Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-11-02
2001-06-26
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S183000, C435S091100, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06251661
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to seamless capsules in which biopolymer can be synthesized, and a production of the seamless capsules.
BACKGROUND OF THE INVENTION
Hitherto, there have been known many techniques for amplifying DNA and RNA and for synthesizing protein. The techniques, for example, include a technique for amplifying DNA fragments of a total volume of 5 to 100 &mgr;L in a plastic reactor, using DNA polymerase as an enzymatic catalyst, or one for synthesizing protein in the. similar volume without an organism.
In the present specification, the amplifying technique performed in a volume of lower than several microliters (e.g. about 5 &mgr;L) is referred to as “reaction in microspace”. The amplifying technique called as “reaction in microspace” can complete the reaction in a very small volume and increase its reactivity.
Recently, there has been intensely studied a method for amplifying DNA fragments using enzyme in an artificially created microspace, because it is more effective than the methods conducted through chemical reaction or using organisms The method conducted through chemical reaction has a limit in reactivity and is very difficult to synthesize long DNA fragments. In the case of the amplification using organisms, it is very difficult to synthesize long DNA fragments containing nucleotide analogues because of employing merely nucleic acids naturally oriented. The amplification using organisms also has some problems, for example, the difficulty in screening from the organism to find useful substance. If a conventional small test tube is used as a reactor, it takes a long time to dispense the starting materials into each test tube. In view of the size of the amplifying and screening facilities, the amplification in the test tube needs a large space for reacting and screening.
The method using the artificially created microspace, however, is simple and useful and does not have the above-mentioned problems, in comparison with the methods conducted through chemical reaction or using organisms.
One example of the artificially created microspace is a microcapsule formed from polypyrrole. The polypyrrole capsule is used in the amplification of DNA fragments by encapsulating DNA fragment amplifying components with polypyrrole film using interfacial polymerization. However, the polypyrrole belongs to synthetic polymer and therefore does not have sufficient biocompatibility.
Another method is proposed in which liposome is employed as encapsulating film instead of polypyrrole. Liposome is a bilayer lipid membrane which can be artificially obtained and has similar composition to the cell membrane. It is therefore believed to have high biocompatibility. However, in encapsulating with the liposome, it is difficult to control a size of the capsules. In addition, since the liposome capsules are very soft and fragile, the capsules are easily broken by physical impact given during working, e.g. pinching with a tweezers, and the like.
OBJECT OF THE INVENTION
An object of the present invention is to provide a seamless capsule formed from a novel outer covering material, in order to synthesize or amplify a DNA fragment, a RNA and a peptide, as well as a protein and the like. The seamless capsule can solve the above problems in both the polypyrrole capsule and the liposome capsule.
DEFINITION OF TERMS
Herein, the term “amplification” includes not only amplification of DNA by polymerase chain reaction, that is abbreviated as “PCR”, but also includes a transcription of the amplified DNA into RNA and a reverse transcription of RNA into DNA. The term “synthesis” includes the amplification defined above, and a synthesis of protein using the amplified DNA or RNA.
A “cell free protein synthesizing system” means a cell extraction extracted from an organism containing well-known three types of RNA polymerases (i.e. type I, II and III) or the other polymerase, which includes approximately all of components necessary for synthesizing protein.
SUMMARY OF THE INVENTION
The present invention provides a seamless capsule for synthesizing biopolymer, comprising:
(a) an aqueous mixture for synthesizing biopolymer,
(b) a seamless capsule layer encapsulating said aqueous mixture, formed from polysaccharide or protein, and,
(c) a viscous liquid intermediate layer, present between said aqueous mixture and said seamless capsule layer, which is immiscible with water,
wherein said seamless capsule has a diameter of 0.01 to 10.0 mm.
In the present invention, the intermediate layer is formed from a viscous liquid intermediate layer which is immiscible with water and is present between the aqueous mixture for synthesizing biopolymer and the seamless capsule layer by using the technique as described in Japanese Kokai Publication Hei 5 (1993)-31352. The presence of the viscous liquid intermediate layer makes it possible to encapsulate the aqueous mixture for synthesizing biopolymer within the capsule layer.
In more detail, the present invention provides a seamless capsule which comprises the aqueous mixture for synthesizing biopolymer, composed of a template DNA or RNA, primers, substrates and DNA polymerase; the seamless capsule layer; and the intermediate layer. In this seamless capsule, DNA or RNA is amplified by the PCR.
The present invention also provides a seamless capsule which comprises the aqueous mixture for synthesizing biopolymer, composed of a template DNA or RNA, lower molecular components for synthesizing a protein, and a cell free protein synthesizing system; the seamless capsule layer; and the intermediate layer. In this capsule, protein is synthesized.
Furthermore, the present invention provides a seamless capsule which comprises the aqueous mixture for synthesizing biopolymer, composed of a template DNA or RNA, primers, substrates, lower molecular components for synthesizing a protein, DNA polymerase and a cell free protein synthesizing system; the seamless capsule layer; and the intermediate layer. In this capsule, amplification of DNA by the PCR is followed by synthesis of a protein with the amplified DNA.
The present invention can also provide a method for producing a seamless capsule for synthesizing biopolymer therein, comprising simultaneously extruding three different solutions through three nozzles arranged concentrically into a cooling solution, wherein the innermost nozzle extrudes an aqueous mixture for synthesizing biopolymer, the intermediate nozzle extrudes a viscous liquid immiscible with water and the outer nozzle extrudes a capsule layer-forming solution formed from polysaccharide or protein.
The seamless capsule layer of the seamless capsule of the present invention is formed from a polysaccharide, such as curdlan and/or agarose; or a protein. These have good biocompatibility and high light transmittance.
The seamless capsule of the present invention can be formed uniformly by suitably controlling the size of the capsule and the amount of an aqueous mixture during producing The seamless capsule is distinguished from the conventional liposome capsule in good thermostability and high physical strength.
REFERENCES:
patent: 5330835 (1994-07-01), Kikuchi et al.
patent: 5-31352 (1993-02-01), None
Hatano Yumi
Kamaguchi Ryosei
Sunohara Hideki
Urabe Itaru
Yamamoto Keizo
Merchant & Gould P.C.
Morishita Jintan Co., Ltd.
Sisson Bradley L.
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