U.S. patent application number 10/435633 was filed with the patent office on 2003-10-02 for method for synthesizing dna.
This patent application is currently assigned to Takara Shuzo Co., Ltd.. Invention is credited to Asada, Kiyozo, Fujita, Tomoko, Hagiya, Michio, Kato, Ikunoshin, Miyake, Kazue, Mukai, Hiroyuki, Okawa, Mariko, Sagawa, Hiroaki, Sato, Yoshimi, Takeda, Osamu, Uemori, Takashi.
Application Number | 20030186312 10/435633 |
Document ID | / |
Family ID | 27530277 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186312 |
Kind Code |
A1 |
Uemori, Takashi ; et
al. |
October 2, 2003 |
Method for synthesizing DNA
Abstract
A DNA synthesis method with a shortened time period required for
DNA synthesis by polymerase chain reaction (PCR), characterized in
that a DNA polymerase is used in an amount effective for providing
more than 10 ng of amplified DNA fragments of about 2 kb per 50
.mu.l of a reaction mixture, when PCR is carried out under the
following conditions (A) and (B): (A) reaction mixture: 50 .mu.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.degree. C., 1 second-66.degree. C., 7
seconds; a kit for DNA synthesis usable for the DNA synthesis
method; and an article of manufacture of a PCR agent. According to
the present invention, the procedures in the genetic engineering
studies and industries involved with PCR can be speeded up.
Inventors: |
Uemori, Takashi; (Otsu-shi,
JP) ; Sato, Yoshimi; (Kurita-gun, JP) ; Okawa,
Mariko; (Otsu-shi, JP) ; Fujita, Tomoko;
(Osaka, JP) ; Miyake, Kazue; (Uji-shi, JP)
; Takeda, Osamu; (Hikone-shi, JP) ; Sagawa,
Hiroaki; (Kusatsu-shi, JP) ; Hagiya, Michio;
(Otsu-shi, JP) ; Mukai, Hiroyuki; (Moriyama-shi,
JP) ; Asada, Kiyozo; (Koga-gun, JP) ; Kato,
Ikunoshin; (Uji-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Takara Shuzo Co., Ltd.
|
Family ID: |
27530277 |
Appl. No.: |
10/435633 |
Filed: |
May 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10435633 |
May 12, 2003 |
|
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09786684 |
May 22, 2001 |
|
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09786684 |
May 22, 2001 |
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PCT/JP99/04815 |
Sep 6, 1999 |
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Current U.S.
Class: |
435/6.12 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/686 20130101;
C12N 15/1003 20130101; C12Q 1/686 20130101; C12Q 2527/125
20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 8, 1998 |
JP |
10-254277 |
Oct 9, 1998 |
JP |
10-288534 |
Oct 12, 1998 |
JP |
10-289879 |
Oct 12, 1998 |
JP |
10-289880 |
Dec 18, 1998 |
JP |
10-361456 |
Claims
What is claimed:
1. A kit for carrying out a DNA synthesis method with a shortened
time period for DNA synthesis by polymerase chain reaction (PCR),
comprising: a PCR reagent mixture which is prepared in accordance
with instructions of said kit comprising a DNA polymerase in an
amount sufficient to provide more than 10 ng of amplified DNA
fragments of about 2 kb per 50 .mu.l of a reaction mixture, wherein
the amount of DNA polymerase added to said reaction mixture is
pre-determined for each specific type of DNA polymerase by carrying
out a test PCR reaction under the following conditions (A) and (B):
(A) reaction mixture: 50 .mu.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 (SEQ ID NO: 10) and Eco-2
(SEQ ID NO: 11); and having a composition suitable for said DNA
polymerase; and (b) reaction conditions: 35 cycles of PCR, wherein
one cycle is at 99.degree. C., 1 second-66.degree. C., 7
seconds.
2. The kit according to claim 1, comprising two or more kinds of
DNA polymerases.
3. The kit according to claim 2, wherein one DNA polymerase
comprises 3'.fwdarw.5' exconuclease activity, and the other DNA
polymerase comprises substantially no 3'.fwdarw.5' exonuclease
activity.
4. The kit according to any one of claims 1 to 3, wherein said PCR
reagent mixture comprises an acidic substance and/or a salt
thereof.
5. The kit according to claim 4, wherein said acidic substance is
an acidic macromolecular substance.
6. The kit according to claim 5, wherein said acidic macromolecular
substance comprises a sugar chain backbone.
7. The kit according to claim 4, wherein said acidic substance
and/or a salt thereof is selected from the group consisting of
sulfated-fucose-containing polysaccharides, dextran sulfate,
carrageenan, heparin, rhamnam sulfate, chondroitin sulfate,
dermatan sulfate (chondroitin sulfate B), heparan sulfate,
hyaluronic acid, alginic acid, pectin, polyglutamic acids,
polyacrylic acids, polyvinyl sulfates, polystyrene sulfates, DNA
and salts thereof.
8. The kit according to any one of claims 1 to 3, wherein said PCR
reagent mixture further comprises at least one substance selected
from the group consisting of spergualins, degraded products thereof
and salts thereof.
9. The kit according to any one of claims 1 to 3, wherein said PCR
reagent mixture further comprises a salt formed from an acidic
substance and spergualins or degraded products thereof.
Description
TECHNICAL FIELD
[0001] 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
[0002] 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.
[0003] 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.
[0004] 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)].
[0005] The method is a method using a mixture of DNA polymerases
for PCR, the mixture comprising DNA polymerase having 3'.fwdarw.5'
exonuclease activity (for example, .alpha.-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.
[0006] 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.
[0007] 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.
1TABLE A Enzyme: TaKaRa Ex Taq*.sup.1 [1.25 U/50 .mu.l (PCR Reagent
Mixture)] Template DNA: E. coli Genomic DNA*.sup.2 [100 ng/50 .mu.l
(PCR Reagent Mixture)] Amplification of 94.degree. C. 30 sec. 1 kbp
Fragment 55.degree. C. 30 sec. {close oversize bracket} 30 cycles
(about 90 minutes) 72.degree. C. 1 min. Amplification of 98.degree.
C. 10 sec. {close oversize bracket} 30 cycles (about 268 minutes)
10 kbp Fragment 68.degree. C. 8 min. Amplification of 98.degree. C.
10 sec. {close oversize bracket} 30 cycles (about 478 minutes) 20
kbp Fragment 68.degree. C. 15 min. *.sup.1Product manufactured by
Takara Shuzo Co., Ltd. *.sup.2genome DNA set for LA PCR .TM.,
manufactured by Takara Shuzo Co., Ltd.
[0008] 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.
[0009] 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.
[0010] 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
[0011] 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.
[0012] 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 .mu.l of
a reaction mixture, when PCR is carried out under the following
conditions (A) and (B):
[0013] (A) reaction mixture: 50 .mu.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
[0014] (B) reaction conditions: 35 cycles of PCR, wherein one cycle
consists of 99.degree. C., 1 second-66.degree. C., 7 seconds.
[0015] 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 .mu.l of a reaction mixture, when PCR is carried out under
the following conditions (A) and (B):
[0016] (A) reaction mixture: 50 .mu.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
[0017] (B) reaction conditions: 35 cycles of PCR, wherein one cycle
consists of 99.degree. C., 1 second-66.degree. C., 7 seconds.
[0018] 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
[0019] (1) DNA Synthesis Method of the Present Invention
[0020] 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 .mu.l of
reaction mixture, when PCR is carried out under the following
conditions (A) and (B):
[0021] (A) reaction mixture: 50 .mu.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
[0022] (B) reaction conditions: 35 cycles of PCR, wherein one cycle
consists of 99.degree. C., 1 second-66.degree. C., 7 seconds.
[0023] 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.
[0024] In the present invention, the term "effective amount of DNA
polymerase" means an amount of a DNA polymerase which is an amount
corresponding to a sufficient activity such that an amount of a DNA
fragment of about 2 kb is more than 10 ng per 50 .mu.l volume of a
reaction mixture, when a 35 cycle PCR is carried out by using 50
.mu.l of a reaction mixture containing 1 ng of E. coli genomic DNA,
and 10 pmol each of primers Eco-1 and Eco-2 (nucleotide sequences
of primers Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of
Sequence Listing, respectively), wherein one cycle consists of
99.degree. C., 1 second-66.degree. C., 7 seconds. Therefore, as
long as an amount of DNA polymerase is an amount capable of
carrying out PCR under these conditions, an amount of its protein
is not limited. The use of this effective amount of DNA polymerase
can speed up the PCR.
[0025] The term "E. coli genomic DNA" described in the present
specification includes, for example, genomic DNA prepared from E.
coli strain JM109 (Escherichia coli JM109) according to the method
described in "IDENSHI.cndot.TANPAKUSHITSU JIKKENSOUSA BLOTTING
(Gene.cndot.Protein Experimental Manipulation and Blotting)"
coauthored by Yoshiytuki Kuchino, Hisamaru Hirai, and Ikunosuke
Sakuragi, 1987, published by Soft Science Company. In addition, the
details of the preparation method are described in Bio View, 13,
6-5 (1994, published by Takara Shuzo Co., Ltd.). Furthermore, the
genomic DNA is available as genome DNA set for LA PCR.TM. from
Takara Shutzo Co., Ltd.
[0026] Incidentally, as a composition of a reaction mixture, there
may be used a reaction mixture having a composition suitable for
DNA polymerase used. Here, the term "composition suitable for DNA
polymerase" means a composition capable of providing optimum
conditions such as optimum kinds of buffers, optimum pH, optimum
salt concentration (magnesium salt, and the like), optimum dNTPs
concentration, optimum amount of primers and other additives.
[0027] Here, an enzyme activity unit of DNA polymerase is expressed
as an index an ability of catalyzing the incorporation of
nucleotides (for example, labeled dNTPs) into template DNA. Such a
method for determining an activity is described in, for example,
DNA Polymerase from Escherichia coli, authored by Richardson C. C.,
page 264, Procedures in Nucleic Acids Research, 1966, published by
D. R. Harper & Row Co., Ltd., edited by Cantoni G. L. et al.
For example, as to conditions when an activity of Taq DNA
polymerase, which is DNA polymerase derived from Thermus aquaticus
is determined by the method, the following conditions are
exemplified.
[0028] (a) template DNA: activated salmon sperm DNA;
[0029] (b) a composition of a reaction mixture: 25 mM TAPS (pH 9.3
at 25.degree. C.), 50 mM potassium chloride, 2 mM magnesium
chloride, 1 mM .beta.-mercaptoethanol, 200 .mu.M each of dATP, dGTP
and dTTP, and 100 .mu.M [.alpha.-.sup.32P] dCTP, a total volume of
50 .mu.l;
[0030] (c) a method for determining an activity: To a reaction
mixture having the composition of (b) containing the template DNA
of (a) is added a sample containing Taq DNA polymerase, and the
resulting mixture is incubated at 74.degree. C. for 10 minutes.
Thereafter, acid-insoluble substances are collected, and the
radioactivity contained in the acid-insoluble substances are
determined.
[0031] Activated salmon sperm DNA used in the above method for
determining an activity is prepared as follows.
[0032] {circle over (1)} Salmon testes DNA (manufactured by Sigma
Co., Ltd.) is allowed to swell with sterilized water.
[0033] {circle over (2)} Seventy units per microliter of DNase I
(manufactured by Takara Shuzo Co. Ltd.) is diluted with 150 mM
sodium chloride so as to be diluted within a range of 500
times-4000 times, preferably within a range of 1500 times-3000
times.
[0034] {circle over (3)} To 20 mg of the above swelled DNA are
added 50 mM Tris-hydrochloric acid buffer (pH 7.5), 5 mM magnesium
chloride, 0.05% BSA, 50 .mu.l of the above diluted DNase I, so as
to make up a volume of 10 ml of a reaction mixture.
[0035] {circle over (4)} After treatment at 37.degree. C. for 10
minutes, enzyme is inactivated by treating at 77.degree. C. for 10
minutes. To a partial portion thereof, perchloric acid (HClO.sub.4)
is added, so as to have a final concentration of about 0.4 M, and
the resulting mixture is centrifuged, thereby providing the
resulting supernatant as an A.sub.260 sample. The remaining portion
is purified by subjecting it to phenol extraction,
chloroform/isoamyl alcohol extraction, and ethanol
precipitation.
[0036] {circle over (5)} Absorbance of UV.sub.260 of DNA before the
above enzyme treatment (A.sub.260 control) and that of the
A.sub.260 sample obtained in the above item {circle over (4)} are
determined to calculate decomposition rate using (A.sub.260
sample)/(A.sub.260 control).times.100 (%). In this case, as a
substrate for determining the above incorporating activity, there
can be used an enzyme-treated DNA having a decomposition rate of
preferably from about 3% to about 9%, more preferably about 4.5% to
about 7.5%.
[0037] Incidentally, depending on the kinds of enzymes to be tested
for incorporating activity, it is reasonable to select an
enzyme-treated DNA having the most optimal decomposition rate in a
preliminary experiment using an enzyme-treated DNA having each
decomposition rate.
[0038] Usually, one unit (hereinafter described as "1 U") of DNA
polymerase activity determined by the above method, namely, an
activity for incorporating dNTPs into DNA (hereinafter, simply
described as dNTPs-incorporating activity) is defined as "an amount
of enzyme capable of incorporating 10 nmol of dNTPs per 30 minutes
into acid-insoluble substances".
[0039] Conventionally, in PCR using a DNA polymerase, it is
standard to add a DNA polymerase in an amount of 1.25 U to 2.5 U as
a dNTPs-incorporating activity in 50 .mu.l of a reaction mixture.
Although not particularly limited in the present invention, DNA
amplification with a short time period conventionally not achieved,
namely, speedup of PCR, can be achieved by carrying out PCR with
addition of DNA polymerase in an amount of 4 to 20 U as a
dNTPs-incorporating activity per 50 .mu.l of a reaction
mixture.
[0040] An example of rapid PCR of the present invention is shown in
Table B. By comparison of Table A with Table B mentioned above, an
effect of shortening a time period by the rapid PCR of the present
invention is obvious. According to the present invention, there is
provided rapid PCR with a shortened total time period for PCR.
2TABLE B Enzyme: Polymerase A Template DNA: E. coli Genomic DNA
[100 ng /50 .mu.l (PCR Reagent Mixture)] Amplification of
98.degree. C. 5 sec. {close oversize bracket} 30 cycles (about 20
minutes) 1 kbp Fragment 66.degree. C. 2 sec Amplification of
98.degree. C. 5 sec. {close oversize bracket} 30 cycles (about 53
minutes) 10 kbp Fragment 68.degree. C. 70 sec. Amplification of
98.degree. C. 5 sec. {close oversize bracket} 30 cycles (about 93
minutes) 20 kbp Fragment 68.degree. C. 150 sec. In Table B, as
polymerase A, there is used DNA polymerase in an amount of 5 U as a
dNTPs-incorporating activity per 50 .mu.l (PCR reagent mixture). In
addition, in Table B, E. coli genomic DNA is the same as that in
Table A.
[0041] When the effective amount of DNA polymerase used in the
rapid PCR of the present invention is used in amplification of a
DNA fragment under conditions for the rapid PCR of the present
invention, there is provided an amount of amplified products of the
same level as that of carrying out PCR using TaKaRa Ex Taq in an
amount of 1.25 U as a dNTPs-incorporating activity per 50 .mu.l as
shown in Table A. Therefore, while the activity unit for an
effective amount of DNA polymerase used in the present invention
shows a higher activity unit in the dNTPs-incorporating activity
than that of the prior arts, an activity Unit expressed by PCR
performance, namely an activity unit expressed by comparison of
amounts of amplified products in PCR processes (PCR effective
ratio), is of the same level as that of the prior arts.
[0042] Embodiments of the DNA synthesis method of the present
invention include a method of using one kind of DNA polymerase and
a method of using two or more kinds of DNA polymerases. The above
method of using two or more kinds of DNA polymerases includes
concretely a method of using DNA polymerase having 3'.fwdarw.5'
exonuclease activity and the other DNA polymerase substantially
having no 3'.fwdarw.5' exonuclease activity (Proceedings of the
National Academy of the Sciences of the USA, supra); a method of
using two or more kinds of DNA polymerases each having 3'.fwdarw.5'
exonuclease activity; a method of using .alpha.-type DNA polymerase
and non-.alpha., non-pol I type DNA polymerase; and the like. Here,
"the other DNA polymerase substantially having no 3'.fwdarw.5'
exonuclease activity" includes naturally-occurring DNA polymerase
having no 3'.fwdarw.5' exonuclease activity or DNA polymerase in
which functional portion involved in expression of 3'.fwdarw.5'
exonuclease activity is artificially modified to exhibit no
activity.
[0043] The amount of DNA polymerase used in the present invention
(in other words, the above "effective amount of DNA polymerases")
is an amount exceeding an amount of DNA polymerase standardly used
in the indication "dNTPs-incorporating, activity" as described in
the instruction manuals, and the like, which are attached to a
commercially available enzyme and kit for standard PCR method.
Based on this indication of enzyme activity, an effective amount
may be used, wherein the effective amount is an amount effective
for exhibiting an effect of shortening a time period required in
operation.
[0044] Concretely, it is desired that the amount of DNA polymerase
used in the present invention, in the indication of
dNTPs-incorporating activity, is preferably twice or more, more
preferably four times or more, the amounts of enzyme used in
conventional PCR, from the viewpoint of fully exhibiting the effect
of shortening a time period required in operation, and that the
amount is preferably 30 times or less, more preferably 20 times or
less, the amounts of enzyme used, from the same viewpoint as above.
In addition, when two or more kinds of DNA polymerases are used,
there may be used an effective amount of each enzyme, or an
effective amount as the amount of enzyme for any one of two or more
kinds of DNA polymerases. As a result, as shown in the following
examples, according to the DNA synthesis method of the present
invention, namely rapid PCR, there can be achieved DNA
amplification with a short time period, and confirmation of
amplified DNA by general agarose electrophoresis, even under such
PCR conditions with a shortened time period that amplification of a
desired DNA fragment cannot be confirmed by general agarose
electrophoresis or the like, when DNA polymerase is used in an
amount in the conventional PCR.
[0045] The above "effective amount of DNA polymerase" may be
determined, for example, as follows. Concretely, regarding to an
arbitrary template DNA, PCR is carried out under standard PCR
conditions (standard conditions) such as an amount of enzyme, a
thermal profile, and the like, which are standardly used for the
amplification of the template DNA. Next, an enzyme amount is
determined by adjusting a time period for each reaction step under
standard conditions; setting PCR conditions so as to shorten a time
period required for a whole amplification reaction; thereafter
carrying out PCR, with varying amounts of enzyme, wherein the
enzyme amount is an amount obtainable in substantially the same
level in PCR performance as an amount of amplified products when a
standard amount of the enzyme used, so that the enzyme amount
determined or an amount not less than the enzyme amount can be
defined as an effective amount of DNA polymerase, which is used for
the rapid PCR of the present invention. Here, "amount of amplified
products" is quantified by, for example, subjecting a given amount
of a sample obtained after termination of PCR to electrophoresis;
staining the resulting gel after electrophoresis by ethidium
bromide to visualize the band ascribed to amplified products; and
thereafter measuring fluorescent intensity of the band with the use
of instrument, such as an image analysis device (image analyzer) or
a densitometer, the instrument being capable of quantifying an
amount of a DNA fragment contained in the band which is separated
on the gel. Furthermore, an amount of a DNA fragment may be
quantified by a known DNA quantification method after purification
of amplified products in the sample.
[0046] In the rapid PCR of the present invention, the reaction time
which is set for each step is not particularly limited, as long as
the reaction time is relatively short, as compared to the reaction
time under the standard conditions, and exhibits the same level of
the enzyme activity as represented by the PCR performance. In the
present invention, it is possible to set PCR conditions such that
the reaction time for all processes, namely a total time period for
PCR is one-half to one-quarter or less of the conventional total
time period for PCR. For instance, in the case of amplification of
a 1 kbp fragment, while conventional one cycle requires 3 minutes,
one cycle in the rapid PCR of the present invention can be
shortened to 40 seconds, so that the total time period for PCR is
two-ninth of the conventional one. In the case of amplification of
a 2 kbp fragment, while conventional one cycle requires about 9
minutes, one cycle in the rapid PCR of the present invention can be
shortened to about 1.8 minutes, so that the total time period for
PCR is about one-fifth of the conventional one. In the case of
amplification of a 10 kbp fragment, while conventional one cycle
requires about 16 minutes, one cycle in the rapid PCR of the
present invention can be shortened to about 3 minutes, so that the
total time period for PCR is about one-fifth of the conventional
one.
[0047] Conventionally, as enzymes possessing excellent DNA
synthesizing speed and capable of amplifying DNA in a short time
period, there has been known an enzyme composition comprising DNA
polymerase (KOD DNA polymerase) derived from Pyrrococcus sp. KOD1
(Japanese Patent Laid-Open No. Hei 10-42874, trade name: KOD Dash
DNA polymerase, manufactured by TOYOBO CO., LTD.). However, no
amplified fragments can be visibly confirmed and the amount of the
amplified product as quantified by high-sensitive image analyzer is
10 ng or less, even if the enzyme composition were used under the
above conditions in a standard amount used of 2.5 U/50 .mu.l as
described in the article of manufacture in accordance with the
instruction manual, and thereafter the resulting reaction mixture
is subjected to general agarose electrophoresis. On the other hand,
when the enzyme is used at an "effective amount of the DNA
polymerase" as defined in the present invention, even under the
above conditions, amplified fragments can be confirmed by agarose
electrophoresis, and the amount of its amplified product is an
amount exceeding 10 ng.
[0048] The DNA polymerase which can be used for the DNA synthesis
method of the present invention (namely the rapid PCR) is not
particularly limited, and includes, for instance, pol I-type DNA
polymerases (E. coli DNA polymerase I, Klenow fragment, Taq DNA
polymerase and the like), .alpha.-type DNA polymerases
[.alpha.-type DNA polymerase derived from the above-mentioned
Pyrococcus furiosus, DNA polymerase derived from Thermococcus
litralis (VENT DNA polymerase), DNA polymerase derived from
Pyrococcus sp. KOD 1 (KOD DNA polymerase), DNA polymerase derived
from Pyrococcus sp. GB-D (DEEP VENT DNA polymerase), and the like],
and non-.alpha., non-pol I type DNA polymerases not belonging to
any of these polymerases. Incidentally, each of the pol I type DNA
polymerases and .alpha.-type DNA polymerases refers to a group of
enzymes classified by the homology on the amino acid sequences
thereof, and the feature on the amino acid sequences is described
in Nucleic Acids Research 15, 4045-4057 (1991).
[0049] In addition, the non-.alpha., non-pol I type DNA polymerase
includes, for instance, DNA polymerase derived from Pyrococcus
furiosus disclosed in WO97/24444 Pamphlet.
[0050] The DNA polymerase which can be used for the method of the
present invention is not limited to a single kind of a DNA
polymerase, and two or more kinds of DNA polymerases can be also
used as a DNA polymerase composition by, for instance, mixing a DNA
polymerase possessing 3'.fwdarw.5' exonuclease activity with
another DNA polymerase possessing substantially no 3'.fwdarw.5'
exonuclease activity. A mixing ratio of both the enzymes is not
particularly limited, and it may be a mixing ratio appropriate for
the rapid PCR of the present invention depending upon the kinds of
both the enzymes. It may be used in a ratio of the DNA polymerase
possessing 3'.fwdarw.5' exonuclease activity to the other DNA
polymerase possessing substantially possessing no 3'.fwdarw.5'
exonuclease activity in the range of 9:1-1:500. As an example of a
polymerase composition thereof, there can be suitably used TaKaRa
Ex Taq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.).
[0051] Generally in PCR, DNA amplification is carried out by
three-step reaction of dissociation (denaturation) of a double
stranded template DNA to a single stranded one, annealing of a
primer to a single stranded template DNA, and a complementary
strand synthesis (extension) from the primer. In addition, DNA
amplification is also carried out in a so-called "shuttle PCR"
["PCR Hou Saizensen (PCR Method Frontier)," "Proteins, Nucleic
Acids, Enzymes" an extra number 41, No. 5, 425-428 (1996)], which
is a two-step reaction in which the annealing step of the primer
and the extension reaction step among the three-step reactions
described above are carried out at the same temperature. In the DNA
synthesis method of the present invention, since the time period
required for the above extension step can be particularly
shortened, in any of the above-mentioned three-step reaction and
two-step reaction, the time period required for an entire synthesis
reaction can be shortened.
[0052] In the DNA synthesis method of the present invention, when
the DNA synthesis reaction is carried out, the DNA synthesis can be
further efficiently carried out by subjecting to the DNA synthesis
reaction in the presence of a substance possessing the action of
enhancing the DNA-synthesizing activity owned by the DNA
polymerase, namely a substance for achieving higher performance in
the rapid PCR.
[0053] As one substance for achieving higher performance in the
rapid PCR, there is included a substance having electrically
negative charges or a salt thereof, particularly an acidic
substance or a salt thereof.
[0054] PCR is carried out in the presence of an effective amount of
the acidic substance and/or a salt thereof, whereby the conditions
for speedup can be universalized. In other words, high-performance
rapid PCR can be carried out without being affected by the nature
of the template (for instance, GC content, or the like). In
addition, at least one kind selected from spergualins, degradation
products and salts thereof may be selected.
[0055] When the rapid PCR of the present invention is carried out,
the details on the function of an effective amount of an acidic
substance and/or a salt thereof, for instance, an acidic substance
having a sugar backbone structure, are unknown. When an effective
amount of the DNA polymerase for carrying out the rapid PCR of the
present invention is used, since excess DNA polymerase is trapped
with the acidic substance during the DNA synthesis reaction, the
high-performance rapid PCR can be further achieved by supplying the
most appropriate DNA polymerase for PCR to a template DNA by the
effects of the acidic substance.
[0056] The action of the acidic substance or a salt thereof of
enhancing DNA-synthesizing activity can be evaluated by the size of
the DNA strand newly synthesized per unit time period or the amount
of the amplified products in PCR. In the DNA synthesis method of
the present invention, the above-mentioned acidic substance or a
salt thereof is used in an amount effective for exhibiting its
action. The effective amount can be evaluated by comparing, for
instance, the amount of the amplified products in the case where
PCR is carried out by using a reaction mixture in which various
amounts of the above acidic substance or a salt thereof are added,
with the amount of the amplified products in the case where PCR is
carried out without adding these substances. The amount of the
amplified products can be quantified, for instance, by subjecting a
given amount of the reaction mixture after PCR to electrophoresis,
staining the gel after electrophoresis by ethidium bromide or the
like, and determining the intensity of fluorescence of the band
ascribed to the amplified products by using an imaging analyzer or
the like.
[0057] The acidic substance possessing an action of enhancing
DNA-synthesizing activity is not particularly limited. For
instance, acidic macromolecular substances such as acidic
polysaccharides can be used. In addition, polyglutamates,
polyacrylates, polyvinyl sulfates, polystyrene sulfates, and DNAs
not serving as a template for a desired DNA synthesis can be also
used. Incidentally, in the present specification, the acidic
substance also encompasses a salt of the above acidic substances,
as long as it possesses an action for enhancing DNA-synthesizing
activity. The acidic polysaccharides which can be used in the
present invention include, for instance, sulfate group-containing
sulfated polysaccharides representatively exemplified by
sulfated-fucose-containing polysaccharides, dextran sulfate,
carrageenan, heparin, heparan sulfate, rhamnam sulfate, chondroitin
sulfate, and dermatan sulfate (chondroitin sulfate B); polyuronic
acids such as hyaluronic acid, alginic acid and pectin, and the
like. As the above sulfated-fucose-containing polysaccharides,
there can be used sulfated-fucose-containing polysaccharide-F or
sulfated-fucose-containing polysaccharide-U. Here, the term
"sulfated-fucose-containing polysaccharide-F" refers to a
sulfated-fucose-containing polysaccharide substantially containing
no uronic acid, obtainable from the plant of Phaeophyceae, for
instance, by the method disclosed in WO97/26896 Pamphlet, or by the
method disclosed in WO097/47208 Pamphlet. In addition, the term
"sulfated-fucose-containing polysaccharide-U" refers to a
sulfated-fucose-containing polysaccharide containing uronic acid,
obtainable by the method described in the above Pamphlets.
[0058] The salt of the above acidic substance is not particularly
limited, as long as it possesses an action of enhancing
DNA-synthesizing activity, and a water-soluble salt is preferable.
The water-soluble salt includes, for instance, alkali metal salts
such as sodium dextran sulfate, sodium alginate, sodium
polyglutaminate, sodium heparin, potassium dextran sulfate and
lithium heparin.
[0059] The above acidic substance may be, for instance, naturally
occurring products, or chemically or enzymatically synthesized
products, as long as the acidic substance is a substance keeping
the action of enhancing the DNA-synthesizing activity. The above
acidic substance may be any of unpurified products containing the
same, partially purified products or purified products. Further,
the acidic substance may be subjected to an appropriate
modification in a range in which an action of enhancing
DNA-synthesizing activity is kept. In addition, the acidic
substance used in the present invention may be a substance obtained
by subjecting it to degradation procedures so that the molecular
weight of the above-mentioned acidic substance is in an appropriate
form for exhibiting an action of enhancing DNA-synthesizing
activity, or the acidic substance may be a substance obtained by
further subjecting the product after the degradation procedures to
a fractionation by molecular weight, as long as the acidic
substance possesses an action of enhancing DNA-synthesizing
activity. In the present invention, an acidic substance having a
molecular weight of several thousands or more can be preferably
used. Further, these substances can be used alone or in
admixture.
[0060] The above acidic substance possessing an action of enhancing
DNA-synthesizing activity is added for the purpose of efficiently
exhibiting an activity of the DNA polymerase, or of keeping its
activity in the rapid PCR of the present invention. The amount
added can be optimized depending upon the kinds of the acid
substances, and the acidic substance may be added at 0.1 ng to 100
.mu.g, preferably at 1 ng to 10 .mu.g, per 50 .mu.l of the reaction
mixture. The action of the acidic substance is not particularly
limited, and it is considered to be on the bases of holding the DNA
polymerase on its molecule, thereby suppressing the nonspecific
interaction of the DNA polymerase to a template DNA, and of
providing an optimal amount of the DNA polymerase for the template
DNA. In other words, the DNA synthesis reaction efficiently
progresses by optimizing the interaction between the template DNA
and the DNA polymerase, the interaction To increasing with the
progress of the DNA synthesis reaction.
[0061] Further, there are exhibited effects that the influences of
amplified regions, nucleotide sequences of the primer, and the like
are reduced, thereby stably obtaining amplified products.
[0062] The DNA polymerase in which the above acidic substance
enhances its activity is not particularly limited. For instance,
the above acidic substance can be applied to the DNA synthesis
method using various DNA polymerases mentioned above.
[0063] In the present invention, there may be added and used to a
PCR mixture spergualins and/or salts thereof for the purpose of
efficiently exhibiting the activity of the DNA polymerase in the
rapid PCR of the present invention, or maintaining its
activity.
[0064] The spergualins possessing an action of enhancing
DNA-synthesizing activity are not particularly limited. Examples
thereof include a 15-deoxyspergualin compound represented by the
following general formula (I):
Gu-(CH.sub.2).sub.6--CONHCH(OR)CONH(CH.sub.2).sub.4NH--(CH.sub.2).sub.3--N-
H.sub.2 (I)
[0065] wherein Gu is guanidino group, and R is hydrogen atom or
methyl group, or a salt thereof, and the like.
[0066] As the above spergualins, for instance, 15-deoxyspergualin,
where R of the above general formula (I) is hydrogen atom, or a
salt thereof, is preferable.
[0067] In addition, the salt of the spergualins may be a salt with
an inorganic acid or a salt with an organic acid, as long as the
salt is a substance capable of exhibiting an action of enhancing
DNA polymerase activity.
[0068] Incidentally, the above spergualins are derivatives of
spergualin isolated from producing bacteria of the genus Bacillus,
which is a substance known to possess anti-tumor activity,
immunoenhancement activity, and immunosuppression activity
depending upon the kinds of the derivatives (Japanese Patent
Laid-Open Nos. Sho 58-62152, Sho 61-129119 and Sho 64-90164).
Therefore, these spergualins can be prepared by readily purifying
by a known method, or synthesizing by a known method.
[0069] A process for preparing the above 15-deoxyspergualin or a
salt thereof is disclosed, for instance, in Japanese Examined
Patent Publication No. Sho 61-23183 or Example 6 of U.S. Pat. No.
4,603,015, and the like.
[0070] The above spergualins may be naturally occurring products or
chemically or enzymatically synthesized products, as long as
spergualins are a substance keeping an action of enhancing
DNA-synthesizing activity. The above spergualins may be any of
unpurified products containing the same, partially purified
products or purified products. Further, the above spergualins may
be subjected to appropriate modifications or a degraded product in
a range in which an action of enhancing DNA-synthesizing activity
is kept. Further, these substances can be used alone or in
admixture.
[0071] In the present specification, the degradation product of the
spergualins is not particularly limited, as long as the degradation
product is a substance keeping an action of enhancing
DNA-synthesizing activity. The degradation product includes, for
instance, a substance formed by hydrolyzing the spergualins at room
temperature under strongly alkali condition using sodium hydroxide,
a substance formed by hydrolyzing the spergualins with heating
under weakly alkali conditions such as buffers for PCR, and the
like. The substance formed by hydrolysis under each of the above
conditions is not particularly limited. When the above
15-deoxyspergualin compound represented by the general formula (I)
is used, the substance includes, for instance, a compound
represented by the general formula (II):
Gu-(CH.sub.2).sub.6--CONH.sub.2 (II)
[0072] wherein Gu is the same group as that of the general formula
(I);
[0073] a compound represented by the general formula (III):
HO--CH(OR)CONH(CH.sub.2).sub.4NH--(CH.sub.2).sub.3--NH.sub.2
(III)
[0074] wherein R is the same group as that of the general formula
(I);
[0075] a compound represented by the general formula (IV):
OHC--CONH(CH.sub.2).sub.4NH--(CH.sub.2).sub.3--NH.sub.2 (IV)
[0076] and the like. The degradation product of the above
spergualins also encompasses a salt of the degradation product of
the spergualins, as long as it possesses an action of enhancing
DNA-synthesizing activity.
[0077] The amount of the above spergualins used is not particularly
limited, as long as it is in a range capable of exhibiting an
action of enhancing DNA-synthesizing activity. The amount used may
be an amount so as to have an optimal concentration depending upon
the kinds and amounts of the template DNA used, the length of a
region to be amplified, and the kinds of the DNA polymerases. For
instance, in the case of 15-deoxyspergualin trihydrochloride, it
may be added so as to have a final concentration of 0.1 .mu.M to
500 .mu.M, preferably 20 .mu.M to 100 .mu.M.
[0078] The action of the spergualins of the present invention is
not particularly limited, and it is thought to efficiently exhibit
the activity of the DNA polymerase or to hold the DNA polymerase,
thereby suppressing nonspecific interaction of the enzyme to the
DNA. Also, it is thought to act to the complex of template DNA and
the primer, thereby facilitating the primer extension reaction.
[0079] When the above spergualins and the acidic substance are used
in combination, both parties may react to form a salt, and it may
be a substance possessing an action of enhancing DNA-synthesizing
activity.
[0080] When the above spergualins and the acidic substance are used
in combination, although there are no particular limitations in the
amount, as long as the amount is in a range capable of exhibiting
an action of enhancing DNA-synthesizing activity, it may be an
amount so as to have optimal coexisting ratio depending upon the
kinds and amounts of the template DNA used, the length of a region
to be amplified, the kinds of the DNA polymerases, and the
like.
[0081] Incidentally, the above spergualins or a salt thereof, and a
mixture comprising the above substance having electrically negative
charges or a salt thereof and the above spergualins or a salt
thereof can be used as DNA-synthesizing activity enhancers.
[0082] Regarding the DNA synthesis method described above, the
method of the present invention also encompasses the detail
description of the method, for instance, a method for preparing a
PCR reagent mixture, a provision of printed matters describing
information such as recommended reaction conditions and an act of
instructing the method of the present invention through electronic
media such as internet.
[0083] (2) Kit Usable for DNA Synthesis Method of Present
Invention
[0084] Rapid PCR can be carried out by using the kit of the present
invention. The kit as mentioned above is not particularly limited,
as long as it is a kit usable in the reaction with the DNA
synthesis by PCR method, and includes a kit for rapid PCR for
carrying, out in vitro DNA synthesis reaction.
[0085] The kit of the present invention is a kit usable in in vitro
DNA synthesis, wherein the PCR reagent mixture prepared in
accordance with the instruction of the kit comprises "an effective
amount of the DNA polymerase" used in the DNA synthesis method
described in item (1) above, i.e. an effective amount of a DNA
polymerase such that an amount of a DNA fragment of about 2 kb is
more than 10 ng per 50 .mu.l of a reaction mixture, when 35 cycles
of PCR is carried out by using 50 .mu.l volume of a reaction
mixture containing 1 ng of E. coli genomic DNA, and 10 pmol each of
primers Eco-1 and Eco-2, wherein one cycle consists of 99.degree.
C., 1 second-66.degree. C., 7 seconds.
[0086] The PCR reagent mixture prepared by using the kit of the
present invention is not particularly limited, and for instance,
the reagent mixture comprises 4 to 10 U as dNTPs-incorporating
activity of a DNA polymerase for rapid PCR in 50 .mu.l of the
reaction mixture, including, for instance, TaKaRa EX Taq DNA
polymerase. In addition, as the composition for the reaction
mixture, a reagent mixture having a composition appropriate for the
DNA polymerase used may be used.
[0087] The above "instruction" refers to a printed matter
describing a method of use of the kit, for instance, a method for
preparing a PCR reagent mixture, recommended reaction conditions,
and the like, which may take a form, besides pamphlet or leaflet
type of instruction manuals, of a label attached to the kit, a
packaging material enclosing the kit, and the like. Further, it
also encompasses information disclosed or provided through
electronic media such as internet. Regarding the preparation of the
PCR reagent mixture, the kit of the present invention encompasses a
kit to which instructions for the use of the amount of the above
DNA polymerase and/or the addition of the above acidic substance or
a salt thereof are attached, or a kit for which the method of the
present invention is disclosed and provided through electronic
media such as internet.
[0088] In addition, the kit may contain an acidic substance or a
salt thereof possessing an action of enhancing DNA-synthesizing
activity of the DNA polymerase. As the acidic substance or a salt
thereof, ones described in item (1) above can be used. The acidic
substance or a salt thereof as mentioned above efficiently allows
to exhibit the DNA polymerase activity or to hold the enzyme,
whereby the interaction between the DNA and the enzyme can be
properly regulated. Therefore, the performance of the kit of the
present invention is further improved.
[0089] In addition, the kit may comprise at least one kind selected
from the group consisting of spergualins, degradation products
thereof, and salts thereof.
[0090] When the above spergualins and the acidic substance are used
in combination, the both parties may react to form a salt, and they
may be in any forms as long as they are substances possessing an
action of enhancing DNA-synthesizing activity.
[0091] The DNA polymerase included in the present invention is not
particularly limited, and includes various kinds of DNA polymerases
for rapid PCR shown in item (I) above.
[0092] The kit may comprise a reagent necessary for the reaction of
the DNA polymerase such as dNTPs, magnesium chloride, and buffer
components for keeping the reaction mixture at an appropriate pH.
The above DNA polymerase, the acidic substance and other reagents
may be contained in the kit in a state where each is present as an
independent component, or a state in which some of the components
are combined, including, for instance, a state in which the
components are added to the reaction buffer and the like.
[0093] One embodiment of the kit of the present invention includes
a composition comprising, besides the above DNA polymerase, various
components necessary for DNA synthesis by PCR method, including,
for instance, dNTP, magnesium chloride, buffer components for
keeping the reaction mixture at an appropriate pH, and the like.
The composition may further comprise the above acidic substance.
The composition described above can be prepared by adding a primer
for amplifying a desired DNA fragment and template DNA, and further
adding, as occasion demands, water or buffer, whereby a reaction
mixture can be prepared. Furthermore, when the DNA fragment to be
amplified by the kit is determined, the composition may comprise a
primer appropriate for the amplification of the fragment. By the
use of the composition described above, the DNA synthesis reaction,
namely the rapid PCR, can be extremely conveniently and rapidly
carried out.
[0094] When applied to manipulations such as PCR and sequencing
utilizing PCR, DNA labeling, cDNA synthesis, and site-directed
mutagenesis, the kit of the present invention exhibits an excellent
effect that the time period required for the manipulations can be
shortened. For instance, when the above kit is applied to PCR, the
time period required for amplification of DNA of the same chain
length is shorter than that by conventional PCR method or LA-PCR
method. Therefore, even under rapid PCR conditions where the
amplification of DNA was impossible in the conventional method, the
amplification of DNA can be carried out. In addition, since the kit
of the present invention can shorten the time period required for
the entire amplification reaction, there is exhibited an excellent
effect that gene diagnostic method or the like can be carried out
at a shorter time period by the use of the gene diagnostic method
on the basis of PCR method.
[0095] (3) Article of Manufacture of Rapid PCR Agent of the Present
Invention
[0096] The article of manufacture of a PCR agent of the present
invention is 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. The above PCR agent may comprise a DNA
polymerase and buffer appropriate for the DNA polymerase and/or
dNTP. Therefore, one of ordinary skill in the art can conveniently
carry out the rapid PCR of the present invention by following the
label indicated on the article of manufacture or the instruction
manual attached to the manufacture, and the article of manufacture
is useful in various industrial fields requiring the rapid PCR of
the present invention.
[0097] According to the DNA synthesis method (rapid PCR) of the
present invention, the time period required for the entire
amplification reaction can be shortened. Although it depends upon
the performance of the apparatus used, for instance, a total time
period for PCR required for amplifying a DNA of 2 kb is shortened
to about one-half that of conventional method, and a total time
period for amplifying a DNA of about 20 kb is shortened to about
one-fifth that of conventional method, whereby speed-up of PCR can
be achieved for the first time. The method of the present invention
exhibits an excellent effect that gene diagnostic method can be
carried out in a shorter period of time by, for instance, the use
of gene diagnostic method on the basis of PCR method. The method is
particularly suitable for nested PCR or the like in which PCR is
carried out twice.
[0098] The rapid PCR of the present invention is extremely useful
in the development and manufacture of the techniques requiring a
large amount of PCR manipulations such as a DNA chip. The DNA chip
comprises a glass chip of a size of thumb and about 10000 kinds of
DNAs, wherein the DNAs are immobilized on the glass chip. In order
to manufacture the DNA chip, it is necessary that DNAs requiring
spots are amplified and prepared by amplification with PCR, and the
necessary PCR manipulations therefor would be enormous.
[0099] For instance, if 10000 of DNA chips described above are
supposedly made, there is exhibited an effect that the chips can be
manufactured in a short time period of about 3 weeks according to
the rapid PCR of the present invention, in contrast to an overall
period of about 3 months just for PCR manipulations when the
conventional PCR is employed.
[0100] In addition, the PCR manipulations required for elucidating
an entire sequence of genome of Bacillus subtilis (5000000 bases)
can be achieved in about 3 weeks according to the rapid PCR of the
present invention, in contrast to about 3.5 months in the
conventional PCR, thereby showing a great difference.
[0101] In the rapid PCR of the present invention, PCR can be
carried out by using conventional PCR apparatus without using
specialized apparatus. The rapid PCR of the present invention is
excellent in the rapidness and the reactivity, and is an extremely
useful technique as high-sensitivity PCR.
[0102] The present invention will be hereinbelow described in
further detail by means of the working examples, without intending
to restrict the scope of the present invention to these working
examples.
[0103] In the following working examples, the activities of the
commercially available DNA polymerases were represented on the
basis of the indicated units in "dNTPs-incorporating activity"
described in instruction manual for each enzyme product. In
addition, unless specified otherwise, the reaction solution
comprising a commercially available enzyme was prepared in
accordance with the manual for each enzyme, or prepared by using a
reaction buffer attached thereto. PCR was carried out by using
TaKaRa PCR Thermal Cycler PERSONAL (manufactured by Takara Shuzo
Co., Ltd.) unless specified otherwise.
EXAMPLE 1
[0104] (1) Preparation of Primers
[0105] Nine kinds of primers .lambda.1 to .lambda.5 and .lambda.7
to .lambda.10 were synthesized on the basis of the nucleotide
sequences of .lambda.DNA. The nucleotide sequences for the primers
.lambda.1 to .lambda.5 and .lambda.8 to .lambda.10 are respectively
shown in SEQ ID NOs: 1 to 8 of Sequence Listing. Further, the
nucleotide sequence for .lambda.7 is shown in SEQ ID NO: 9 of
Sequence Listing. The sizes of DNA fragments amplified by PCR with
.lambda.DNA as a template depending upon the combinations of these
primers are shown in Table 1.
3 TABLE 1 Size of Amplified Primer Pair DNA Fragment
.lambda.1/.lambda.2 0.5 kb .lambda.1/.lambda.3 1 kb
.lambda.1/.lambda.4 2 kb .lambda.1/.lambda.5 4 kb
.lambda.1/.lambda.7 8 kb .lambda.1/.lambda.8 10 kb
.lambda.1/.lambda.9 12 kb .lambda.1/.lambda.10 15 kb
[0106] (2) Preparation of Polymerase A and Polymerase B
[0107] Using TaKaRa EX Taq DNA polymerase having a concentration of
5 U/.mu.l, its concentrated preparation was prepared. Each of a
preparation having a concentration of 10 U/.mu.l (referred to as
"Polymerase A") and a preparation having a concentration of 20
U/.mu.l (referred to as "Polymerase B") was prepared to use in the
subsequent examples.
[0108] (3) Rapid PCR Using Polymerase A and Polymerase B
[0109] According to the instruction manual attached to TaKaRa EX
Taq DNA polymerase, the DNA polymerase is usually used in an amount
of 1.25 U per 50 .mu.l of a PCR reagent mixture. In this example,
rapid PCR was studied by varying the amount of each of the DNA
polymerases.
[0110] PCR was carried out by preparing a PCR reagent mixture
comprising .lambda.DNA as a template, and primers .lambda.1 and
.lambda.4 as a primer pair. The composition of the PCR reagent
mixture is shown below.
[0111] Composition of PCR Reagent Mixture:
[0112] 50 mM Tris-acetate (pH 8.5), 0.2 mM each of dATP, dCTP, dGTP
and dTTP, 3 mM magnesium acetate, 50 mM potassium acetate, 1 pg of
.lambda.DNA, 10 pmol each of primers .lambda.1 and .lambda.4. To
the above PCR reagent mixture were added 1.25 U of TaKaRa EX Taq
DNA polymerase and 0.5 .mu.l of Polymerase A or 0.5 .mu.l of
Polymerase B, each mixture making up a final volume of 50
.mu.l.
[0113] Rapid PCR was carried out in 35 cycles for a total time
period of about 31.5 minutes, wherein one cycle of reaction
comprises a process consisting of 98.degree. C., 5
seconds-66.degree. C., 10 seconds. After the termination of
reaction, 5 .mu.l of the resulting sample was electrophoresed on 1%
agarose gel containing ethidium bromide in an amount of 0.00005%,
thereby confirming a desired amplified fragment of about 2 kb. The
results thereof are shown in Table 2.
4 TABLE 2 Amplification Enzyme Used Results 1.25 U/50 .mu.l of PCR
- TaKaRa EX Taq Polymerase A ++ Polymerase B ++ ++: Intensive
amplification being observed; +: Amplification being observed;
.+-.: Slight amplification being observed; and -: No amplification
being observed.
[0114] As shown in Table 2, there could be confirmed that rapid PCR
can be carried out by using Polymerase A [5 U/50 .mu.l (PCR reagent
mixture)] or Polymerase B [10 U/50 .mu.l (PCR reagent
mixture)].
EXAMPLE 2
[0115] (1) Preparation of Primers
[0116] Five kinds of primers Eco-1, Eco-2, Eco-2-2, Eco-5 and Eco-6
were synthesized on the basis of the nucleotide sequence of E. coli
genomic DNA. The nucleotide sequences of the primers Eco-1, Eco-2,
Eco-2-2, Eco-5 and Eco-6 are respectively shown in SEQ ID NOs: 10
to 14 of Sequence Listing. The sizes of DNA fragments depending
upon the combinations of these primers are shown in Table 3 being
amplified by PCR with E. coli DNA as a template.
5 TABLE 3 Size (kb) of Amplified Primer Pair DNA Fragment
Eco-1/Eco-2 2 Eco-2-2/Eco-5 8 Eco-1/Eco-6 20
[0117] (2) Rapid PCR when Polymerase A was Used
[0118] Rapid PCR using Polymerase A was studied. Rapid PCR was
carried out by preparing a PCR reagent mixture containing each of
the combinations of primers Eco-1 and Eco-2, primers Eco-2-2 and
Eco-5, and primers Eco-1 and Eco-6 as a primer pair using as a
template E. coli genomic DNA in genome DNA set (manufactured by
Takara Shuzo Co., Ltd.) for LA PCR.TM.. The composition of the PCR
reagent mixture is shown below.
[0119] Composition of PCR Reagent Mixture:
[0120] 50 mM Tris-acetate (pH 8.5), 0.2 mM each of dATP, dCTP, dGTP
and dTTP, 3 mM magnesium acetate, 50 mM potassium acetate, 10 pmol
each of the combinations of primers Eco-1 and Eco-2, primers
Eco-2-2 and Eco-5, and primers Eco-1 and Eco-6. Incidentally, when
the size of the fragment to be amplified are 2 kb and 8 kb, 1 ng of
E. coli genomic DNA was used, or when the size was 20 kb, 20 ng of
E. coli genomic DNA was used. To the above PCR reagent mixture were
added 0.5 .mu.l of Polymerase A and 2.5 .mu.g of sodium alginate,
each making up a final volume of 50 .mu.l.
[0121] PCR was carried out in a fast mode under temperature
conditions as shown in Table 4 by using TaKaRa PCR Thermal Cycler
PERSONAL (manufactured by Takara Shuzo Co., Ltd.). After the
termination of reaction, 5 .mu.l of each resulting sample was
electrophoresed on 1% agarose gel containing ethidium bromide in an
amount of 0.00005%, thereby confirming an amplified fragment. The
results thereof are shown in Table 4.
6 TABLE 4 PCR Reaction Total Amplification Size (kb) of Temp.
Number Period Results Amplified Conditions of of Time Polymerase
DNA Fragment (1 Cycle) Cycles (minute) A 2 99.degree. C./1 s, 35
About + 66.degree. C./6 s 25.1 99.degree. C./1 s, 35 About ++
66.degree. C./7 s 25.7 99.degree. C./1 s, 35 About +++ 66.degree.
C./8 s 26.3 8 99.degree. C./1 s, 35 About + 68.degree. C./60 s 56.6
99.degree. C./1 s, 35 About +++ 68.degree. C./70 s 62.4 99.degree.
C./1 s, 35 About ++++ 68.degree. C./80 s 68.3 20 99.degree. C./1 s,
35 About ++ 68.degree. C./150 s 108.5 99.degree. C./1 s, 35 About
+++ 68.degree. C./165 s 117.3 + to ++++: Extent of amplification is
shown in 4 grades. -: No amplification being observed.
[0122] As shown in Table 4, when Polymerase A was used, there was
confirmed that a fragment of 2 kb, 8 kb or 20 kb was amplified
which was anticipated under any of rapid PCR conditions. Further,
when similar PCR was carried out with 0.5 .mu.l of Polymerase B,
there was found to have the same results as in Polymerase A.
[0123] (3) Comparison with DNA Polymerases Marketed by Other
Manufacturers
[0124] Among the rapid PCR experiments shown in Item (2) of Example
2, with respect to a product experiments allowing to amplify a DNA
fragment of 2 kb, its amount of amplified fragment was quantified.
As a control, KOD dash DNA Polymerase (manufactured by TOYOBO CO.,
LTD.) was used. With respect to KOD dash DNA Polymerase, there were
quantified an amount of amplified fragment, with a PCR reagent
mixture having an amount of used enzyme of 2.5 U, a standard unit
amount of the used enzyme described in its instruction manual, per
50 .mu.l of the PCR reagent mixture, and also with each of PCR
reagent mixtures having the unit amount increased to 5 U or 10
U.
[0125] Rapid PCR was carried out in 35 cycles, wherein one cycle
comprises a process consisting of 99.degree. C., 1
second-66.degree. C., 7 seconds. PCR was carried out in a fast mode
by using TaKaRa PCR Thermal Cycler PERSONAL (manufactured by Takara
Shuzo Co., Ltd.). The standard values in the fast mode of Thermal
Cycler are shown in Table 5.
7 TABLE 5 Standard Definition of Standard Value Value Heat Maximum
heating rate when heating from 35.degree. .gtoreq.1.5.degree. C./s
rate to 94.degree. C. Cool Maximum cooling rate when cooling from
94.degree. .gtoreq.1.5.degree. C./s rate to 40.degree. C. Over
Indicating how many degrees block temperature .ltoreq.1.0.degree.
C. temp is raised from the set temperature and then stabilized
Under Indicating how many degrees block temperature
.ltoreq.2.0.degree. C. temp is lowered from the set temperature and
then stabilized
[0126] When the temperature conditions were set as described above,
a step of one cycle required about 45 seconds, and a total time
period of PCR was about 25.7 minutes. Eight microliters of each
sample after termination of reaction was electrophoresed on 1%
agarose gel containing ethidium bromide in an amount of 0.00005%,
thereby confirming an amplified fragment. As a result, when
Polymerase A and 5 U and 10 U of KOD dash DNA Polymerase were used,
a desired amplified fragment of about 2 kb could be confirmed. On
the other hand, when 2.5 U of KOD dash DNA Polymerase was used, a
desired amplified fragment could not be confirmed. Therefore, a
desired amplified fragment was detected and quantified by using an
image analyzer FM-BIO (manufactured by Takara Shuzo Co., Ltd.)
having a further enhanced detection sensitivity, with a DNA
molecular weight marker at a known amount as a control, while
adjusting the detection sensitivity. As a result, there could be
confirmed a slight amplification even in 2.5 U of KOD dash DNA
Polymerase. The quantified values are shown in Table 6.
8 TABLE 6 Quantified Used Enzyme Value (ng) Polymerase A 153 2.5 U
KOD dash .ltoreq.10 5 U KOD dash 28 10 U KOD dash 88
[0127] As shown in Table 6, in the quantification by the image
analyzer, the amount of amplified DNA when Polymerase A was used
was about 153 ng, per 50 .mu.l of the sample after the
reaction.
[0128] On the other hand, in the quantification by the image
analyzer, even in the case of KOD dash DNA polymerase, an amplified
product of an amount more than 10 ng could not be obtained in a
standard used amount [2.5 U/50 .mu.l (PCR reagent mixture)]
described in its instruction manual. However, when the amount of
used enzyme was doubled or quadrupled, 28 ng and 88 ng of amplified
products, respectively, were obtained. In other words, not only in
Polymerase A but also in KOD dash DNA polymerase, the amount of
amplified DNA was increased by increasing its amount. In addition,
a PCR reagent mixture was prepared from the PCR reagent mixture for
Polymerase A used in Item (2) of Example 2 without adding sodium
alginate. When rapid PCR was carried out under the above reaction
conditions and by using the PCR reagent mixture using Polymerase A,
there was confirmed that an amplified fragment of the amount more
than 10 ng was obtained.
[0129] Therefore, under the above conditions, amplified fragments
could be visually confirmed by general agarose electrophoresis,
regardless of the kinds of the DNA polymerases, by using an amount
of DNA polymerase capable of obtaining an amplified product of an
amount more than 10 ng. Therefore, it was clarified that rapid PCR
of which amount of the resulting amplified product was more than 10
ng could be carried out.
EXAMPLE 3
[0130] A total time period of PCR in rapid PCR, the time period
being required for obtaining the same amount of the amplified
product using Polymerase A, was studied for 3 kinds of commercially
available thermal cyclers on the basis of basic protocol conditions
using TaKaRa EX Taq DNA polymerase in an amount of 1.25 U/50 .mu.l
(PCR reagent mixture) as described in the instruction manual for
TaKaRa EX Taq DNA polymerase. The basic protocol conditions were
set by referring to conditions described at page 6 of TaKaRa PCR
Enzymes (published by Takara Shuzo Co., Ltd., May Edition, 1998)
when amplifying a fragment of 20 kb; or to conditions for TaKaRa LA
Taq DNA polymerase described at page 8 of the manual attached to
TaKaRa LA PCR Kit Ver. 2.1 (manufactured by Takara Shuzo Co., Ltd.)
when amplifying fragments of a size other than the above. As the
thermal cyclers, there were used 3 equipments, namely TaKaRa PCR
Thermal Cycler MP (manufactured by Takara Shuzo Co., Ltd.; referred
to as "MP" in Table 7); TaKaRa PCR Thermal Cycler PERSONAL
(manufactured by Takara Shuzo Co., Ltd.; referred to as "PP" in
Table 7); and GeneAmp PCR System 9600 (manufactured by
Perkin-Elmer; referred to as "9600" in Table 7").
9TABLE 7 Temperature Conditions for One Cycle (Upper Row) Size
[Total Period of Time for PCR] (kb) of (Lower Row) Amplified Equip-
1.25 U/50 .mu.l (PCR DNA ments Reagent Mixture) Fragment Used
TaKaRa EX Taq Polymerase A 2 PP 98.degree. C./10 s, 68.degree.
C./60 s 98.degree. C./5 s, 66.degree. C./10 s [About 58 min.]
[About 27 min.] MP 98.degree. C./10 s, 68.degree. C./60 s
98.degree. C./1 s, 66.degree. C./10 s [About 67 min.] [About 37
min.] 9600 98.degree. C./10 s, 68.degree. C./60 s 98.degree. C./1
s, 66.degree. C./10 s [About 70 min.] [About 36 min.] 8 PP
98.degree. C./10 s, 68.degree. C./5 min. 98.degree. C./5 s,
68.degree. C./75 s [About 178 min.] [About 58 min.] MP 98.degree.
C./10 s, 68.degree. C./5 min. 98.degree. C./1 s, 68.degree. C./75 s
[About 187 min.] [About 69 min.] 9600 98.degree. C./10 s,
68.degree. C./5 min. 98.degree. C./1 s, 68.degree. C./75 s [About
190 min.] [About 67 min.] 20 PP 98.degree. C./10 s, 68.degree.
C./15 min. 98.degree. C./5 s, [About 478 min.] 68.degree. C./3 min.
[About 110 min.] MP 98.degree. C./10 s, 68.degree. C./15 min.
98.degree. C./1 s, [About 487 min.] 68.degree. C./3 min. [About 121
min.] 9600 98.degree. C./10 s, 68.degree. C./15 min. 98.degree.
C./1 s, [About 490 min.] 68.degree. C./3 min. [About 119 min.]
[0131] The kinds of the template used, the amount of the template,
the primer pair, the amount of the enzyme and the composition for
the PCR reagent mixture were made to be the same as those in
Example 2. PCR was carried out for 30 cycles under each of the
conditions listed in Table 7, and thereafter the amounts of the
amplified products were quantified by the method described in Item
(3) of Example 2. Incidentally, TaKaRa PCR Thermal Cycler PERSONAL
was set at normal mode when using 1.25 U, and set at fast mode when
using Polymerase A.
[0132] As a result, differences in the amounts of the amplified
products obtained under each of conditions using TaKaRa EX Taq DNA
polymerase in an amount of 1.25 U/50 .mu.l (PCR Reagent Mixture)
and Polymerase A were not found. In other words, the enzyme
activity indicated by each PCR performance was of the same level.
On the other hand, it has been shown from the results of Table 7
that a time period required for an entire amplification reaction,
namely, a total time period of PCR, is markedly shortened to about
1/2 to about 1/5 by using Polymerase A. Thus, it has been shown
that rapid PCR could be carried out. From the above, when an
effective amount of the DNA polymerase of the present invention was
used, it was shown that even with the shortened set time period, an
amplification ratio per one cycle was of the same level to that
under standard reaction conditions. Incidentally, as a control, PCR
was carried out under the reaction conditions used for Polymerase A
mentioned above by using TaKaRa EX Taq DNA polymerase in an amount
of 1.25 U/50 .mu.l (PCR Reagent Mixture). As a result, no
amplification could be confirmed in any of the primer pairs
used.
EXAMPLE 4
[0133] Comparison of the amounts of amplified products was made
when PCR was carried out using TaKaRa Taq DNA polymerase and
Polymerase A under reaction conditions appropriate for each
polymerase. An amount of a product of about 500 bp resulting from
amplification with .lambda.DNA as a template and primers .lambda.1
and .lambda.2 as the primer pair was used as an index.
[0134] a) TaKaRa Taq DNA Polymerase Reaction System: A PCR reagent
mixture having a final volume of 50 .mu.l comprising 1 ng or 100 pg
of .lambda.DNA, 10 pmol each of primers .lambda.1 and .lambda.2,
1.25 U of TaKaRa Taq DNA polymerase, and dATP, dCTP, dGTP and dTTP
each at a final concentration of 0.2 mM was prepared by using
TaKaRa PCR Amplification Kit and using the attached reaction buffer
in accordance with the instruction manual for the kit. PCR System
9600 was used as the thermal cycler. PCR was carried out in 25
cycles, wherein one cycle comprises a process consisting of
94.degree. C., 30 seconds-55.degree. C., 30 seconds-72.degree. C.,
30 seconds, one cycle being set at about 167 seconds. After
termination of the reaction, 8 .mu.l of each sample was
electrophoresed on 1% agarose gel containing ethidium bromide in an
amount of 0.00005%, thereby confirming an amplified fragment.
Further, an amount of an amplified fragment was quantified with a
DNA molecular weight marker at a known amount as a control, with
adjusting the detection sensitivity by using an image analyzer
FM-BIO.
[0135] b) Polymerase A Reaction System: A PCR reagent mixture
having a final volume of 50 .mu.l comprising 50 mM Tris-acetate (pH
8.5), 0.2 mM each of dATP, dCTP, dGTP and dTTP, 3 mM magnesium
acetate, 50 mM potassium acetate, 1 ng or 100 pg of .lambda.DNA, 10
pmol each of primers .lambda.1 and .lambda.2, 0.5 .mu.l of
Polymerase A and 2.5 .mu.g of sodium alginate was prepared. PCR
System 9600 was used as the thermal cycler. PCR was carried out in
25 cycles, wherein one cycle comprises a process consisting of
98.degree. C., 5 seconds-55.degree. C., 5 seconds-72.degree. C., 5
seconds. Eight microliters of the sample after termination of the
reaction was electrophoresed on 1% agarose gel containing ethidium
bromide in an amount of 0.00005%, thereby confirming an amplified
fragment. Further, an amplified fragment was quantified with a DNA
molecular weight marker at a known amount as a control, with
adjusting the detection sensitivity by using an image analyzer
FM-BIO. The quantified value was compared with the quantified value
of the amplified product obtained for the TaKaRa Taq DNA polymerase
having a concentration of 1.25 U.
[0136] As a result, the amount of the amplified product was larger
in a case where 1 ng of .lambda.DNA was used as a template for
either of the enzymes used. In addition, there was found no
differences in the amounts of the amplified products for the same
amount of the template between TaKaRa Taq DNA polymerase and
Polymerase A, showing that substantially the same amount of the DNA
fragment was amplified for both PCRs. In other words, the enzyme
activity represented by PCR performance was of the same level for
both PCRs. Therefore, in a reaction consisting of the same number
of cycles, while the extension reaction time in the TaKaRa Taq DNA
polymerase system was 30 seconds at 72.degree. C., the extension
reaction of 5 seconds at 72.degree. C. was enough in the Polymerase
A Reaction System.
[0137] In other words, there could be confirmed that rapid PCR
could be carried out in which about 167 seconds per one cycle in a
convention process was shortened to about 92 seconds per one cycle,
a total time period of PCR being shortened to about 1/2.
EXAMPLE 5
[0138] Enhancement of Taq DNA Polymerase-Synthesizing Activity by
Acidic Substance
[0139] (1) Preparation of Sulfated-Fucose-Containing
Polysaccharide-F
[0140] The sulfated-fucose-containing polysaccharide-F used in the
subsequent examples was purified by the following process. A
preparation example of sulfated-fucose-containing polysaccharide-F
is given hereinbelow.
[0141] Gagome seaweed was sufficiently dried, and thereafter 20 kg,
a weight on a dry basis, of the Gagome seaweed was pulverized.
Next, the resulting dry powder was suspended in 900 liters of tap
water containing 7.3 kg of calcium chloride.dihydrate, and the
temperature was raised to 90.degree. C. over a period of 40 minutes
with stirring. The extraction was carried out for one hour with
keeping at 90.degree. to 95.degree. C. Thereafter, the resulting
solution was cooled to 20.degree. C., stopped stirring, and allowed
to stand overnight, to give an extract.
[0142] Next, solid-liquid separation was carried out using a
centrifuge (Model CNA, manufactured by Westfalia Separator Inc.).
About 900 liters of supernatant of the solid-liquid separation was
obtained from the above extract by using the centrifuge.
Three-hundred and sixty liters of the supernatant was filtered with
SPARKLER FILTER (manufactured by Nippon Senshoku Kikai)
incorporated with a filter of a size of 3 .mu.m (manufactured by
Nippon Shokuhin Rozai). The filtrate was concentrated to a volume
of 20 liters by an UF membrane ("FE10-FC-FUS0382," manufactured by
DAICEL CHEMICAL INDUSTRIES, LTD.) having a fractionated molecular
weight of 30000. Thereafter, 20 liters of tap water was added to
the resulting concentrate, and the dilution was concentrated again
to a volume of 20 liters. The dilution-concentration procedures as
described above were repeated five time, to give liters of a
concentrate.
[0143] To 700 ml of the above concentrate were added so as to give
a final concentration of 0.2 M calcium chloride and 20 mM sodium
acetate. Thereafter, the resulting mixture was dialyzed against 20
mM sodium acetate equilibrated buffer (pH 6.0) containing 0.2 M
calcium chloride. The solution after the dialysis treatment was
applied to 3500 ml of DEAE-Sepharose FF column (column inner
diameter: 9.7 cm) equilibrated with 10 liters of the above
equilibrated buffer, and washed with 5 liters of the equilibrated
buffer. The elution was carried out under 3-step gradient
conditions given hereinbelow.
[0144] Incidentally, the flow rate of the chromatography was set at
3500 ml/1 hour.
[0145] Gradient Conditions:
[0146] 1] linear gradient of 0 to 0.5 M sodium chloride (amount of
eluent: 4.5 liters)
[0147] 2] linear gradient of 0.5 to 1.0 M sodium chloride (amount
of eluent: 4.5 liters)
[0148] 3] linear gradient of 1.0 to 2.0 M sodium chloride (amount
of eluent: 4.5 liters)
[0149] The eluent was collected 250 ml per one fraction. Each
fraction was subjected to sugar quantification by phenol sulfuric
acid method, and to uromic acid quantification by
carbazole-sulfuric acid method. As a result, fractions of Fraction
Nos. 40 to 53, which were fractions having high sugar contents and
low contents of uronic acid, were obtained. The fractions of
Fraction Nos. 40 to 53 are referred to "sulfated-fucose-containing
polysaccharide-F fractions." Each of the sulfated-fucose-containing
polysaccharide-F fractions was concentrated with a ultrafiltration
membrane of 100000, and thereafter the concentrate was dialyzed
against 50 mM sodium citrate, and further dialyzed overnight
against distilled water. Subsequently, the mixture was lyophilized,
to give 1.696 g of sulfated-fucose-containing polysaccharide-F from
the sulfated-fucose-containing polysaccharide-F fraction.
[0150] (2) Effects of Acidic Substance Against Taq DNA
Polymerase
[0151] Using as acidic substances the sulfated-fucose-containing
polysaccharide-F obtained by the process described in Item (1) of
Example 5, dextran sulfate powder (manufactured by Onco), or sodium
alginate (100 to 150 centipoises, manufactured by Wako Pure
Chemicals), the effects of these acidic substances on the activity
of TaKaRa Taq DNA polymerase (manufactured by Takawa Shuzo Co.,
Ltd.) were examined.
[0152] A PCR reagent mixture comprising .lambda.DNA as a template,
primers .lambda.1 and .lambda.7 as a primer pair and TaKaRa Taq DNA
polymerase as a DNA polymerase was prepared, and PCR was carried
out. The PCR reagent mixture was prepared so as to have the
following composition.
[0153] Composition of PCR Reagent Mixture:
[0154] Buffer for TaKaRa Taq DNA polymerase, 10 U of TaKaRa Taq DNA
polymerase, 100 pg of .lambda.DNA, 0.2 mM each of dATP, dCTP, dGTP
and dTTP, and 5 pmol each of primers .lambda.1 and .lambda.7 (final
volume being 25 .mu.l). Further, 0.25 ng of the
sulfated-fucose-containing polysaccharide-F, 0.25 ng of dextran
sulfate powder, or 0.5 .mu.g of sodium alginate were added as
acidic substances to the above PCR reagent mixture.
[0155] The reaction was carried out in 30 cycles for a total time
period of PCR of about 110 minutes, wherein one cycle of reaction
comprises a process consisting of 98.degree. C., 5
seconds-68.degree. C., 3 minutes. After the termination of
reaction, 5 .mu.l of the resulting sample was electrophoresed on 1%
agarose gel containing ethidium bromide in an amount of 0.00005%,
thereby confirming an amplified fragment. The results thereof are
shown in Table 8.
10 TABLE 8 Amount Amplification Acidic Substance Added Results
Sulfated-Fucose- 0.25 ng ++ Containing Polysaccharide-F Dextran
Sulfate 0.25 ng ++ Powder Sodium Alginate 0.5 .mu.g ++ No Addition
.+-. ++: Intensive amplification being observed; +: Amplification
being observed; .+-.: Slight amplification being observed; and -:
No amplification being observed.
[0156] As shown in Table 8, there was confirmed that an expected 8
kb fragment was excellently amplified in case where any kinds of
acidic substances were added, and thus rapid PCR could be carried
out. Incidentally, in this Example, when template DNA was increased
from 100 pg to 1 ng, the amount of a desired amplified product was
improved even in a case of no addition of the acidic substance. On
the other hand, even when the reaction was carried out under the
above PCR conditions by using 1.25 U of TaKaRa Taq DNA polymerase
and 1 ng of template DNA, a desired amplified product could not be
obtained. By adding the acidic substance, it was shown that rapid
PCR of an even higher performance could be carried out.
EXAMPLE 6
[0157] Rapid PCR by Combination of DNA Polymerase for LA-PCR with
Acidic Substance
[0158] Rapid PCR in the presence of the acidic substances was
studied in LA-PCR using a combination of a DNA polymerase having
3'.fwdarw.5' exonuclease activity and a DNA polymerase without
having the activity.
[0159] (1) Rapid PCR by Combination of Polymerase A with Acidic
Substance
[0160] Using .lambda.DNA as template, a PCR reagent mixture
comprising .lambda.DNA as a template, primers .lambda.1 and
.lambda.8 as a primer pair was prepared, and PCR was carried out.
The composition for a PCR reagent mixture is as follows.
[0161] Composition of PCR Reagent Mixture:
[0162] 50 mM Tris-acetate (pH 8.5), 0.2 mM each of dATP, dCTP, dGTP
and dTTP, 3 mM magnesium acetate, 50 mM potassium acetate, 10 pg of
.lambda.DNA, 0.5 .mu.l of Polymerase A and 10 pmol each of primers
.lambda.1 and .lambda.8 (final volume being 25 .mu.l). Further, 2.5
.mu.g of sodium alginate was added to the above PCR reagent
mixture. As a control, a PCR reagent mixture without adding sodium
alginate was also prepared.
[0163] Reaction Conditions: The reaction was carried out in 30
cycles, wherein one cycle of reaction comprises a process
consisting of 98.degree. C., 5 seconds-68.degree. C., 75 seconds.
The reaction was carried out in fast mode using TaKaRa PCR Thermal
Cycler PERSONAL (manufactured by Takara Shuzo Co., Ltd.).
[0164] After the termination of reaction, 6 .mu.l each of the
resulting sample was electrophoresed on 1% agarose gel containing
ethidium bromide in an amount of 0.00005%, thereby confirming an
amplified fragment of about 10 kb. The results thereof are shown in
Table 9.
11 TABLE 9 Acidic Amplification Substance Results Addition of ++
Sodium Alginate No Addition - ++: Intensive amplification being
observed; +: Amplification being observed; .+-.: Slight
amplification being observed; and -: No amplification being
observed.
[0165] As shown in Table 9, when sodium alginate was added,
amplification of a fragment of about 10 kb was confirmed. On the
other hand, when there was no addition of sodium alginate, an
amplified fragment of about 10 kb could not be confirmed under the
above conditions, but when similar PCR was carried out by
increasing the amount of template DNA from 10 pg to 1 ng, a desired
amplified fragment could be confirmed. Incidentally, even when the
amount of template DNA was changed to 1 ng, a desired fragment
could not be confirmed in PCR using a standard used amount of
TaKaRa EX Taq DNA polymerase. It was shown that rapid PCR could be
carried out with a further higher performance by addition of the
acidic substance.
[0166] (2) Rapid PCR by Combination of Polymerase B with Acidic
Substance
[0167] A PCR reagent mixture comprising .lambda.DNA as a template
and primers .lambda.1 and .lambda.9 as a primer pair was prepared,
and PCR was carried out. The composition for a PCR reagent mixture
is as follows.
[0168] Composition of PCR Reagent Mixture:
[0169] Buffer for TaKaRa EX Taq DNA polymerase, 0.2 mM each of
dATP, dCTP, dGTP and dTTP, 10 pg of .lambda.DNA, 0.5 .mu.l of
Polymerase B and 5 pmol each of primers .lambda.1 and .lambda.9
(final volume being 25 .mu.l). Further, 0.75 ng of
sulfated-fucose-containing polysaccharide-F or 5 .mu.g of sodium
alginate was added, respectively, to the above PCR reagent
mixture.
[0170] The reaction was carried out in 30 cycles, wherein one cycle
of reaction comprises a process consisting of 98.degree. C., 5
seconds-68.degree. C., 3 minutes. After the termination of
reaction, 5 .mu.l of the resulting sample was electrophoresed on 1%
agarose gel containing ethidium bromide in an amount of 0.00005%,
thereby confirming, an amplified fragment. The results thereof are
shown in Table 10.
12 TABLE 10 Amount Amplification Acidic Substance Added Results
Sulfated-Fucose- 0.75 ng ++ Containing Polysaccharide-F Sodium
Alginate 5 .mu.g +++ No Addition + + to +++: Extent of
amplification is shown in 3 grades.
[0171] As shown in Table 10, in case where any of the acidic
substances were added, there was confirmed that the amount of a
desired amplified fragment of 12 kb was improved, as compared to
the case where the acidic substance was not added.
EXAMPLE 7
[0172] The effects of the amount of DNA polymerase used for PCR and
the acidic substance and its effects on the reaction time were
studied.
[0173] A PCR reagent mixture comprising .lambda.DNA as a template
and primers .lambda.1 and .lambda.8 as a primer pair was prepared,
and PCR was carried out. In this PCR, TaKaRa EX Taq DNA polymerase
and Polymerase B were used. The composition for a PCR reagent
mixture is shown below.
[0174] Composition of PCR Reagent Mixture:
[0175] Buffer for TaKaRa EX Taq DNA polymerase, 0.2 mM each of
dATP, dCTP, dGTP and dTTP, 10 pg of .lambda.DNA, 1.25 U of TaKaRa
EX Taq DNA polymerase or 0.5 .mu.l of Polymerase B and 10 pmol each
of primers .lambda.1 and .lambda.8 (final volume being 50
.mu.l).
[0176] Further, to the above PCR reagent mixture was added 2.5
.mu.g of sodium alginate, to prepare a PCR reagent mixture.
[0177] The reaction was carried out in 30 cycles for a total period
for PCR of about 80 minutes, wherein one cycle of reaction
comprises a process consisting of 98.degree. C., 5
seconds-68.degree. C., 2 minutes. After the termination of
reaction, 8 .mu.l of the resulting sample was electrophoresed on 1%
agarose gel containing ethidium bromide in an amount of 0.00005%,
thereby confirming an amplified fragment. The results thereof are
shown in Table 11.
13 TABLE 11 Amount Amplification Enzyme Used Added Results 1.25
U/50 .mu.l No Addition - (PCR Reagent Mixture) of TaKaRa EX Taq
Polymerase B No Addition + Polymerase B 2.5 .mu.g ++ ++: Intensive
amplification being observed; +: Amplification being observed;
.+-.: Slight amplification being observed; and -: No amplification
being observed.
[0178] As shown in Table 11, in both cases where Polymerase B was
used and where sodium alginate was added to Polymerase B,
amplification of an expected fragment of 10 kb was confirmed, and
thus rapid PCR could be carried out. Further, the amounts of the
amplified products were quantified by using an image analyzer
FM-BIO (manufactured by Takara Shuzo Co., Ltd.), and as a result,
the amount of the amplified product of the case where sodium
alginate was added to Polymerase B was about five times as that
compared to the case where no sodium alginate was added, and thus
more rapid PCR could be achieved. On the other hand, when 1.25 U of
TaKaRa EX Taq DNA polymerase, a standard used amount as described
in TaKaRa EX Taq instruction manual, was used alone, amplification
by rapid PCR could not be confirmed.
EXAMPLE 8
[0179] The effects of the combination of spergualins and acidic
substances on the DNA polymerase were studied.
[0180] (1) A PCR reagent mixture comprising .lambda.DNA as a
template and primers .lambda.1 and .lambda.8 as a primer pair was
prepared, and rapid PCR was carried out. In this PCR, Polymerase B
was used. As the spergualins, 15-deoxyspergualin trihydrochloride
was used, and as the acidic substances, sodium alginate
(manufactured by Wako Pure Chemicals) was used. The composition for
a PCR reagent mixture is shown below.
[0181] Composition of PCR Reagent Mixture:
[0182] 50 mM Tris-acetate buffer (pH 8.5), 0.2 mM each of dATP,
dCTP, dGTP and dTTP, 3 mM magnesium acetate, 50 mM potassium
acetate, 10 pg of .lambda.DNA, 10 pmol each of primers .lambda.1
and .lambda.8. To the above PCR reagent mixture was added 0.5 .mu.l
of Polymerase B, and further 15-deoxyspergualin trihydrochloride
and sodium alginate were added in combination at concentrations
shown in Table 10, making up a final volume of 50 .mu.l. In
addition, as controls, there were respectively prepared a PCR
reagent mixture in which 2.5 .mu.g of sodium alginate was added
without adding 15-deoxyspergualin trihydrochloride, and a PCR
reagent mixture in which both 15-deoxyspergualin trihydrochloride
and sodium alginate were not added.
[0183] Rapid PCR was carried out in 30 cycles for a total time
period of PCR of about 65.5 minutes, wherein one cycle of reaction
comprises a process consisting of 98.degree. C., 5
seconds-68.degree. C., 90 seconds. After the termination of
reaction, 8 .mu.l of the resulting sample was electrophoresed on 1%
agarose gel containing ethidium bromide in an amount of 0.00005%,
thereby confirming an amplified fragment. The results thereof are
shown in Table 12.
14 TABLE 12 Spergualins/ Acidic Substances Amplification (Amount
Added) Results 15-Deoxyspergualin/ Sodium Alginate 120 .mu.M/2.5
.mu.g ++++ 100 .mu.M/2.5 .mu.g ++++ 80 .mu.M/2.5 .mu.g +++ 60
.mu.M/2.5 .mu.g ++ 20 .mu.M/2.5 .mu.g + No Addition/2.5 .mu.g + No
Addition/No Addition - + to ++++: Extent of amplification is shown
in 4 grades. -: No amplification being observed.
[0184] As shown in Table 12, in a system where 15-deoxyspergualin
trihydrochloride and sodium alginate were added in combination, the
DNA amplification reaction of a 10 kb fragment was enhanced, and
thus it was confirmed that rapid PCR for a total time period of PCR
of about 65.5 minutes could be achieved. Incidentally, in this
example, when the amount of template DNA was increased from 10 pg
to 1 ng, a desired amplified product could be confirmed, even in a
system where both the spergualin and the acidic substance were not
added. On the other hand, even though PCR was carried out under the
above conditions using 1.25 U of TaKaRa Taq DNA polymerase and 1 ng
of template DNA, a desired amplified product could not be obtained.
It was shown that rapid PCR with a further higher performance could
be achieved by the addition of the acidic substance.
[0185] (2) The effects of the combination of spergualins and acidic
substances on the DNA polymerase were studied in a case where
template DNA was E. coli genomic DNA. The composition for a PCR
reagent mixture is shown below.
[0186] Composition of PCR Reagent Mixture:
[0187] The composition for the PCR reagent mixture was the same as
that in Item (1) of Example 8, except for using 5 ng of E. coli
genomic DNA (manufactured by Takara Shuzo Co., Ltd.) and 10 pmol
each of primers of Eco-1 and Eco-6. To the above PCR reagent
mixture was added 0.5 .mu.l of Polymerase A, and further were added
in combination 2.5 .mu.g of sodium alginate and 15-deoxyspergualin
trihydrochloride so as to have a final concentration of 1 .mu.M, 5
.mu.M or 10 .mu.M, respectively, making up a final volume of 50
.mu.l. In addition, as controls, there were respectively prepared a
PCR reagent mixture in which 2.5 .mu.g of sodium alginate was only
added without adding 15-deoxyspergualin trihydrochloride, and a PCR
reagent mixture in which both 15-deoxyspergualin trihydrochloride
and sodium alginate were not added.
[0188] Rapid PCR was carried out in 30 cycles for a total time
period of PCR of about 110 minutes, wherein one cycle of reaction
comprises a process consisting of 98.degree. C., 5
seconds-68.degree. C., 3 minutes. After the termination of
reaction, 8 .mu.l of the resulting sample was electrophoresed on 1%
agarose gel containing ethidium bromide in an amount of 0.00005%,
thereby confirming an amplified fragment. The results thereof are
shown in Table 13.
15 TABLE 13 Spergualins/ Acidic Substances Amplification (Amount
Added) Results 15-Deoxyspergualin/ Sodium Alginate 10 .mu.M/2.5
.mu.g ++ 5 .mu.M/2.5 .mu.g ++ 1 .mu.M/2.5 .mu.g ++ No Addition/2.5
.mu.g .+-. No Addition/No Addition - ++: Intensive amplification
being observed; +: Amplification being observed; .+-.: Slight
amplification being observed; and -: No amplification being
observed.
[0189] As shown in Table 13, in a system where 15-deoxyspergualin
trihydrochloride and sodium alginate were added in combination, the
DNA amplification reaction of a 20 kb fragment was enhanced, and
thus it was confirmed that rapid PCR for a total time period of PCR
of about 110 minutes could be achieved. Incidentally, in this
example, when the amount of template DNA was increased from 5 ng to
20 ng, a desired amplified product could be confirmed, even in a
system where both the spergualins and the acidic substances were
not added. On the other hand, even though PCR was carried out under
the above conditions using 1.25 U of TaKaRa Taq DNA polymerase and
1 ng of template DNA, a desired amplified product could not be
obtained. It was shown that rapid PCR with a further higher
performance could be achieved by the addition of the acidic
substance.
EXAMPLE 9
[0190] Preparation of Kit
[0191] (1) Kit in Which Polymerase A or Polymerase B is Used
[0192] A kit (20 reactions) for rapid PCR of the present invention
was constructed.
[0193] The composition for the kit is shown below:
16 10 .times. Reaction Buffer 50 .mu.l 500 mM Tris-Acetate (pH 8.5)
500 mM Potassium Acetate 30 mM Magnesium Acetate 2.5 mM dNTPs Mix
80 .mu.l (2.5 mM Each of dATP, dCTP, dGTP and dTTP) DNA Polymerase
Enzyme Solution 10 .mu.l Polymerase A or Polymerase B
[0194] A PCR reagent mixture was prepared using the above kit. E.
coli genomic DNA was used as a template. The composition for the
PCR reagent mixture is shown below:
17 Composition of PCR Reagent Mixture: 10 .times. Reaction Buffer 5
.mu.l dNTPs Mix 4 .mu.l DNA Polymerase Enzyme Solution 0.5 .mu.l E.
coli genomic DNA 1 ng Eco-1 Primer 10 pmol Eco-2 Primer 10 pmol
Sterilized Distilled Water Final Volume 50 .mu.l
[0195] The PCR reagent mixture was reacted under the PCR conditions
shown in Item (3) of Example 2, and as a result, a desired
amplified fragment of about 2 kbp could be confirmed.
[0196] (2) Kit in Which Polymerase A or Polymerase B is Used and
Further Comprises Acidic Substance
[0197] A kit (20 reactions) for rapid PCR of the present invention
was constructed.
[0198] The composition for the kit is shown below:
18 10 .times. Reaction Buffer 50 .mu.l 500 mM Tris-Acetate (pH 8.5)
500 mM Potassium Acetate 30 mM Magnesium Acetate 25 .mu.g Sodium
Alginate (100 to 150 centipoises) 2.5 mM dNTPs Mix 80 .mu.l (2.5 mM
Each of dATP, dCTP, dGTP and dTTP) DNA Polymerase Enzyme Solution
10 .mu.l Polymerase A or Polymerase B
[0199] A PCR reagent mixture was prepared using the above kit.
.lambda.DNA was used as a template. The composition for the PCR
reagent mixture is shown below:
19 Composition of PCR Reagent Mixture: 10 .times. Reaction Buffer 5
.mu.l dNTPs Mix 4 .mu.l DNA Polymerase Enzyme Solution 0.5 .mu.l
.lambda.DNA 1 ng .lambda.1 Primer 10 pmol .lambda.8 Primer 10 pmol
Sterilized Distilled Water Final Volume 50 .mu.l
[0200] The PCR reagent mixture was reacted under the PCR conditions
shown in Example 7, and as a result, a desired amplified fragment
of about 10 kbp could be confirmed.
[0201] (3) Kit Comprising Polymerase A or Polymerase B, Acidic
Substances, and Spergualins
[0202] A kit (20 reactions) for rapid PCR of the present invention
was constructed.
[0203] The composition for the kit is shown below:
20 10 .times. Reaction Buffer 100 .mu.l 500 mM Tris-Acetate (pH
8.5) 500 mM Potassium Acetate 30 mM Magnesium Acetate 0.04% Sodium
Alginate (100 to 150 centipoises) 1 mM 15-Deoxyspergualin
Trihydrochloride 2.5 mM dNTPs Mix 160 .mu.l (2.5 mM Each of dATP,
dCTP, dGTP and dTTP) DNA Polymerase Enzyme Solution 10 .mu.l
Polymerase B Concentrate
[0204] A PCR reagent mixture was prepared using the above kit.
.lambda.DNA was used as a template. The composition for the PCR
reagent mixture is shown below:
21 Composition of PCR Reagent Mixture: 10 .times. Reaction Buffer 5
.mu.l dNTPs Mix 8 .mu.l DNA Polymerase Enzyme Solution 0.5 .mu.l
.lambda.DNA 10 pg .lambda.1 Primer 10 pmol .lambda.8 Primer 10 pmol
Sterilized Distilled Water Final Volume 50 .mu.l
[0205] The PCR reagent mixture was reacted tinder the PCR
conditions shown in Item (1) of Example 8, and as a result, a
desired amplified fragment of about 10 kbp could be confirmed.
[0206] Industrial Applicability
[0207] Rapid PCR which is useful in the field of genetic
engineering is provided by the DNA synthesis method and the kit for
the DNA synthesis reaction of the present invention, so that there
is exhibited an excellent effect that the procedures in the genetic
engineering studies and industries which are involved with PCR can
be speeded up. In addition, the above-mentioned DNA synthesis
method, the kit for the DNA synthesis reaction and the article of
manufacture of a PCR agent of the present invention are extremely
useful in speeding up and activation of procedures in the entire
field in which PCR method can be used. Further, according to the
present invention, there can be achieved handling of a large amount
of DNA amplified samples in a short period of time and the
preparation of a large amount of PCR products. Therefore, the
present invention can be utilized in a variety of fields such as
the field of genetic diagnosis on the basis of PCR method, DNA
chips for diagnosing genes, and the like.
Sequence CWU 1
1
14 1 35 DNA Artificial Sequence Artificial sequence derived from
lambda DNA. 1 gatgagttcg tgtccgtaca actggcgtaa tcatg 35 2 25 DNA
Artificial Sequence Artificial sequence derived from lambda DNA. 2
ggttatcgaa atcagccaca gcgcc 25 3 23 DNA Artificial Sequence
Artificial sequence derived from lambda DNA. 3 gcgtaccttt
gtctcacggg caa 23 4 22 DNA Artificial Sequence Artificial sequence
derived from lambda DNA. 4 gatagctgtc gtcataggac tc 22 5 23 DNA
Artificial Sequence Artificial sequence derived from lambda DNA. 5
cttaaccagt gcgctgagtg act 23 6 28 DNA Artificial Sequence
Artificial sequence derived from lambda DNA. 6 ttgccacttc
cgtcaaccag gcttatca 28 7 29 DNA Artificial Sequence Artificial
sequence derived from lambda DNA. 7 tgtccgtcag ctcataacgg tacttcacg
29 8 28 DNA Artificial Sequence Artificial sequence derived from
lambda DNA. 8 atatctggcg gtgcaatatc ggtactgt 28 9 28 DNA Artificial
Sequence Artificial sequence derived from lambda DNA. 9 gacaatctgg
aatacgccac ctgacttg 28 10 35 DNA Artificial Sequence Artificial
sequence derived from E. coli. 10 ggtggcgatg caaatgcaat cttcgttgcc
ccaac 35 11 35 DNA Artificial Sequence Artificial sequence derived
from E. coli. 11 ttatgtatgc cgcgtatcag cttcatgtct ggctc 35 12 35
DNA Artificial Sequence Artificial sequence derived from E. coli.
12 gagccagaca tgaagctgat acgcggcata cataa 35 13 35 DNA Artificial
Sequence Artificial sequence derived from E. coli. 13 atcatctaac
ctgttctgga aaacgcttgc gcagc 35 14 35 DNA Artificial Sequence
Artificial sequence derived from E. coli. 14 tgcaaatact tctgcgccaa
tgcggcattt gaagt 35
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