-02-27
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C528S422000, C528S423000, C525S540000, C527S312000, C527S200000
Reexamination Certificate
active
06194157
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for separating a target biological substance.
BACKGROUND ART
The following technologies have been reported as methods for extracting DNA or RNA from a particular single cell or a clump of cells, or from tissue or a portion of tissue.
(1) In one method, a certain portion of cells, a tissue, or a chromosome is cut away using a micro needle while viewed under a microscope. DNA or RNA is extracted from the removed portion. (by S. Noguchi, et al in Cancer Research 54, p1849-1853, 1994 and by Z. Zhuang, et al in American Journal of Pathology vol. 146, No. 3, p620-625)
(2) In another method, a certain cell or tissue is pierced with a micro capillary tube while viewed under a microscope and the contents of the certain cell or tissue are withdrawn through the micro capillary tube. (Proc. Natl. Acad. Sci. USA Vol. 92, p3814-3818, 1995 and in Proc. Natl. Acad. Sci. USA Vol. 89, p3010-3014, 1992)
(3) In a further method, a laser is used to burn off portions other than a target cell or tissue. Afterward, the target cell or tissue is collected and DNA or RNA is extracted from the collected cell or tissue. (Genomics vol. 11, p364-373, 1991)
However, the method using the micro capillary tube requires accurate manipulation of the micro capillary tube under a microscope and so requires a highly-skilled and experienced technician. For this reason, it is difficult to collect the contents of the target cell or tissue only without contamination. Also, although easier manipulation can be anticipated with the method using a laser, it is difficult to completely burn away portions other than the target cell or tissue using a laser. As a result, it is difficult to avoid contamination of the target cell or tissue from surrounding material. For this reason, when DNA or RNA is extracted using the above-described methods and afterward amplified, the contamination will result in an undesirably high background.
It is a purpose of the present invention to provide a simple method for easily separating biological substances, such as certain cells, tissues, biopolymers or low weight biomolecules, for example, biomolecules such as DNA, RNA, protein, or glucose, with little impurities.
DISCLOSURE OF INVENTION
In order to attain the above-described objective, the present invention is characterized by using a photoresist to separate biological substances.
The present invention is also characterized by embedding or encasing a biological sample, such as a cell or a tissue, in photoresist.
Photoresist is a photosensitive resin that is widely used in the production of transistors and integrated circuits.
The present invention provides a method characterized by first fixing a biological sample using a photoresist. Afterward, in order to retrieve a particular biological substance, such as a target cell, biopolymer, or low weight biomolecule, from the biological sample embedded in the photoresist, either the portion of the photoresist covering the biological substance, or all other portions of the photoresist, are selectively exposed to light such as a visible light, ultraviolet light, X-rays, or an electron beam, thereby differentiating the characters of the exposed and unexposed portions of the photoresist. The biological substance embedded in the photoresist is then collected.
According to another aspect, the present invention provides a method characterized by first fixing a biological sample using a positive type photoresist. Afterward, in order to retrieve a particular biological substance, such as a target cell, biopolymer, or low weight biomolecule, from the biological sample embedded in the photoresist, the portion of the photoresist covering the biological substance is exposed to light such as visible light, ultraviolet light, X-rays, or an electron beam, thereby decomposing the photoresist or changing properties of the photoresist (sometimes referred to collectively as “changing properties of the photoresist,” hereinafter). Then, the biological substance embedded in the changed photoresist is collected.
According to still another aspect, the present invention provides a method characterized by first fixing a biological sample using an amphoteric type photoresist. Afterward, in order to retrieve a particular biological substance, such as a target cell, biopolymer, or low weight biomolecule, embedded in the photoresist, a region of the photoresist other than its portion covering the biological substance is exposed to light such as visible light, ultraviolet light, X-rays, or an electron beam, thereby differentiating solubility of the exposed portion of the photoresist with respect to a solvent from solubility of the unexposed portion of the photoresist with respect to the solvent. Next, the exposed photoresist portion and the portion of the biological sample embedded in the exposed photoresist portion are removed based on the difference in solubility. Afterward, the remaining unexposed portion of the photoresist is dissolved in another appropriate solvent and the biological substance embedded in the unexposed portion is collected.
According to still another aspect, the present invention provides a method characterized by first covering a biological sample with a negative type photoresist. Afterward, the biological sample is fixed by solidifying the photoresist (using solvent casting method). Afterward, in order to retrieve a particular biological substance, such as a target cell, biopolymer, or low weight biomolecule, a region of the photoresist other than the portion covering the biological substance is exposed to light such as visible light, ultraviolet light, X-rays, or an electron beam, thereby making the exposed photoresist portion insoluble with respect to a solvent. Next, the biological substance, which is covered in the portion of the photoresist that is not made insoluble with respect to the solvent, is collected.
Thus, the present invention is characterized by using a photoresist for separating a target biological substance.
That is, according to one aspect, the present invention provides a method for separating a biological substance, the method comprising the steps of:
(a) covering, with a photoresist, a biological sample that includes a biological substance;
(b) fixing the biological sample by solidifying the photoresist;
(c) selectively exposing the solidified photoresist to light either at a portion that covers the biological substance or at another portion; and
(d) selectively dissolving, using a solvent, at least one of the portion of the photoresist that is exposed to light during the step (c) and the portion that is not exposed to light, and collecting the biological substance embedded in the photoresist.
No particular limitations are placed on the photoresist used in the present invention. That is, a positive type photoresist, an amphoteric type photoresist, or a negative type photoresist could be used.
A positive type photoresist is a photoresist having a character wherein portions exposed to light become soluble with respect to a solvent.
According to another aspect, therefore, the present invention provides a method for separating a biological substance, the method comprising the steps of:
(a) covering, with a positive type photoresist, a biological sample that includes a biological substance;
(b) fixing the biological sample by solidifying the photoresist (a solvent casting method);
(c) exposing the solidified photoresist to light at a portion that covers the biological substance; and
(d) dissolving, using a solvent, the portion of the photoresist that is exposed to light during the step (c), and collecting the biological substance embedded in the photoresist.
A negative type photoresist is a photoresist having a character wherein portions exposed to light become insoluble with respect to a solvent.
Accordingly, in still another aspect, the present invention provides a method for separating a biological substance, the method comprising the steps of:
(a) covering, with a negative type photoresist, a biological sample that includes a b
Ito Toshiaki
Tachiiri Yoshiaki
Tsuchiya Hiroshi
Chakrabarti Arun
Fredman Jeffrey
Hamamatsu Photonics K.K.
Oliff & Berridg,e PLC
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