U.S. patent application number 12/954828 was filed with the patent office on 2011-07-14 for method for enhancing polymerase activity.
Invention is credited to Ikunoshin KATO, Eiji KOBAYASHI, Hiroyuki MUKAI, Yoshimi SATO, Yuki UEDA, Takashi UEMORI.
Application Number | 20110171717 12/954828 |
Document ID | / |
Family ID | 39318393 |
Filed Date | 2011-07-14 |
United States Patent
Application |
20110171717 |
Kind Code |
A1 |
KOBAYASHI; Eiji ; et
al. |
July 14, 2011 |
METHOD FOR ENHANCING POLYMERASE ACTIVITY
Abstract
A polymerase activity is effectively enhanced by adding an
anionic surfactant, in particular an anionic surfactant having a
polyethoxyl group, to a reaction mixture containing a
polymerase.
Inventors: |
KOBAYASHI; Eiji; (Otsu-shi,
JP) ; UEDA; Yuki; (Otsu-shi, JP) ; SATO;
Yoshimi; (Otsu-shi, JP) ; UEMORI; Takashi;
(Otsu-shi, JP) ; MUKAI; Hiroyuki; (Otsu-shi,
JP) ; KATO; Ikunoshin; (Otsu-shi, JP) |
Family ID: |
39318393 |
Appl. No.: |
12/954828 |
Filed: |
November 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11866148 |
Oct 2, 2007 |
7846703 |
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12954828 |
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60848401 |
Oct 2, 2006 |
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Current U.S.
Class: |
435/194 |
Current CPC
Class: |
C12N 9/1241
20130101 |
Class at
Publication: |
435/194 |
International
Class: |
C12N 9/12 20060101
C12N009/12 |
Claims
1. A method for enhancing a polymerase activity, the method
comprising adding an anionic surfactant having a polyethoxyl group
to a reaction mixture containing a polymerase.
2. The method according to claim 1, wherein the anionic surfactant
has a sulfonate group or a carboxylate group.
3. The method according to claim 1, wherein the anionic surfactant
is selected from the group consisting of allyl alcohol
1,2-butoxylate-block-ethoxylate ammonium sulfate, glycolic acid
ethoxylate 4-nonylphenyl ether, glycolic acid ethoxylate oleyl
ether and poly(ethylene glycol)4-nonylphenyl 3-sulfopropyl ether as
well as salts thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/866,148, filed Oct. 2, 2007, which claims the priority
of U.S. provisional patent application No. 60/848,401, filed Oct.
2, 2006, the contents of each of which are incorporated herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method for enhancing a
polymerase activity as well as a composition and a reaction mixture
used for said method, which are useful in the field of genetic
engineering.
[0004] 2. Description of Related Art
[0005] A polymerase is one of the most important enzymes in the
field of genetic engineering. Various additives such as DMSO,
glycerol, formamide and poly(ethylene glycol) have been reported to
enhance a polymerase activity. A surfactant is also one of
additives for enhancing a polymerase activity. It is known that a
nonionic surfactant stabilizes a thermostable DNA polymerase
(Japanese Patent No. 2719528 (corresponding to U.S. Pat. No.
6,127,155)). Furthermore, it has been reported that a cationic
surfactant, polyethoxylated amine, stabilizes a polymerase
(Japanese Patent No. 3673175 (corresponding to U.S. Pat. No.
6,242,235)). In this document, it is predicted that high affinity
ionic bonds protect a protein from denaturation by other active
substances, and it is shown that ionic surfactants stabilize a
polymerase better than nonionic surfactants. Specifically, the
charged groups on the ionic surfactant electrostatically interact
with the charged residues on the surface of the protein, the
hydrophobic region in the ionic surfactant hydrophobically binds to
the hydrophobic site in the protein, and the protein is protected
due to the noncovalent crosslinking action. However, it is feared
that a cationic surfactant may interact with an anionic nucleic
acid molecule as a substrate for a polymerase to reduce the
reactivity of the polymerase, although it depends on the
concentration of the added surfactant.
[0006] Anionic surfactants such as sodium dodecyl sulfate (SDS),
sodium deoxycholate, sodium N-lauroyl sarcosinate are known to
inhibit a polymerase activity (Weyant, R. S. et al., Bio
Techniques, 1990, Vol. 9, p. 308-309). Such anionic surfactants
inhibit a polymerase activity even at low concentrations (e.g.,
0.01% of SDS). Thus, if a polymerase reaction mixture is
contaminated with an anionic surfactant used in pretreatment such
as a nucleic acid extraction step, the polymerase activity is
considerably inhibited.
[0007] With the progress in genetic engineering techniques,
large-scale reading of nucleotide sequences and replication of a
large amount of nucleic acid are routinely conducted. The influence
of enhancement of a polymerase activity is great even if the degree
is low. Thus, an additive that enhances a polymerase activity more
effectively than conventional additives has been desired.
Furthermore, an additive having an effect that is not brought by
conventional additives has also been required.
SUMMARY OF THE INVENTION
[0008] The present invention has been made in view of the
above-mentioned prior art. The main object of the present invention
is to provide a method for enhancing a polymerase activity more
effectively.
[0009] As a result of intensive studies, the present inventors have
found that a polymerase activity can be effectively enhanced by
adding an anionic surfactant having a polyethoxyl group to a
reaction mixture containing a polymerase. Thus, the present
invention has been completed.
[0010] The first aspect of the present invention relates to a
method for enhancing a polymerase activity, the method comprising
adding an anionic surfactant having a polyethoxyl group to a
reaction mixture containing a polymerase. According to the first
aspect, the anionic surfactant may have a sulfonate group or a
carboxylate group. Furthermore, the anionic surfactant may be
selected from the group consisting of allyl alcohol
1,2-butoxylate-block-ethoxylate ammonium sulfate, glycolic acid
ethoxylate 4-nonylphenyl ether, glycolic acid ethoxylate oleyl
ether and poly(ethylene glycol)4-nonylphenyl 3-sulfopropyl ether as
well as salts thereof.
[0011] The second aspect of the present invention relates to a
composition comprising a polymerase and an anionic surfactant
having a polyethoxyl group. According to the second aspect, the
anionic surfactant may have a sulfonate group or a carboxylate
group. Furthermore, the anionic surfactant may be selected from the
group consisting of allyl alcohol 1,2-butoxylate-block-ethoxylate
ammonium sulfate, glycolic acid ethoxylate 4-nonylphenyl ether,
glycolic acid ethoxylate oleyl ether and poly(ethylene
glycol)4-nonylphenyl 3-sulfopropyl ether as well as salts
thereof.
[0012] A polymerase activity can be enhanced more effectively using
the method and the composition of the present invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0013] FIG. 1 shows the effect of the method of the present
invention.
[0014] FIG. 2 shows the effect of the method of the present
invention.
[0015] FIG. 3 shows the effect of the method of the present
invention.
[0016] FIG. 4 shows the effect of the method of the present
invention.
[0017] FIG. 5 shows the effect of the method of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0018] As used herein, "a polymerase" is an enzyme that extends a
nucleic acid strand. It attaches a deoxyribonucleoside triphosphate
(dNTP) or a ribonucleoside triphosphate (NTP) to a 3' end of a
nucleic acid strand (polymerizes) to synthesize a nucleic acid
strand into which the corresponding nucleoside monophosphate is
incorporated. Polymerases include DNA-dependent DNA polymerases,
RNA-dependent DNA polymerases and DNA-dependent RNA
polymerases.
[0019] As used herein, "a polymerase activity" means an activity of
attaching a deoxyribonucleoside triphosphate or a ribonucleoside
triphosphate to a nucleic acid strand to synthesize a nucleic acid
strand into which the corresponding nucleoside monophosphate is
incorporated, i.e., an activity of catalyzing extension of a
nucleic acid.
[0020] As used herein, "enhancement of a polymerase activity" means
increase in an enzymatic activity of a polymerase. Specifically,
enhancement of a polymerase activity means: increase in an activity
of incorporating a nucleoside monophosphate into a nucleic acid by
a polymerase; increase in an amplification product generated by a
polymerase chain reaction (PCR); increase in a length of a nucleic
acid strand extended by a polymerase; or increase in accuracy of an
incorporated nucleoside triphosphate (dNTP or NTP). Enhancement of
a polymerase activity according to the present invention also
includes increase in an enzymatic activity of a polymerase by
stabilizing a polymerase protein. Furthermore, enhancement of a
polymerase activity according to the present invention includes
increase in a polymerization activity of a polymerase by
suppressing an action of inhibiting a polymerase activity by a
contaminant or the like.
[0021] As used herein, "a surfactant" is a compound having both a
hydrophobic part and a hydrophilic part in a single molecule. It
gathers on a boundary between oil and water or between air and
water and reduces the boundary tension to stabilize the boundary.
"An anionic surfactant" is a surfactant that is dissociated in an
aqueous solution into an ion, and of which the atomic group
exerting the surface activity forms an anion. "A cationic
surfactant" is a surfactant that is dissociated in an aqueous
solution into an ion, and of which the atomic group exerting the
surface activity forms a cation.
[0022] 1. Method for Enhancing Polymerase Activity
[0023] The present invention provides a method for enhancing a
polymerase activity, comprising adding an anionic surfactant having
a polyethoxyl group to a reaction mixture containing a
polymerase.
[0024] There is no specific limitation concerning the anionic
surfactant to be added as long as it has a polyethoxyl group
(polyoxyethylene group) and enhances a polymerase activity. A
polyethoxyl group (polyoxyethylene group) has a structure in which
an ethoxyl group (oxyethylene group) is repeatedly connected. It
may be a polyethoxyl group having a structure in which for example
1 or more, preferably 1 to 40, more preferably 2 to 25, still more
preferably 7 to 20 ethoxyl groups are polymerized. A polyethoxyl
group is represented by a chemical formula
(CH.sub.2CH.sub.2O).sub.n, wherein n is 1 or more, preferably 1 to
40, more preferably 2 to 25. An anionic surfactant in which a
polyethoxyl group is located between a hydrophilic part and a
hydrophobic part of the surfactant can be used according to the
present invention.
[0025] A surfactant having a sulfonate group, a carboxylate group
or a phosphate group, which forms an anion in an aqueous solution,
as the hydrophilic part of the anionic surfactant can be preferably
used according to the present invention. There is no specific
limitation concerning the hydrophobic part of an anionic surfactant
used according to the present invention. It may be a linear or
branched hydrocarbon or it may contain an aromatic ring.
[0026] For example, the anionic surfactant used according to the
present invention may be represented by the following general
formula:
R.sub.1--R.sub.2--(CH.sub.2CH.sub.2O).sub.n--R.sub.3--R.sub.4
(Formula)
wherein one of R.sub.1 and R.sub.4 is a hydrophobic group and the
other is a hydrophilic group. The hydrophobic groups include
linear, branched and cyclic hydrocarbons. The hydrocarbons include
both saturated and unsaturated ones. The hydrophilic group is a
group that forms an anionic ion in an aqueous solution. R.sub.2 and
R.sub.3 are linkers that may be optionally included and may be, for
example, --(CH.sub.2).sub.m-- or --O--(CH.sub.2).sub.m--. n is 1 or
more, preferably 1 to 40, more preferably 2 to 25. m is 0 or more,
preferably 0 to 3.
[0027] Examples of anionic surfactants used according to the
present invention include allyl alcohol
1,2-butoxylate-block-ethoxylate ammonium sulfate, glycolic acid
ethoxylate 4-nonylphenyl ether, glycolic acid ethoxylate oleyl
ether and poly(ethylene glycol)4-nonylphenyl 3-sulfopropyl ether or
salts thereof as shown in Table 1 below. For example, the salt may
be a sodium salt, a potassium salt, an ammonium salt, a lithium
salt, a magnesium salt or a calcium salt.
TABLE-US-00001 TABLE 1 ##STR00001## ##STR00002## ##STR00003##
##STR00004##
[0028] One anionic surfactant having a polyethoxyl group or a
mixture of plural (preferably two or three) kinds of anionic
surfactants each having a polyethoxyl group may be added according
to the method of the present invention.
[0029] The concentration of an anionic surfactant having a
polyethoxyl group according to the method of the present invention
may be appropriately selected to enhance a polymerase activity. The
surfactant is added at a concentration of generally 10% to 0.0001%,
preferably 1% to 0.001%, more preferably 0.1% to 0.01%. In cases
where plural kinds of anionic surfactants are used, they are added
so that the final concentration of the anionic surfactants (the sum
of the concentrations of the anionic surfactants to be used) falls
within the above-mentioned concentration range.
[0030] The method for enhancing a polymerase activity of the
present invention can be applied to a reaction mixture containing a
DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase or a
DNA-dependent RNA polymerase. There is no specific limitation
concerning the DNA polymerase to which the method of the present
invention is applied. Examples thereof include pol I-type (pol
I-like) DNA polymerases (Escherichia coli DNA polymerase I, Klenow
fragment, Thermus aquaticus-derived DNA polymerase (Tag
polymerase), Thermus filiformis-derived thermostable DNA polymerase
(Tfi DNA polymerase), etc.), .alpha.-type (.alpha.-like) DNA
polymerases (Pyrococcus furiosus-derived .alpha.-type
(.alpha.-like) DNA polymerase, Thermococcus litralis-derived DNA
polymerase (VENT DNA polymerase), Pyrococcus sp.-derived DNA
polymerase (Pyrobest (registered trademark) DNA polymerase (Takara
Bio), KOD DNA polymerase), Pyrococcus sp. GB-D-derived DNA
polymerase (DEEP VENT DNA polymerase), the DNA polymerases
disclosed in WO 2005/118815, PrimeSTAR (registered trademark) HS
DNA polymerase (Takara Bio), etc.), and non-.alpha., non-pol I-type
DNA polymerases which do not belong to the above. The pol I-type
DNA polymerase or the .alpha.-type DNA polymerase refers to a group
of enzymes classified based on the amino acid sequence homology.
The features of the amino acid sequences are described in Nucleic
Acids Research, Vol. 15, p. 4045-4057 (1991). The origin of the DNA
polymerase may be a prokaryote (e.g., a bacterium or an
archaebacterium), a eukaryote, a phage or a virus. In general, an
.alpha.-type (.alpha.-like) DNA polymerase has a 3'-5' exonuclease
activity. The DNA polymerase used according to the present
invention may or may not have a 3'-5 exonuclease activity. A
polymerase activity can be enhanced according to the method of the
present invention not only in cases where the polymerase is used
alone but also in cases where a mixture of plural DNA polymerases
is used.
[0031] An RNA-dependent DNA polymerase is called a reverse
transcriptase. The present invention can be applied to a reaction
mixture containing AMV (avian myeloblastosis virus)- or MMLV
(Moloney murine leukemia virus)-derived reverse transcriptase or
the like.
[0032] A DNA-dependent RNA polymerase is usually called an RNA
polymerase. The present invention can be applied to a reaction
mixture containing an RNA polymerase derived from a phage (e.g.,
SP6 or T7 RNA polymerase), or an RNA polymerase derived from a
mammal (e.g., H1 or U6 RNA polymerase).
[0033] The effect of the method for enhancing a polymerase activity
of the present invention can be confirmed using the enzymatic
activity of the polymerase as an index. For example, a polymerase
activity enhanced by the addition of an anionic surfactant can be
confirmed as described in Example 1 below by measuring the amount
of a nucleoside monophosphate incorporated into a nucleic acid
strand per unit time by the action of the polymerase. Furthermore,
the amount of an amplified nucleic acid generated according to a
nucleic acid amplification method (e.g., PCR) may be measured. A
nucleic acid strand generated according to a nucleic acid
amplification method (e.g., PCR) may be observed using agarose gel
electrophoresis to confirm the effect of amplifying a nucleic acid
strand. In addition, the enhancement of a polymerase activity can
be confirmed by measuring the length of a nucleic acid strand
extended by the polymerase, examining the increase in accuracy of
an incorporated nucleic acid, or the like. The method for enhancing
a polymerase activity of the present invention includes any method
other than the above with which an effect preferable for a
polymerase activity is achieved. There is no specific limitation
concerning the mode or the conditions of the reaction according to
the present invention as long as the enhancement of a polymerase
activity is observed. A case where increase in a polymerization
activity of a polymerase is observed in a chained nucleic acid
amplification method such as PCR (polymerase chain reaction) is
particularly useful according to the present invention.
[0034] A case is included in the present invention if a polymerase
activity is enhanced upon the addition of an anionic surfactant
having a polyethoxyl group according to the method of the present
invention as compared with the activity without the addition.
Enhancement of preferably 5% or more, more preferably 10% or more,
sill more preferably 15% or more, most preferably 20% or more is
observed.
[0035] The method of the present invention is effective
particularly if the concentration of a polymerase or the
concentration a nucleic acid as a template in a reaction mixture is
low.
[0036] According to the method of the present invention, a
composition in which a polymerase and an anionic surfactant having
a polyethoxyl group have been mixed together and stored beforehand
may be used, or they may be mixed together immediately before the
initiation of a reaction to prepare a reaction mixture.
[0037] The method for enhancing a polymerase activity of the
present invention is not restricted by the principle of
enhancement. It may be considered that a polymerase activity is
enhanced, for example, by: acting on a polymerase to increase the
catalytic activity; suppressing nonspecific interaction of a
polymerase with a template nucleic acid; providing the optimal
amount of an enzyme to a template nucleic acid; stabilizing a
polymerase protein; suppressing inactivation of a polymerase;
acting on a template nucleic acid to maintain its conformation in a
state with which a polymerase reaction readily proceeds; or
increasing the efficiency of annealing of a primer to a template
nucleic acid. However, the present invention is not restricted by
such a principle as long as an action preferable for a polymerase
activity is consequently achieved.
[0038] 2. Composition Comprising Polymerase and Anionic Surfactant
Having Polyethoxyl Group
[0039] The composition of the present invention is a composition
comprising a polymerase and an anionic surfactant having a
polyethoxyl group which is used for the above-mentioned method for
enhancing a polymerase activity. The composition of the present
invention can be used to enhance a polymerase activity.
[0040] The polymerase utilized for the present invention may be
produced using genetic engineering techniques, or it may be
purified from a naturally-occurring organism. Many polymerases are
commercially available and they can also be utilized for the
present invention. A surfactant that is not preferable for the
application to the present invention which may be contained can be
removed utilizing chromatography, salting-out, dialysis or the
like.
[0041] The composition of the present invention may further
comprise a component necessary for a polymerase reaction. For
example, the composition may comprise a buffering agent, a nucleic
acid that functions as a primer, a deoxyribonucleoside
triphosphate, a ribonucleoside triphosphate or the like.
[0042] In another embodiment of the present invention, a reaction
buffer for a thermostable DNA polymerase as described below can be
used. For example, Tris or phosphate may be used as a buffering
component. The concentration may range from about 5 to 150 mM,
preferably from about 10 to 100 mM, and the pH at 25.degree. C. may
be about 7.0 to 10.0, preferably about 8.0 to 9.0. K.sup.+ or
Na.sup.+ can be used as a monovalent salt. The concentration may
range from about 1 to 20 mM, preferably from 2 to 10 mM. Mg.sup.2+,
Mn.sup.2+ or Co.sup.2+ can be used as a divalent cation. The
concentration may range from about 1.0 to 20.0 mM. The
concentration of each dNTP may range from about 0.05 to 2.0 mM,
preferably from about 0.1 to 1.0 mM.
[0043] In another embodiment of the present invention, a reaction
buffer for T4 DNA polymerase as described below can be used. For
example, Tris or phosphate may be used as a buffering component.
The concentration may range from about 5 to 150 mM, preferably from
about 10 to 100 mM, and the pH at 25.degree. C. may be about 7.0 to
9.0, preferably about 7.5 to 8.5. K.sup.+ or Na.sup.+ can be used
as a monovalent salt.
[0044] The concentration may range from about 10 to 100 mM,
preferably from 30 to 70 mM. Mg.sup.2+, Mn.sup.2+ or Co.sup.2+ can
be used as a divalent cation. The concentration may range from
about 1.0 to 20.0 mM. For example, DTT may be used as a reducing
agent. The concentration may be about 0.1 to 5 mM, preferably about
0.3 to 1 mM. The concentration of each dNTP may range from about
0.05 to 2.0 mM, preferably from about 0.1 to 1.0 mM.
EXAMPLES
[0045] The following Examples illustrate the present invention in
more detail, but are not to be construed to limit the scope
thereof.
[0046] Among the procedures described herein, basic procedures were
carried out as described in J. Sambrook et al. (eds.), Molecular
Cloning: A Laboratory Manual 3rd ed., 2001, Cold Spring Harbor
Laboratory.
Referential Example 1
[0047] A synthetic gene of SEQ ID NO:1 encoding Tfi DNA polymerase
was prepared based on the information about the sequence of a
thermostable DNA polymerase from Thermus filiformis (Tfi DNA
polymerase) (accession no. AF030320) with genetic modification for
optimal expression in Escherichia coli. The synthetic gene of SEQ
ID NO:1 was cloned at the HincII site in pUC18. An about 2.5-kbp
DNA fragment obtained by treatment with BspHI and BamHI was cloned
between the NcoI and BamHI sites in an expression vector pTV118N.
The thus obtained vector for expressing Tfi DNA polymerase was used
to transform Escherichia coli JM109, the resulting transformant was
cultured, and the cells were collected by centrifugation and then
disrupted by sonication. A supernatant obtained by centrifuging the
cell homogenate was subjected to nucleic acid removal using
polyethyleneimine and heating followed by column purification using
phenyl-Sepharose, heparin-Sepharose and HiTrapQ in this order. A
fraction containing Tfi DNA polymerase at a high concentration
obtained as a result of the procedure was dialyzed and then
subjected to exchange with a surfactant-free configuration buffer
(20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50%
glycerol).
Example 1
Preparation of Sample
[0048] In this Example, the activity of Tfi DNA polymerase was
measured as follows.
[0049] Briefly, a sample to be subjected to activity measurement
was reacted at 75.degree. C. for 5 minutes in 50 .mu.l of a
reaction mixture for activity measurement (18 mM Tris-hydrochloride
buffer (pH 9.0), 13.5 mM magnesium chloride, 1.8 mM
2-mercaptoethanol, 36 .mu.M each of dATP, dGTP, dCTP and dTTP, 9
.mu.Ci/ml of [.sup.3H]-methyl TTP, 0.18 mg/ml of active-type salmon
sperm DNA).
[0050] After reaction, 40 .mu.l of the reaction mixture was spotted
onto DE81 paper (Whatman). The paper was washed four times with 5%
Na.sub.2PO.sub.4 followed by water and ethanol. After drying, the
radioactivity remaining on DE81 paper was measured using a liquid
scintillation counter. An amount of the enzyme that incorporated 10
nmol of [.sup.3H]-methyl TMP into DNA in 30 minutes according to
the above-mentioned enzymatic activity measurement method was
defined as 1 U of the enzyme.
Example 2
[0051] One of the anionic surfactants as shown in Table 2 was added
to surfactant-free Tfi DNA polymerase (containing 0.1% bovine serum
albumin) at a final concentration of 0.5% and the activity was
measured according to the method as described in Example 1.
TABLE-US-00002 TABLE 2 Sample number Abbreviation Surfactant 1 LSS
N-Lauroylsarcosine-sodium salt 2 LDS Lithium dodecyl sulfate 3
SOCHO Sodium cholate 4 SDC Sodium deoxycholate 5 SDS Sodium dodecyl
sulfate 6 PNSE Poly(ethylene glycol)4-nonylphenyl 3-sulfopropyl
ether potassium salt 7 ABA Allyl alcohol
1,2-butoxylate-block-ethoxylate, ammonium sulfate end-capped
solution 8 GAE-Na Glycolic acid ethoxylate 4-nonylphenyl ether
sodium salt 9 GAE-K Glycolic acid ethoxylate 4-nonylphenyl ether
potassium salt 10 GEO Glycolic acid ethoxylate oleyl ether
[0052] The measurement results are shown in FIG. 1. In FIG. 1, the
longitudinal axis represents the relative activity defining the
activity without the addition of surfactant as 1. The horizontal
axis represents the surfactant with the abbreviation as shown in
Table 2. The anionic surfactants of sample numbers 6 to 10 enhanced
the Tfi DNA polymerase activity, whereas the anionic surfactants of
sample numbers 1 to 5 inhibited the same. Thus, it was shown that
the surfactants of sample numbers 6 to 10 enhanced the polymerase
activity.
Example 3
[0053] The effect of enhancing an activity of Tfi DNA polymerase by
an anionic surfactant, poly(ethylene glycol) 4-nonylphenyl
3-sulfopropyl ether potassium salt (PNSE), was examined using a
reaction of amplifying human DCLRE1A gene by PCR. The PCR reaction
was conducted using synthetic DNA primers having nucleotide
sequences of SEQ ID NOS:2 and 3 for amplifying a 2-kbp DNA fragment
from human DCLRE1A gene as primers for an amplification reaction in
a reaction system of a total volume of 50 .mu.L containing 50 mM
Tris-HCl (pH 8.4), 15 mM (NH.sub.4).sub.2SO.sub.4, 5 mM KCl, 0.2 mM
each of dNTPs, 1.5 mM MgCl.sub.2, 0.2 .mu.M each of synthetic DNA
primers of SEQ ID NOS:2 and 3, 1.25 units of Tfi DNA polymerase (in
surfactant-free configuration buffer) and 100 ng of human genomic
DNA as a template. PNSE was added to the reaction system at a final
concentration of 0, 0.01 or 0.1%. A PCR reaction was conducted
using Thermal Cycler DICE (Takara Bio) under the following PCR
conditions: 94.degree. C. for 2 minutes; 30 cycles of 94.degree. C.
for 30 seconds, 60.degree. C. for 30 seconds and 68.degree. C. for
2 minutes; and finally 68.degree. C. for 10 minutes. After
reaction, 3 .mu.L each of the reaction mixtures was subjected to
electrophoresis on 1% LO3 agarose (Takara Bio). The results are
shown in FIG. 2. Specifically, FIG. 2 shows results of PCR
amplification of human DCLRE1A gene from human genomic DNA. Lane M:
molecular weight marker; Lanes 1 to 3: results of amplification
with the addition of PNSE at final concentrations of 0, 0.1 and
0.01%, respectively. Based on the results in FIG. 2, the effect of
enhancing the Tfi DNA polymerase activity by PNSE was
confirmed.
Example 4
[0054] The effect of enhancing an activity of PrimeSTAR (registered
trademark) HS DNA polymerase by an anionic surfactant, PNSE, was
examined using a DNA amplification reaction with .lamda. phage
genomic DNA as a template by PCR. A surfactant had been removed
from the polymerase using Q-Sepharose column chromatography before
use. Furthermore, a DNA polymerase sample in which PNSE was added
at a final concentration of 0.2% following the above procedure was
also prepared. The PCR reaction was conducted using synthetic DNA
primers having nucleotide sequences of SEQ ID NOS:4 and 5 for
amplifying a 2-kbp DNA fragment from A phage genomic DNA as primers
for an amplification reaction in a reaction system of a total
volume of 50 .mu.L containing 1.times. PrimeSTAR Buffer (Takara
Bio), 0.2 mM dNTP mix, 0.2 .mu.M each of synthetic DNA primers of
SEQ ID NOS:4 and 5, 0.25 unit of PrimeSTAR HS DNA polymerase
(without a surfactant or with the addition of 0.2% PNSE) and 0.1,
1, 10 or 100 pg of .lamda. phage genomic DNA as a template. A PCR
reaction was conducted using Thermal Cycler DICE (Takara Bio) under
the following PCR conditions: 30 cycles of 98.degree. C. for 10
seconds, 60.degree. C. for 5 seconds and 72.degree. C. for 2
minutes. After reaction, 3 .mu.L each of the reaction mixtures was
subjected to electrophoresis on 1% LO3 agarose (Takara Bio). The
results are shown in FIGS. 3A and 3B. Specifically, FIGS. 3A and 3B
show results of PCR reactions using .lamda. phage genomic DNA as a
template and PrimeSTAR HS DNA polymerase with or without 0.2% PNSE
in the configuration buffer. Lane M: molecular weight marker; Lanes
1 to 5: results of reactions using 0, 0.1, 1, 10 and 100 pg of
.lamda. phage genomic DNA as a template, respectively. Based on the
results in FIGS. 3A and 3B, the effect of enhancing the PrimeSTAR
HS DNA polymerase activity by PNSE was confirmed.
Example 5
[0055] The effect of enhancing an activity of Pyrobest (registered
trademark) DNA polymerase by an anionic surfactant, PNSE, and a
nonionic surfactant, Triton X-100, was examined using a reaction of
amplifying human DCLRE1A gene by PCR. The PCR reaction was
conducted using synthetic DNA primers having nucleotide sequences
of SEQ ID NOS:2 and 3 for amplifying a 2-kbp DNA fragment from
human DCLRE1A gene as primers for an amplification reaction in a
reaction system of a total volume of 50 .mu.L containing 120 mM
Tris-acetate (pH 8.5), 6 mM (NH.sub.4).sub.2SO.sub.4, 10 mM KOAc,
0.2 mM each of dNTPs, 1 mM Mg(OAc).sub.2, 0.01% BSA, 0.2 .mu.M each
of synthetic DNA primers of SEQ ID NOS:2 and 3, 0.5 U of Pyrobest
DNA polymerase (Takara Bio) from which the surfactant had been
removed by loading onto heparin-Sepharose column chromatography
followed by Q-Sepharose column chromatography and 0.1, 1 or 10 ng
of human genomic DNA as a template. PNSE or Triton X-100 (Nacalai
Tesque) was added to the reaction system at a final concentration
of 0 or 0.005%. A PCR reaction was conducted using Thermal Cycler
DICE (Takara Bio) under the following PCR conditions: 30 cycles of
98.degree. C. for 10 seconds and 68.degree. C. for 2 minutes. After
reaction, 3 .mu.L each of the reaction mixtures was subjected to
electrophoresis on 1% LO3 agarose (Takara Bio). The results are
shown in FIG. 4. FIGS. 4A, 4B and 4C show results of PCR reactions
using human genomic DNA as a template without a surfactant, with
0.005% PNSE or with 0.005% Triton X-100 in the PCR reaction
mixtures. Specifically, FIG. 4 shows results of PCR amplification
of human DCLRE1A gene from human genomic DNA. Lane M: molecular
weight marker; Lanes 1 to 4: results of PCR reactions using 0, 0.1,
1 and 10 ng of human genomic DNA as a template, respectively. Based
on the results in FIGS. 4A and 4B, the effect of enhancing the
Pyrobest DNA polymerase activity by PNSE was confirmed.
Furthermore, it was confirmed based on the results of FIGS. 4B and
4C that the effect of enhancing the Pyrobest DNA polymerase
activity by 0.005% PNSE was higher than that by 0.005% Triton
X-100.
Example 6
[0056] The effect of enhancing an activity of Advantage GC
polymerase mix (a mixture of two DNA polymerases) by an anionic
surfactant, PNSE, was examined using a reaction of amplifying human
DCLRE1A gene by PCR. The PCR reaction was conducted using synthetic
DNA primers having nucleotide sequences of SEQ ID NOS:2 and 3 for
amplifying a 2-kbp DNA fragment from human DCLRE1A gene as primers
for an amplification reaction in a reaction system of a total
volume of 25 .mu.L containing 1.times. TITANIUM (trademark) Taq PCR
Buffer (Clontech), 0.2 mM each of dNTPs, 0.4 .mu.M each of
synthetic DNA primers of SEQ ID NOS:2 and 3, .times.0.2 (one fifth
of usually used amount) Advantage GC polymerase mix (Clontech) and
0.05, 0.5, 5 or 50 ng of human genomic DNA as a template. PNSE was
added to the reaction system at a final concentration of 0 or
0.01%. A PCR reaction was conducted using Thermal Cycler DICE
(Takara Bio) under the following PCR conditions: 95.degree. C. for
1 minute; 30 cycles of 95.degree. C. for 30 seconds and 68.degree.
C. for 2 minutes; and 68.degree. C. for 3 minutes. After reaction,
3 .mu.L each of the reaction mixtures was subjected to
electrophoresis on 1% LO3 agarose (Takara Bio). The results are
shown in FIG. 5. FIGS. 5A and 5B show results of PCR reactions
using human genomic DNA as a template with or without 0.01% PNSE in
the PCR reaction mixtures. Specifically, FIG. 5 shows results of
PCR amplification of human DCLRE1A gene from human genomic DNA.
Lane M: molecular weight marker; Lanes 1 to 5: results of PCR
reactions using 0, 0.05, 0.5, 5 and 50 ng of human genomic DNA as a
template, respectively. Based on the results in FIGS. 5A and 5B,
the effect of enhancing the Advantage GC polymerase mix activity by
PNSE was confirmed.
[0057] The present invention provides a method for enhancing a
polymerase activity. The present invention further provides a
composition with an enhanced polymerase activity. The present
invention is a technique that can be widely applied to nucleic acid
amplification, nucleotide sequence reading and the like in the
field of genetic engineering, and can be used to increase the
operation efficiency.
[0058] All publications and patent documents cited herein are
hereby incorporated by reference in their entity for all purposes
to the same extent as if each were so individually denoted.
Sequence Listing Free Text
[0059] SEQ ID NO:1: DNA fragment containing DNA polymerase gene
from Thermus filiformis
[0060] SEQ ID NO:2: Synthetic primer for amplification of human
DCLRE1A gene.
[0061] SEQ ID NO:3: Synthetic primer for amplification of human
DCLRE1A gene.
[0062] SEQ ID NO:4: Synthetic primer for amplification of lambda
phage.
[0063] SEQ ID NO:5: Synthetic primer for amplification of lambda
phage.
Sequence CWU 1
1
512526DNAArtificial SequenceDNA fragment containing DNA polymerase
gene from Thermus filiformis 1catatgatca tgaccccact gtttgatctg
gaggaaccgc cgaaacgcgt gctgctggtg 60gatggccacc acctggccta tcgcaccttc
tatgccctga gcctcaccac ctcgcgtggt 120gagccggtgc agatggtcta
tggcttcgca cgcagcctcc tcaaagcctt gaaagaggat 180ggccaggcgg
tggtcgtggt ctttgatgcc aaagccccgt cgttccgcca cgaggcctat
240gaggcctata aagcaggtcg cgcaccgacc ccggaggatt tcccgcgtca
gctcgccttg 300gtcaaacgcc tggtggatct gctgggcctg gtccgcctgg
aggcaccggg ctatgaggcg 360gatgatgtcc tgggcaccct ggccaaaaaa
gccgaacgcg agggcatgga ggtgcgcatc 420ctcacgggcg atcgcgattt
cttccagctc ctctcggaga aagtctcggt cctcctgccg 480gatggcaccc
tggtcacccc gaaagatgtc caggagaaat atggcgttcc gccggagcgc
540tgggtggatt tccgcgcact cacgggcgat cgctcggata acatcccggg
tgtggcgggt 600attggcgaga aaaccgccct gcgcctcctc gcagagtggg
gcagcgtgga aaacctcctg 660aaaaacctgg atcgcgtgaa accggattcg
ctccgtcgca aaattgaggc gcacctggag 720gatctccacc tctcgttaga
tctggcacgc atccgcaccg atctcccgct ggaggtggat 780tttaaagccc
tgcgccgtcg caccccggat ctggagggcc tgcgtgcctt tttggaggag
840ctggagttcg gcagcctcct ccacgagttc ggcctcctgg gtggcgagaa
accgcgtgag 900gaggcaccgt ggccgccacc ggaaggcgcc ttcgtgggct
tcctcctgtc gcgcaaagag 960ccgatgtggg cggagctgct ggccctggcg
gcagcctcgg agggtcgcgt ccaccgcgca 1020accagcccgg ttgaggccct
ggccgatctc aaagaggccc gtggcttcct ggccaaagat 1080ctggccgttt
tggccctgcg cgagggcgtg gcccttgatc cgacggatga tccgctcctg
1140gtggcctatc tccttgatcc ggccaacacc cacccggagg gcgtggcacg
tcgctatggc 1200ggtgagttca cggaggatgc agcggagcgc gcactcctct
cggagcgcct cttccagaac 1260ctctttccgc gtctgtcgga gaaactcctc
tggctctatc aggaagtgga gcgtccgctc 1320tcgcgcgtct tggcccacat
ggaggcccgt ggcgtgcgcc tggatgtccc gctgctggag 1380gccctctcgt
ttgagctgga gaaagagatg gagcgcctgg agggcgaggt cttccgtttg
1440gcaggtcacc cgttcaacct caactcgcgc gatcagctgg aacgcgtcct
ctttgatgag 1500ctgggcctca ccccggtggg tcgcacggag aaaacgggca
aacgctcgac cgcccagggt 1560gccctggagg ccctccgcgg tgcccacccg
atcgtggagc tcatcctcca gtatcgcgag 1620ctgtcgaaac tcaaaagcac
ctatcttgat ccgctgccgc gtctcgtcca cccgcgtacg 1680ggacgcctcc
acacccgctt caaccagacg gccacggcca cgggacgcct gtcgagctct
1740gatccgaacc tgcaaaacat cccggtgcgc accccgttgg gccagcgcat
ccgcaaagcc 1800ttcgtggccg aggagggctg gctcctgttg gcagcggatt
attcgcagat tgagctccgc 1860gtcctggccc acctctcggg cgatgagaac
ctgaaacgcg tcttccgcga gggcaaagat 1920atccataccg agaccgcagc
ctggatgttc ggcttagatc cggcactggt tgatccgaaa 1980atgcgtcgcg
cagccaaaac ggtcaacttc ggcgtcctct atggcatgtc ggcccaccgc
2040ctctcgcagg agctcggcat tgattataaa gaggcggagg cctttattga
gcgctatttc 2100cagagcttcc cgaaagtgcg cgcatggatt gaacgcaccc
tggaggaggg tcgcacgcgt 2160ggctatgtgg agaccctgtt cggccgtcgt
cgctatgtgc cggatctggc ctcgcgcgtc 2220cgctcggtgc gcgaggcagc
ggagcgcatg gccttcaaca tgccggtgca gggcaccgcc 2280gcagatctga
tgaaaatcgc gatggtcaaa ctcttcccgc gtctgaaacc gctgggcgcc
2340cacctcctcc tccaagtgca cgatgagctg gtcctggagg tgccggagga
tcgtgccgag 2400gaggccaaag ccctggtcaa agaggtcatg gagaacgcct
atccgctgga tgtgccgctc 2460gaggtggagg tgggcgtggg tcgcgattgg
ctggaggcga aacaggattg ataaggatcc 2520aagctt 2526225DNAArtificial
SequenceSynthetic primer for amplification of human DCLRE1A gene.
2ccttatgatc tggcatgtac tggtg 25325DNAArtificial SequenceSynthetic
primer for amplification of human DCLRE1A gene. 3attaagtgta
ctgactggcg atgtg 25435DNAArtificial SequenceSynthetic primer for
amplification of lambda phage. 4gatgagttcg tgtccgtaca actggcgtaa
tcatg 35522DNAArtificial SequenceSynthetic primer for amplification
of lambda phage. 5gatagctgtc gtcataggac tc 22
* * * * *