Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-08-19
2001-03-27
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06207378
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for amplifying nucleic acid molecules and in particular, to a method for amplifying nucleic acid molecules of a single kind. The present invention also pertains to a method for synthesizing a protein using a cell-free protein-synthesis system. Moreover, the present invention further relates to a method for constituting a protein library using the foregoing methods for amplifying nucleic acid molecules and for synthesizing proteins.
To preferentially amplify nucleic acid molecules of a single kind out of many kinds of nucleic acid molecules, there has been adopted a method which makes use of a combination of a plasmid and a host microorganism such as
Escherichia coli
or yeast. This method makes use of such a characteristic of the plasmid that only one plasmid molecule is taken in a host microorganism such as
E. coli
or yeast and comprises the steps of introducing a plasmid into a host microorganism by the calcium chloride method or the electroporation method, isolating-proliferating the microorganism to give a colony of the microorganism containing nucleic acid molecules of a single kind and to thus amplify the nucleic acid molecules of a single kind.
However, for instance, the linkage of a desired nucleic acid molecule to a plasmid, the introduction of the plasmid into a host microorganism and the isolation and proliferation of the microorganism require very complicated operations and as a result, they require the use of an advanced technology and a great deal of expenses and time. In particular, DNA molecules of a single kind should be isolated from vast kinds of DNA molecules in the field wherein industrially useful proteins are produced by altering the base sequences of genetic DNA's coding for amino acid sequences, producing variant proteins whose amino acid sequences are changed by using the resulting DNA variants, but it is very difficult to practice such operations.
Moreover, in the cell-free protein-synthesis system, the operations required for the synthesis of proteins are quite simple as compared with the protein-synthesis system which makes use of, for instance, organisms or living cells such as cultured cells and for this reason, there has recently been desired for the development of such a cell-free system. In addition, the PCR (polymerase chain reaction) technique is an extremely effective and simple means for amplifying DNA genes. Under such circumstances, there has been desired for the development of a technique, which comprises the combination of these two techniques, for easily and directly synthesizing a large amount of proteins using the DNA's which code for the proteins and which are produced by the PCR technique without using any plasmid DNA.
There has already been known a cell-free protein-synthesis system using DNA's produced by the PCR technique (Proc. Natl. Acad. Sci. USA, 1997, 94, pp. 412-417). However, it has been difficult to synthesize a protein in a desired amount and therefore, it has been an important subject to improve the yield of proteins synthesized by this technique.
SUMMARY OF THE INVENTION
Accordingly, it is a first object of the present invention to provide a method for easily and effectively amplifying nucleic acid molecules of a single kind.
It is a second object of the present invention to provide a method for easily producing a protein in a sufficient amount, which makes use of a cell-free protein-synthesis system.
It is a third object of the present invention to provide a method for efficiently and easily constituting a protein library.
The inventors of this invention have conducted various studies to accomplish the foregoing objects, have found that only desired nucleic acid molecules of a single kind can efficiently be amplified by isolating and purifying a group of nucleic acid molecules including the desired nucleic acid molecules to be amplified and carrying out PCR (polymerase chain reaction) procedures at a very low molar concentration of the desired nucleic acid molecules using primers each at a sufficiently low concentration and have thus completed the present invention.
Thus, according to a first aspect of the present invention, there is provided a method for amplifying desired nucleic acid molecules by PCR which comprises the steps of isolating and purifying a group of nucleic acid molecules including the desired nucleic acid molecules to be amplified, then carrying out PCR procedures while establishing such a condition that the nucleic acid molecules capable of being amplified, except for primers, present in a PCR reaction solution are constituted by only the desired nucleic acid molecules and that the concentration of each primer used is limited to a level of not more than 100 nM. In this regard, the term “nucleic acid molecules of a single kind” is herein used to mean DNA, RNA or both represented by base sequence.
According to a second aspect of the present invention, there is provided a method for producing a protein in a cell-free protein-synthesis system containing a cell-free extract, which is characterized by using the nucleic acid of a single kind produced by the foregoing method of the present invention as a template. The term “cell-free protein-synthesis system” used herein means both cell-free translation systems which read the information of mRNA's and synthesize proteins or polypeptides and systems each comprising a cell-free transcription system wherein an RNA is synthesized using a DNA as a template and a cell-free translation system.
According to a further aspect of the present invention, there is provided a method for establishing a protein library which comprises the steps of separately carrying out the foregoing nucleic acid-amplification method of the present invention to obtain at least two kinds of nucleic acid molecules and separately carrying out the foregoing protein-production method of the present invention using each of the at least two kinds of the amplified nucleic acids as a template to thus establish a protein library which comprises at least two kinds of proteins encoded by the resulting at least two amplified nucleic acids respectively.
REFERENCES:
patent: 4683195 (1987-07-01), Mullis et al.
Gyllensten p. 300-306, in PCR Protocols, Ed. Innis et al., Academic Press, Inc., 1990.*
Innis et al., p. 3-12, in PCR Protocols, Ed. Innis et al., Academic Press, Inc., 1990.
Nakano Hideo
Okumura Reiko
Ouchi Masashi
Sekiguchi Satoshi
Yamane Tsuneo
Houtteman Scott W.
Nippon Flour Mills Co., Ltd.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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