Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-05-22
2004-01-06
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120
Reexamination Certificate
active
06673578
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for DNA synthesis, a kit usable for the method and an article of manufacture, which are useful in the field of genetic engineering, and are capable of shortening a time period needed for polymerase chain reaction (PCR) method.
BACKGROUND ART
The synthesis of DNA is used for various purposes in the research of the field of genetic engineering. Among them, much of the DNA syntheses are carried out by the enzymatic method utilizing DNA polymerase, except for chemical synthesis of a short strand DNA such as an oligonucleotide. Accordingly, the DNA polymerase is highly valuable as reagents for DNA sequencing, DNA labelling, and site-directed mutagenesis.
In addition, recently, with the development of PCR method, thermostable DNA polymerases have attracted attention, and various kinds of DNA polymerases suitable for PCR method have been developed and commercialized.
Furthermore, there is known a method capable of carrying out an efficient DNA synthesis by using a combination of plural DNA polymerases, wherein the efficient DNA synthesis could not be achieved by a single DNA polymerase [
Proc. Natl. Acad. Sci. USA,
91, 5695-5699 (1994)].
The method is a method using a mixture of DNA polymerases for PCR, the mixture comprising DNA polymerase having 3′→5′ exonuclease activity (for example, &agr;-type DNA polymerase derived from
Pyrrococcus furiosus
) and DNA polymerase not having the exonuclease activity (for example, DNA polymerase derived from
Thermus aquaticus
(Taq DNA polymerase), and is known as LA-PCR method.
According to this method, the yield of amplified DNA is increased, as compared with that by conventional PCR using only one kind of DNA polymerase. The method can also amplify long DNA fragment, which could not be amplified by conventional PCR.
Optimum PCR conditions which have been conventionally and generally performed are shown in Table A. The amplification of each DNA is terminated at a reaction time of about 90 minutes in the 1 kbp amplification, at a reaction time of about 268 minutes in the 10 kbp amplification, and at a reaction time of about 478 minutes in the 20 kbp amplification.
TABLE A
Enzyme: TaKaRa Ex Taq
*1
[1.25 U/50 &mgr;l (PCR Reagent Mixture)]
Template DNA:
E. coli
Genomic DNA
*2
[100 ng/50 &mgr;l (PCR Reagent
Mixture)]
Amplification of
94° C.
30 sec.
1 kbp Fragment
55° C.
30 sec.
30 cycles (about 90 minutes)
72° C.
1 min.
Amplification of
98° C.
10 sec.
30 cycles (about 268 minutes)
10 kbp Fragment
68° C.
8 min.
Amplification of
98° C.
10 sec.
30 cycles (about 478 minutes)
20 kbp Fragment
68° C.
15 min.
*1
Product manufactured by Takara Shuzo Co., Ltd.
*2
genome DNA set for LA PCR ™, manufactured by Takara Shuzo Co., Ltd.
Since the PCR method has an ability to amplify a trace amount of DNA into several millions times the amount in a short time period, the PCR method is applied to all sorts of studies, tests and clinical fields including medical science and agriculture. Particularly, the PCR method is powerful in genetic diagnosis of infectious diseases such as cancers and AIDS, and the like. In addition, its application has been extended even to city life, including confirmation of a guilty party, or a parent-and-child relationship by genetic diagnosis; a gene detection of harmful bacteria in foods, and the like.
However, it has become an important problem to further shorten a reaction time period of PCR and to speed up PCR in food examination for which a quick result is demanded, and in a clinical test in which PCR is required to be carried out on a large scale.
Further, PCR procedures are indispensable for DNA chip preparation, genome analysis and the like, so that it is important to improve its efficiency in view of efficiently carrying out a whole research.
DISCLOSURE OF INVENTION
An object of the present invention is to provide a DNA synthesis method, a kit for use in the synthesis method and an article of manufacture, for carrying out rapid PCR with a more shortened time period as compared with conventional PCR.
A first invention of the present invention relates to a DNA synthesis method with a shortened time period required for DNA synthesis by polymerase chain reaction (PCR), characterized in that DNA polymerase is used in an amount effective for providing more than 10 ng of amplified DNA fragments of about 2 kb per 50 &mgr;l of a reaction mixture, when PCR is carried out under the following conditions (A) and (B):
(A) reaction mixture: 50 &mgr;l volume of a reaction mixture comprising DNA polymerase, 1 ng of genomic DNA from
Escherichia coli,
and 10 pmol each of primers Eco-1 and Eco-2 (nucleotide sequences of the primers Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of Sequence Listing, respectively); and having a composition suitable for the DNA polymerases; and
(B) reaction conditions: 35 cycles of PCR, wherein one cycle consists of 99° C., 1 second -66° C., 7 seconds.
A second invention of the present invention relates a kit for DNA synthesis usable for the DNA synthesis method of the first invention of the present invention, characterized in that a PCR reagent mixture which is prepared in accordance with instructions of the kit, comprises a DNA polymerase in an amount effective for providing more than 10 ng of amplified DNA fragments of about 2 kb per 50 &mgr;l of a reaction mixture, when PCR is carried out under the following conditions (A) and (B):
(A) reaction mixture: 50 &mgr;l volume of a reaction mixture comprising DNA polymerase, 1 ng of genomic DNA from
Escherichia coli,
and 10 pmol each of primers Eco-1 and Eco-2 (nucleotide sequences of the primers Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of Sequence Listing, respectively); and having a composition suitable for the DNA polymerase; and
(B) reaction conditions: 35 cycles of PCR, wherein one cycle consists of 99° C., 1 second-66° C., 7 seconds.
A third invention of the present invention relates to an article of manufacture of a PCR agent, comprising packaging material and a PCR reagent contained within the packaging material, wherein the PCR agent comprises DNA polymerases, and wherein a label or instruction indicates that the PCR reagent can be used for PCR in a short time period, the label being attached to the packaging material, and the instruction being enclosed with the packaging material.
BEST MODE FOR CARRYING OUT THE INVENTION
(1) DNA Synthesis Method of the Present Invention
The DNA synthesis method of the present invention is a DNA synthesis method with a shortened time period for synthesizing DNA by PCR method, namely rapid PCR, characterized in that DNA polymerase is used in an amount effective for providing more than 10 ng of amplified DNA fragments of about 2 kb per 50 &mgr;l of reaction mixture, when PCR is carried out under the following conditions (A) and (B):
(A) reaction mixture: 50 &mgr;l volume of a reaction mixture comprising DNA polymerase, 1 ng of genomic DNA from
Escherichia coli,
and 10 pmol each of primers Eco- 1 and Eco-2 (nucleotide sequences of the primers. Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of Sequence Listing, respectively); and having a composition suitable for the DNA polymerase; and
(B) reaction conditions: 35 cycles of PCR, wherein one cycle consists of 99° C., 1 second-66° C., 7 seconds.
According to the DNA synthesis method of the present invention, since “effective amount of DNA polymerase” is used, there can be shortened a time period required in synthesis reaction of complementary strand corresponding to a given size of DNA fragment (extension step), i.e. there can be shortened a time period required in 1 cycle of reaction. As a result, there are exhibited excellent effects that in PCR, a desired DNA fragment can be amplified in a short time period which has not been achieved conventionally, in other words, rapid PCR can be carried out with a shortened total time period required in PCR.
In the present invention, the term “effective amount of DNA polymerase” mean
Asada Kiyozo
Fujita Tomoko
Hagiya Michio
Kato Ikunoshin
Miyake Kazue
Fredman Jeffrey
Takara Shuzo Co. Ltd.
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