U.S. patent application number 10/188073 was filed with the patent office on 2004-10-28 for method for effecting site-directed mutagenesis.
This patent application is currently assigned to Takara Shuzo Co., Ltd.. Invention is credited to Kato, Ikunoshin, Kita, Akihiko, Tomono, Jun, Tsunasawa, Susumu.
Application Number | 20040214170 10/188073 |
Document ID | / |
Family ID | 16464252 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040214170 |
Kind Code |
A1 |
Tomono, Jun ; et
al. |
October 28, 2004 |
Method for effecting site-directed mutagenesis
Abstract
A method for performing site-directed mutagenesis characterized
in that the method includes the step of carrying out PCR by the use
of a double-stranded DNA vector having one or more amber codons,
the vector resulting from insertion of a target DNA fragment for
site-directed mutagenesis, and at least two kinds of selection
primers; and a kit for site-directed mutagenesis for use in the
above method, characterized in that the kit includes amber codon
reversion primers. According to the present invention, there can be
provided a method for performing site-directed mutagenesis and a
kit, which is useful for genetic engineering or protein
engineering, more simply and rapidly. By using the method and the
kit of the present invention, it is possible to efficiently obtain
a mutation-introduced gene at the desired position by simply
transforming a host with a PCR product obtained by PCR.
Inventors: |
Tomono, Jun; (Muko-shi,
JP) ; Kita, Akihiko; (Mie-gun, JP) ;
Tsunasawa, Susumu; (Otsu-shi, 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: |
16464252 |
Appl. No.: |
10/188073 |
Filed: |
July 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10188073 |
Jul 3, 2002 |
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09214146 |
Dec 29, 1998 |
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6448048 |
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09214146 |
Dec 29, 1998 |
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PCT/JP97/02355 |
Jul 7, 1997 |
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Current U.S.
Class: |
435/6.16 ;
435/252.33; 435/455; 435/488; 435/91.2 |
Current CPC
Class: |
C12N 15/102
20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 435/455; 435/252.33; 435/488 |
International
Class: |
C12Q 001/68; C12P
019/34; C12N 015/85; C12N 015/74; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 11, 1996 |
JP |
8-202851 |
Claims
1. A kit for site-directed mutagenesis comprising a termination
codon reversion primer, PCR reagents, a thermostable DNA polymerase
suitable to carry out LA (Long and Accurate) PCR, and a
suppressor-free (Sup.sup.0) host, wherein said suppressor-free
(Sup.sup.0) host is Escherichia coli.
2. The kit for site-directed mutagenesis according to claim 1,
wherein said kit further comprises a double-stranded DNA vector
having at least one termination codon and a multicloning site for
target DNA insertion.
3. The kit for site-directed mutagenesis according to claim 2,
wherein said double-stranded DNA vector is selected from the group
consisting of: pKF18k-2, pKF19k-2, pKF19kM and pKB101.
4. The kit for site-directed mutagenesis according to claim 2,
wherein said termination codon is located on a drug resistance gene
of said vector.
5. (Canceled).
6. A kit for site-directed mutagenesis comprising a termination
codon reversion primer, PCR reagents, a thermostable DNA
polymerase, suitable to carry out LA (Long and Accurate) PCR, a
suppressor-free (Sup.sup.0) host, wherein said suppressor-free
(Sup.sup.0) host is Escherichia coli, and optionally a
double-stranded DNA vector having at least one termination codon
and a multicloning site for target DNA insertion wherein said kit
allows for rapid site-directed mutagenesis which comprises the
steps of: (1) producing a PCR product from a single PCR
amplification carried out in a reaction tube, wherein in said tube
at least the three following components are present: (a) a
double-stranded DNA vector having at least one termination codon
and having a target DNA fragment for site-directed mutagenesis, (b)
a selection primer that binds specifically to said at least one
termination codon, wherein said selection primer (b) is an
termination codon reversion primer that is designed to revert the
termination codon to a non-termination codon, and (c) a selection
primer that binds specifically to said target DNA fragment, wherein
said selection primer (c) is a mutagenic primer that is designed to
introduce a desired mutation into the target DNA fragment, wherein,
said PCR amplification reaction comprises at least 20 cycles,
wherein in an early cycle, a mixture of at least two PCR products
are produced: (i) a copy of the vector template wherein the desired
mutation is present in the target DNA fragment of the copy, and
(ii) a copy of the vector template wherein the termination codon is
reverted to a non-termination codon in the copy, and, in a later
cycle of said PCR reaction, said selection primer (b) binds to said
PCR product (i) to produce a PCR product (iii) having both the
desired mutation and the reverted non-termination codon, and said
selection primer (c) binds to said PCR product (ii) to produce a
PCR product (iv) having both the desired mutation and the reverted
non-termination codon, so that, the PCR products (iii) and (iv)
bind to the DNA vector and serve as a long-chain PCR primer for the
amplification of a full-length copy of said DNA vector that
possesses both the desired mutation and the reverted
non-termination codon, within said single PCR amplification; and
(2) transforming the suppressor-free (Sup.sup.0) host with the PCR
product of step (1), thereby directly obtaining a desired mutant.
Description
TECHNICAL FIELD
[0001] The present invention concerns a method for performing
site-directed mutagenesis used in genetic engineering techniques,
more easily and efficiently, and a kit for the use in the above
method.
BACKGROUND ART
[0002] In the field of genetic engineering, recently, it is often
difficult to obtain a gene product in large amounts while solely
relying on techniques of cloning gene to express, and successful
cases are few. For this reason, in order to increase a level of
expression of a product of a cloned gene, a technique for
coinciding a frame and a technique for altering base sequences near
the initiation codon without altering amino acid sequences (silent
mutation) are minimally required techniques. In addition, in order
to carry out cloning of genes and produce more useful protein as a
protein to be expressed, a technique is important and essential,
which alters the base sequence of a corresponding codon to delete
or substitute amino acids and thereby modifies specificities of the
protein, such as optimal pH, stability, substrate specificity, Km
value, and the like, in the cases where the protein is an
enzyme.
[0003] As described above, a method for changing a particular base
sequence in a cloned gene as desired, i.e. site-directed
mutagenesis, is essential to carry out structural and functional
analyses of various regulatory regions on genes including RNA, and
protein engineering applications using recombinant DNA techniques.
In addition, in the research field of protein engineering, a method
for performing site-directed mutagenesis is more important, in a
view of more rapid and accurate research by introduction of
mutations and elimination at the DNA level.
[0004] Conventionally, a method for performing site-directed
mutagenesis comprises, for instance, the following procedures:
[0005] (1) First, a desired DNA to be mutation-introduced is
inserted to a vector. Thereafter, the complementary strand of the
desired DNA is dissociated by heat-denaturation, in the case of
using a double-stranded plasmid DNA, or a M13 phage vector is used
to prepare a single-stranded DNA;
[0006] (2) An oligonucleotide designed to introduce a desired
mutation (mutagenic primer) is annealed with the above
single-stranded DNA. Thereafter, a complimentary strand DNA is
synthesized in vitro system by a reaction of DNA polymerase and DNA
ligase;
[0007] (3) Escherichia coli is transformed with the DNA obtained in
the above item (2), and then a clone in which a mutation is
introduced is selected.
[0008] However, since a ratio of a mutant to a parent DNA is
extremely low in the case of carrying out only the above
procedures, it is necessary to efficiently select a clone resulting
from annealing to a mutagenic primer. Therefore, in the step of the
above item (3), there is employed a system for selective removal so
that a clone harboring the parent DNA does not grow.
[0009] Examples of such system are a method utilizing amber
mutation (amber codon) [Nucleic Acids Research, 12, No. 24,
pp.9441-9456 (1984)]; a method utilizing restriction endonuclease
site [Analytical Biochemistry, 200, pp.81-88 (1992); Gene, 102, pp.
67-70 (1991)]; and a method utilizing dut (dUTPase) mutation and
ung (uracil DNA glycosilase) mutation [Proceedings of the National
Academy of Sciences of the USA, 82, pp.488-492 (1985)], etc.
However, in these methods of mutagenesis, their procedures are
complicated, and much time is consumed. In addition, in these
methods, a proportion of obtaining desired clones in which a
mutation is introduced is low.
[0010] On the other hand, a method of site-directed mutagenesis
described in Japanese Patent Laid-Open No. Hei 7-289262 is a method
utilizing amber mutation using DNA polymerase and DNA ligase. As
compared to the above methods, although in the method described in
Japanese Patent Laid-Open No. Hei 7-289262, mutants can be produced
at a high efficiency, the two-step transformation into Escherichia
coli is necessary, thus hampering simple operation for practically
purposes.
[0011] In recent years, methods for site-directed mutagenesis based
on the wide-spread use of PCR techniques have been developed.
[0012] For example, there has been known a method is known, the
method comprising the steps of synthesizing a DNA strand to be
mutation-introduced using three or more kinds of primers, the
primers including a mutagenic primer; thereafter cutting out a DNA
strand with restriction endonucleases and then ligating to another
vector the resulting DNA strand in which a mutation is expected to
be introduced; and transforming a host Escherichia coli with the
resulting vector. In addition, Quik Change.TM. Site-Directed
Mutagenesis Kit, manufactured by Stratagene, can be used to obtain
a mutation-introduced DNA by means of synthesizing a strand with
PCR or DNA polymerase from Pyrococcus furiosus using two kinds of
complementary mutagenic oligonucleotides (mutagenic primers), the
oligonucleotides being capable of hybridizing to double-stranded
DNA to which mutation is to be introduced, thereafter, digesting
with restriction endonuclease DpnI the resulting strand without
mutation, and then transforming a host Escherichia coli with
resultants treated with DpnI. Furthermore, there has been known a
method capable of obtaining a mutation-induced DNA comprising
adding class IIS restriction endonuclease recognition site at
5'-terminal side of each two kinds of mutagenic oligonucleotides
(mutagenic primers), synthesizing strands by PCR, thereafter
digesting with class IIS restriction endonuclease a
mutation-introduced DNA, ligating and then transforming a host
Escherichia coli (U.S. Pat. No. 5,512,463).
[0013] As described above, the conventional method using DNA
polymerase and DNA ligase necessitates such enzyme reactions and a
plurality of steps of transformation for fixing mutation sites,
consuming too much time, thereby making it difficult to increase
efficiency. Also, in the methods utilizing amber mutation or "dut"
and "ung" mutations, single-stranded DNA must be isolated. The
method for removing restriction endonuclease site has drawbacks,
including limited availability of restriction endonucleases.
[0014] In view of the above, a method for performing site-directed
mutagenesis using the recently widely spread PCR technique has been
developed and brought into actual application. However, its
operation is complicated by necessitating three or more kinds of
primers, including mutagenic primers, the primers including two or
more kinds of mutagenic primers, and by necessitating a restriction
endonuclease reaction during the operation, and other aspects.
There have been other various problems including extremely reduced
mutation efficiency that can result from incomplete restriction
endonuclease reaction.
[0015] Therefore, an object of the invention is to provide a simple
and practical method for performing site-directed mutagenesis using
PCR method, and a kit for carrying out the method for performing
site-directed mutagenesis.
[0016] As a result of intensive investigation to develop an
efficient and simple method for performing site-directed
mutagenesis, the present inventors have succeeded to obtain a clone
in which a desired mutation is introduced, only once transforming
Escherichia coli after carrying out PCR. The present invention has
been completed, based on such a fact.
DISCLOSURE OF INVENTION
[0017] Accordingly, the gist of the present invention follows:
[0018] [1] a method for performing site-directed mutagenesis
characterized in that the method comprises the step of carrying out
PCR by the use of a double-stranded DNA vector having one or more
amber codons, the vector resulting from insertion of a target DNA
fragment for site-directed mutagenesis, and at least two kinds of
selection primers;
[0019] [2] the method for performing site-directed mutagenesis
according to the above item [1], wherein the selection primers are
mutagenic primers and amber codon reversion primers;
[0020] [3] the method for performing site-directed mutagenesis
according to the above item [1] or [2], characterized in that the
vector carries a drug resistance gene containing one or more amber
codons;
[0021] [4] the method for performing site-directed mutagenesis
according to any one of the above items [1] to [3], further
comprising a step of transforming a suppressor-free (Sup.sup.0)
host with a PCR product;
[0022] [5] the method for performing site-directed mutagenesis
according to the above item [4], characterized in that the
suppressor-free (Sup.sup.0) host is Escherichia coli;
[0023] [6] a kit for site-directed mutagenesis for use in the
method for performing site-directed mutagenesis according to any
one of the above items [1] to [5], characterized in that the kit
comprises amber codon reversion primers;
[0024] [7] the kit for site-directed mutagenesis according to the
above item [6], characterized in that the kit comprises a vector
having one or more amber codons;
[0025] [8] the kit for site-directed mutagenesis according to the
above items [6] or [7], characterized in that the kit comprises a
suppressor-free (Sup.sup.0) host; and
[0026] [9] the kit for site-directed mutagenesis according to the
above item [8], characterized in that the suppressor-free
(Sup.sup.0) host is Escherichia coli.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a schematic view showing a structure of plasmid
pKF19k.
[0028] FIG. 2 is a schematic view showing a structure of plasmid
pKF19kM.
BEST MODE FOR CARRYING OUT THE INVENTION
[0029] The present invention will be explained in detail below.
[0030] The present invention is a method for performing
site-directed mutagenesis characterized in that the method
comprises the step of carrying out PCR by the use of a
double-stranded DNA vector having one or more amber codons, the
vector resulting from insertion of a target DNA fragment for
site-directed mutagenesis, and at least two kinds of selection
primers. As the selection primers used in the present invention,
there can be used at least two kinds of primers consisting a
mutagenic primer for inducing a mutation at a desired position on
the gene to be mutated; and an amber codon reversion primer for
reverting the amber codons, the amber codon reversion primer being
arranged on the strand opposite to the mutagenic primer. The term
"at least two kinds of primers" as used herein refers to the
mutagenic primers and the amber codon reversion primers, and at
least two kinds of selection primers as described in the present
invention encompass any set of selection primers, as long as these
two kinds of selection primers are included.
[0031] The double-stranded DNA vector used in the present invention
which has one or more amber codons may be any vector, as long as
the vector has one or more amber codons, and can be used without
limitation for the method for performing site-directed mutagenesis
of the present invention. In this case, it is preferred that a
vector having the amber codon(s) on a drug resistance gene is used.
The drug resistance gene is not particularly limited, but the
kanamycin resistance gene, the chloramphenicol resistance gene,
etc., for example, are preferred. In other words, vectors having
one or more amber codons on a drug resistance gene, such as the
kanamycin or chloramphenicol resistance gene, can be preferably
used. For instance, pKF19k (manufactured by Takara Shuzo Co., Ltd.)
can be used. It is also possible to prepare a cassette having amber
codons and then introduce the cassette into a vector. By the use of
such a vector, it is easy to confirm the reversion of amber codons
on the drug resistance gene simply by introducing the PCR product
into a suppressor-free host, such as Escherichia coli and then
growing the resulting host on an agar medium containing a drug
including, for example, kanamycin, chloramphenicol or the like.
[0032] The kinds of mutations that can be introduced by the method
for performing site-directed mutagenesis of the present invention
are not particularly limited. Examples thereof include base
substitution, deletion and insertion. Although the size of mutation
is not subject to limitation, it is preferable that the length of
the mutagenic primer is changed according to the size of mutation.
For example, when the size of mutation is about 1 to 3 bases, the
length of the mutagenic primer is preferably about 20 bases. When
the size of mutation is 4 bases or more, it is preferable that a
mutagenic primer of as long as about 30 bases, including about 15
base pairs on each of the 5'-side and 3'-side with respect to the
position of desired mutation, is used. It is also desirable that
the 3'-terminal of the mutagenic primer is G or C, because the
3'-terminal serves as the origin of polymerase reaction. Taking
these conditions into consideration, a mutagenic primer is
prepared. It is also possible to evaluate a design and purity of
the primer by confirming by means of electrophoresis and the like
whether the desired fragment is surely amplified by PCR using the
primer. In addition, in the present invention, as to the number of
the mutagenic primer, a desired mutation can be introduced by means
of a single kind of mutagenic primer. When introducing different
mutations at one site, however, a number of mutagenic primers
depending on the purposes may be used in mixture to obtain a gene
in which each of the desired mutations is introduced by a single
operation. For example, when base-substitution site-directed
mutagenesis for which alteration of a base at one site from G to A,
T or C, respectively, is carried out, genes resulting from
introducing mutation of from G to A, T or C, respectively, can be
obtained by a single operation by preparing each three kinds of
mutagenic primers, and then by using the mutagenic primers in
mixture.
[0033] In addition, the amber codon reversion primer may be a
primer capable of reverting amber codon portions to a wild type and
the like, without being particularly limited thereto. For example,
when an amber codon is TAG, the amber codon can be reverted by
means of a primer prepared so as to alter the TAG to TCG or CTG. It
is preferable that the length of the amber codon reversion primer
is changed according to the number of amber codons, PCR efficiency,
and the like. The length of the amber codon reversion primer is not
particularly limited. It is preferable that the length is about 20
bases to about 30 bases. Also, it is desirable that the 3'-terminal
of the amber codon, like the mutagenesis primer, is G or C, because
the 3'-terminal serves as the origin of polymerase reaction. It is
also possible to evaluate a design and purity of the primer by
confirming by means of electrophoresis and the like whether the
desired fragment is surely amplified by PCR using the primer. In
addition, it is recommended that one kind of the amber codon
reversion primer is used. When two or more amber codons are
present, it is preferable that an amber codon reversion primer is
prepared so that those amber codons are reverted by one kind of
primer.
[0034] The concentration of each of the mutagenic primer and the
amber codon reversion primer in PCR is not particularly limited. It
is desirable that the respective optimal concentrations during
reaction is studied, since when the concentration of each primers
is too low, amounts of amplification are decreased, and when the
concentration is too high, non-specific reactions are promoted,
which in turn may hamper the onset of the specific amplification
reaction in some case. Usually, it is preferable that the PCR is
carried out within the final concentration range from 0.1 to 1.0
.mu.M for each primer.
[0035] The suppressor-free host, as referred to the present
specification, includes but not limited to preferably Escherichia
coli, for example, Escherichia coli MV1184 (manufactured by Takara
Shuzo Co., Ltd). Any host can be used, as long as the host lacks
suppression ability, and all such hosts are included in the scope
of the suppressor-free host as referred in the present
specification.
[0036] The site-directed mutagenesis by the method of the present
invention can, for example, be carried out by the following
steps:
[0037] (1) A target DNA is inserted into a double-stranded DNA
vector having one or more amber codons.
[0038] (2) The DNA-inserted vector prepared in the above item (1),
a mutagenic primer for introducing a mutation at a desired position
on the gene to be mutated, and an amber codon reversion primer for
reversion of an amber codon arranged on the strand opposite to the
above mutagenic primer are mixed, and then PCR is carried out.
[0039] (3) This PCR product is introduced into a suppressor-free
(Sup.sup.0) host, and a clone having the desired mutation is
selected.
[0040] By carrying out the above steps, a mutation-introduced gene
at the desired position can be obtained efficiently.
[0041] In the present invention, since the PCR product resulting
from PCR amplification serves as a long-chain primer, the PCR
product facilitates the synthesis of full-length DNA during the PCR
process. In addition, the PCR product becomes circular when
introduced directly into an Escherichia coli (in vivo) as a host.
In addition, in a case of selection of a clone including a gene
introducing a mutation at a desired position, the selection is
simplified by a method using a suppression system. Specifically,
amber codon reversion can be confirmed simply by introducing the
PCR product into a host, e.g., suppressor-free Escherichia coli,
culturing the resulting host on a medium containing the drug for
one of the above-described drug resistance genes, and confirming
its growth. In other words, since only an amber codon-reverted
clone can be selected, the probability that the grown strain is a
clone incorporating a mutation-introduced gene at the desired
position.
[0042] According to the method of the present invention, an
introduction of mutations is possible at any position on the gene
to be mutated.
[0043] Although ordinary procedures can be used to carry out PCR by
the method of the present invention, it is preferred that the cares
shown below are considered to avoid erroneous base incorporation by
PCR, adenine (A) addition to the 3'-terminal, etc.
[0044] (I) The gene to be mutated and inserted into a vector is
preferably as short as 2 kbp or less.
[0045] (II) The number of PCR cycles is preferably 20 to 30
cycles.
[0046] (III) The thermostable DNA polymerase for PCR preferably has
a high amplification efficiency and low error rate. For example,
the TaKaRa LA PCR Kit (manufactured by Takara Shuzo Co., Ltd.) can
be used as the PCR kit. In addition, regarding the thermostable DNA
polymerase, it is possible to carry out PCR using TaKaRa Ex Taq
(manufactured by Takara Shuzo Co., Ltd.) to achieve high
amplification efficiency and low error rate. Methods for
transforming hosts, such as Escherichia coli, with PCR product and
other hosts include the calcium chloride method [Journal of
Molecular Biology, 166, pp. 557-580 (1983)] and the electroporation
method [Nucleic Acids Research, 16, pp. 6127-6145 (1988)].
Regarding screening of the transformants obtained, a clone
incorporating a gene of high probability of amber codon reversion,
i.e., a gene of high probability of mutation at the desired
position, can be selected by culturing the transformants on a
medium containing the drug corresponding to one of the
above-mentioned drug resistance genes, and then confirming their
growth.
[0047] Since a simple method for performing site-directed
mutagenesis using an amber codon has been made possible by the
present invention, it is obvious to those skilled in the art that a
termination codon other than the amber codon, i.e., ochre codon or
opal codon, can be also applied in place of the amber codon of the
present invention. Therefore, methods for performing site-directed
mutagenesis using the ochre codon or opal codon in place of the
amber codon of the present invention are, as a matter of course,
encompassed in the scope of the present invention.
[0048] The present invention is hereinafter described in more
detail by means of the following examples, but the present
invention is not limited to those examples.
EXAMPLE 1
[0049] One Base-Substitution Site-Directed Mutagenesis
[0050] 1. Construction of pKF19kM
[0051] A pKF19kM (FIG. 2) was constructed as the plasmid to be used
in the following examples, the pKF19kM resulting from conversion of
G to A at a 30th position downstream from the multicloning site in
the lacZ' gene in pKF19k (manufactured by Takara Shuzo Co., Ltd.,
FIG. 1), harboring the kanamycin resistance gene containing two
amber codons.
[0052] The lacZ' gene exhibits a P-galactosidase activity when
lacZ' gene is introduced into a host cell having the lacZ.DELTA.M15
genotype. Therefore, in the case of the lacZ' gene without base
substitution, the resulting grown colonies show a blue color by
reacting with 5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside
(X-Gal, manufactured by Takara Shuzo Co., Ltd.) as the substrate in
the presence of isopropyl-.beta.-D-thiogalactopyranoside (IPTG,
manufactured by Takara Shuzo Co., Ltd.). By contrast, the mutant
lacZ' gene with base substitution cannot exhibit the inherent
activity, so that the resulting grown colonies show a white
color.
[0053] By utilizing this phenomenon, the efficiency of reversion of
the base substitution in the mutant lacz' gene in pKF19kM to the
active gene by site-directed mutagenesis was calculated from the
numbers of blue colonies and white colonies.
[0054] 2. Synthesis of Mutagenic Oligonucleotide (Mutagenic Primer)
and Oligonucleotide for Reversion of Double Amber Codons in
Kanamycin Resistance Gene (Amber Codon Reversion Primer)
[0055] The 5'-terminal phosphorylated oligonucleotide (mut1) as
shown by SEQ ID NO: 1 in Sequence Listing was synthesized as a
mutagenic oligonucleotide used for reversion of the base
substitution site in the mutant lacZ' gene (reversion from A to G).
In other words, the mut1 was designed to revert the base
substitution by 10th C to result in active lacZ' gene.
[0056] In addition, the 5'-terminal phosphorylated oligonucleotide
as shown by SEQ ID NO: 2 in Sequence Listing was prepared as a
primer (KQ2) for reversion of the double amber codons (two amber
codons) in the kanamycin resistance gene. Specifically, in pKF19kM,
the primer was designed to revert the amber codons (TAG) by TTG at
the 3rd to 5th position and CTG at the 9th to 11th position of
KQ2.
[0057] 3. PCR
[0058] The composition of a reaction mixture is shown in Table 1.
PCR was carried out using a thermal cycler (manufactured by Takara
Shuzo Co., Ltd.). The resulting reaction mixture was subjected to
ethanol precipitation to desalt and concentrate. Thereafter, the
resulting mixture was suspended in 5 .mu.l of sterilized water.
1TABLE 1 pKF19kM 5 ng primers (mut1 and KQ2) final concentration at
5 pmol each TaKaRa Ex Taq (manufactured 5 U by Takara Shuzo Co.,
Ltd.) dNTP mixture final concentration at 200 .mu.M TAPS buffer (pH
9.3, 25.degree. C.) 25 mM KCl 50 mM MgCl.sub.2 2 mM
2-mercaptoethanol 1 mM total volume 50 .mu.l (overlaying mineral
oil thereon) note: TAPS =
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonate
[0059] A cycle was carried out under the condition of 94.degree. C.
for 30 seconds, 55.degree. C. for 2 minutes, and 72.degree. C. for
2 minutes. The above condition was repeated in 20 cycles for Cycle
I and 24 cycles for Cycle II. Incidentally, after completion of
PCR, each of the PCR products from Cycles I and II was subjected to
electrophoresis to confirm that a DNA fragment of a desired size
was amplified.
[0060] 4. Transformation
[0061] A suppressor-free Escherichia coli MV1184 (manufactured by
Takara Shuzo Co., Ltd.) was transformed by the electroporation
method using 2 .mu.l of each PCR product after ethanol
precipitation. Thereafter, the resulting transformant cells were
spread over an LB agar medium [bactotrypton (10 g), bactoyeast
extract (5 g), NaCl (5 g), distilled water (1 liter), pH (7.0)]
containing kanamycin (50 .mu.g/ml), X-Gal (40 .mu.g/ml) and IPTG
(0.2 mM), and the efficiency of site-directed mutagenesis for the
clones obtained was calculated. Incidentally, it should be noted
that when the mutated portion in the mutant lacZ' gene has been
reverted to the active lacZ' gene, the resulting colonies show a
blue color, and when the mutated portion has not been reverted, the
resulting colonies show a white color. The results are shown in
Table 2.
2 TABLE 2 blue white total (blue/total) .times. 100[%] Cycle I 586
314 900 65 Cycle II 2655 891 3546 75
[0062] In other words, clones with reversion to the active gene by
site-directed mutagenesis were obtained at 75% efficiency in Cycle
II.
[0063] 5. Analysis of Site-Directed Mutation Site
[0064] A plasmid DNA was prepared by the alkali lysis method for
each of four blue colonies obtained in item 4 of Example 1. The
base sequence of each of the plasmid DNAs was determined by the
dideoxy method, and then a mutation site was analyzed. As a result,
G reverted from A was observed at a 30th position downstream of the
multicloning site in the lacZ' gene, thereby clearly demonstrating
the induction of the site-specific mutation.
EXAMPLE 2
[0065] Three Bases- or 6 Bases-Deletion Site-Directed
Mutagenesis
[0066] pKF19k (manufactured by Takara Shuzo Co., Ltd., FIG. 1) was
used to delete 3 bases or 6 bases, the pKF19k containing wild type
lacZ' gene and kanamycin resistance gene with two amber codons.
[0067] 1. Synthesis of Mutagenic Oligonucleotide
[0068] The 5'-terminal phosphorylated oligonucleotide was
synthesized so as to lack a BamHI site and BamHI-EcoRI site in the
multicloning site on pKF19k by 3-bases or 6-bases deletion,
respectively. An oligonucleotide used in 3-bases deletion (del1) is
shown by SEQ ID NO: 3 in Sequence Listing. An oligonucleotide used
in 6-bases deletion (del2) is shown by SEQ ID NO: 4 in Sequence
Listing. Specifically, del1 was designed to delete bases at the
positions between 12th G and 13th C of SEQ ID NO: 3 in Sequence
Listing, and del2 was designed to delete bases at the positions
between 12th G and 13th T of SEQ ID NO: 4 in Sequence Listing.
[0069] 2. PCR and Transformation
[0070] Thirty cycles of PCR where each of cycles consisted of the
conditions of 94.degree. C. for 30 seconds, 55.degree. C. for 2
minutes, and 72.degree. C. for 2 minutes was carried out, by using
each of primers dell and del2 prepared in item 1 in Example 2 and
KQ2 primer used in Example 1, according to the composition of Table
1 (in the table, pKF19k was used in place of pKF19kM). The
resulting PCR product was then subjected to ethanol precipitation
in the same manner as in Example 1. Thereafter, the precipitated
PCR product was suspended in 5 .mu.l of sterilized water, and then
Escherichia coli MV1184 was transformed with 2 .mu.l out of the
resulting suspension. Next, the resulting transformants were spread
over an LB agar medium containing 50 .mu.l/ml kanamycin, and each
of 10 strains was selected from grown colonies. Thereafter, plasmid
DNAs were prepared in the same manner as item 5 in Example 1.
Confirmation of mutation was carried out by digesting each of the
above plasmid DNAs with restriction endonuclease BamHI. In other
words, a mutation-introduced plasmid is not digested. The results
are shown in Table 3.
3 TABLE 3 numbers of efficiency non-digested numbers of of mutation
clones assayed strain (%) del1 9 10 90 del2 9 10 90
[0071] It was confirmed that the mutation was introduced in each of
PCR products using primers dell and del2 at 90% efficiency of
mutation, and that 3 bases or 6 bases were deleted in each of PCR
products from the results of sequencing.
Example 3
[0072] Sixty Bases-Deletion Site-Directed Mutagenesis
[0073] 1. Synthesis of Mutagenic Oligonucleotides
[0074] A 5'-terminal phosphorylated oligonucleotide was synthesized
so as to lack a whole multicloning site (60 bases) of pKF19k
(manufactured by Takara Shuzo Co., Ltd.) by deletion. The
oligonucleotide (del3) is shown by SEQ ID NO: 5 in Sequence
Listing. In other words, del3 was designed so as to delete a whole
multicloning site (60 bases) at the position between 10th T and
11th G of SEQ ID NO: 5 in Sequence Listing.
[0075] 2. PCR and Transformation
[0076] Thirty cycles of PCR where each of cycles consisted of the
conditions of 94.degree. C. for 30 seconds, 50.degree. C. for 2
minutes, and 72.degree. C. for 2 minutes were carried out by using
primer del3 prepared in item 1 in Example 3 and KQ2 primer used in
Example 1 according to the composition of Table 1 (in the table,
pKF19k was used in place of pKF19M). The resulting PCR product was
then subjected to ethanol precipitation in the same manner as in
Example 1. Thereafter, the precipitated PCR product was suspended
in 5 .mu.l of sterilized water, and then Escherichia coli MV1184
was transformed with 2 .mu.l out of the resulting suspension. Next,
the resulting transformants were spread over an LB agar medium
containing kanamycin at a concentration of 50 .mu.l/ml, and each of
20 strains was selected from grown colonies. Thereafter, plasmid
DNAs were prepared in the same manner as item 5 in Example 1.
Confirmation of mutation was carried out by digesting each of the
above plasmid DNAs with restriction endonucleases EcoRI and
HindIII. In other words, a mutation-introduced plasmid has not been
digested. The results are shown in Table 4.
4 TABLE 4 numbers of numbers of efficiency non-digested assayed of
mutation clones strain (%) del3 20 20 100
[0077] It was confirmed that the mutation was introduced by PCR
using primer del3 at 100% efficiency of mutation, and that a whole
multicloning site (60 bases) was deleted from the results of
sequencing.
[0078] From these results, it was clarified that deletion of bases
of a comparably long region could be easily carried out.
Example 4
[0079] Deletion and Insertion Site-Directed Mutagenesis
[0080] 1. Synthesis of Mutagenic Oligonucleotides
[0081] The 5'-terminal phosphorylated oligonucleotide was
synthesized so as to introduce an additional EcoRI site at 100th
base downstream from EcoRI site in multicloning site of pKF19k. The
oligonucleotide (eco1) is shown by SEQ ID NO: 6 in Sequence
Listing. In other words, eco1 was designed so as to delete bases
AAT of original sequence corresponding to bases at the positions of
from 9th G to 14th C, and to insert an EcoRI site, GAATTC.
[0082] 2. PCR and Transformation
[0083] Twenty-four cycles of PCR where each of cycles consisted of
the conditions of 94.degree. C. for 30 seconds, 55.degree. C. for 2
minutes, and 72.degree. C. for 2 minutes were carried out, by using
primer eco1 prepared in item 1 in Example 4 and KQ2 primer used in
Example 1, according to the composition of Table 1 (in the table,
pKF19k was used in place of pKF19M). The resulting PCR product was
then subjected to ethanol precipitation in the same manner as
Example 1. Thereafter, the precipitated PCR product was suspended
in 5 .mu.l of sterilized water, and then Escherichia coli MV1184
was transformed with 2 .mu.l out of the resulting suspension. Next,
the resulting transformants were spread over an LB agar medium
containing kanamycin at a concentration of 50 .mu.l/ml, and each of
12 strains was selected from grown colonies. Thereafter, plasmid
DNAs were prepared in the same manner as item 5 in Example 1.
Confirmation of mutation was carried out by digesting the resulting
plasmid with restriction endonuclease EcoRI, and then confirming a
DNA fragment of 100 bases by electrophoresis. In other words, a
mutation-introduced plasmid has two EcoRI sites at an interval of
100 bases, and the above fragment of 100 bases can be confirmed by
digestion with restriction endonuclease EcoRI. The results are
shown in Table 5.
5 TABLE 5 numbers of confirmed numbers of efficiency fragment of
assayed of mutation 100 bases strain (%) eco1 12 12 100
[0084] It was confirmed that the desired mutant plasmid was
obtained by introducing a mutation using primer eco1 at 100%
efficiency, and that EcoRI site was introduced at the desired site
from the results of sequencing.
EXAMPLE 5
[0085] Introduction of Mutation into DNA Fragment Ligated to
pKF19k
[0086] 1. Ligation of Escherichia coli Plasmid Vector pBR322, to
pKF19k
[0087] A vector was prepared by digesting pKF19k with restriction
endonuclease BamHI, and then incorporating Escherichia coli plasmid
pBR322 [manufactured by Takara Shuzo Co., Ltd.; Gene, 22, pp.
277-280 (1983)] having a length of 4361 bases to BaMHI site of the
above digested pKF19k. The resulting vector was named as pKB101.
The site-directed mutagenesis to an insert fragment was made by
using the above vector.
[0088] 2. Synthesis of Mutagenic Oligonucleotide
[0089] The 5'-terminal phosphorylated oligonucleotide was
synthesized so as to introduce an additional BamHI site at 400th
base downstream from BamHI site of pBR322. The oligonucleotide
(bam1) is shown by SEQ ID NO: 7 in Sequence Listing. In other
words, bam1 was designed so as to substitute the original sequence
AGCGCT with BamHI site GGATCC at positions of from 16th G to 21th C
of SEQ ID NO: 7 in Sequence Listing.
[0090] 3. PCR and Transformation
[0091] Twenty-five cycles of PCR where each of cycles consisted of
the condition of 94.degree. C. for 30 seconds, 55.degree. C. for 30
seconds, and 72.degree. C. for 6 minutes were carried out, by using
primer bam1 prepared in item 2 in Example 5 and KQ2 primer used in
Example 1 according to the composition of Table 1 (in the table,
pKB101 was used in place of pKF19M). The resulting PCR product was
then subjected to ethanol precipitation in the same manner as in
Example 1. Thereafter, the precipitated PCR product was suspended
in 5 .mu.l of sterilized water, and then Escherichia coli MV1184
was transformed with 2 .mu.l out of the resulting suspension. Next,
the resulting transformants were spread over an LB agar medium
containing kanamycin at a concentration of 50 .mu.l/ml, and each of
6 strains was selected from grown colonies. Thereafter, plasmid
DNAs were prepared in the same manner as item 5 in Example 1.
Confirmation of mutation was carried out by digesting the resulting
plasmid with restriction endonuclease BamHI, and then confirming a
DNA fragment of 400 bases by electrophoresis., In other words, a
mutation-introduced plasmid has two BamHI sites at an interval of
400 bases, and the above fragment of 400 bases can be confirmed by
digestion with restriction endonuclease BamHI. The results are
shown in Table 6.
6 TABLE 6 numbers of confirmed numbers of efficiency fragment of
assayed of mutation 400 bases strain (%) bam1 5 6 83
[0092] It was confirmed that the desired mutant plasmid was
obtained at 80% or more efficiency for using a DNA fragment ligated
to pKF19k, and that BamHI site was introduced at the desired site
from the results of sequencing.
Example 6
[0093] Preparation of Kit for Site-Directed Mutagenesis
[0094] A kit for performing site-directed mutagenesis (for 20 runs)
was constructed as a set of host Escherichia coli, a vector and
oligonucleotides used as control (Table 7).
7 TABLE 7 .mu.l KQ2 primer (for reversion of amber codon; 5
pmol/.mu.l) 20 mut1 primer (mutagenic primer; 5 pmol/.mu.l) 5
pKF19kM (5 ng/.mu.l) 5 pKF18k and pKF19k (10 OD/ml) 10 each
Escherichia coli MV1184 100 (stored in 10% glycerol solution)
Remark: pKF18k and pKF19k being manufactured by Takara Shuzo Co.,
Ltd.
[0095] Incidentally, a kit shown in Table 7 can be combined with a
commercially available kit for PCR.
[0096] In addition, a kit for performing site-directed mutagenesis
(for 20 runs) was constructed, the kit comprising a set of a
reaction mixture for carrying out PCR, a dNTP mixture, and
thermostable DNA polymerase (Table 8).
8TABLE 8 .mu.l TaKaRa Ex Taq (5 U/.mu.l; manufactured by Takara
Shuzo 20 Co., Ltd.) 10 .times. Ex Taq buffer (manufactured by
Takara Shuzo Co., Ltd.) 200 dNTP mixture (2.5 mM each) 240 KQ2
primer (for amber codon reversion; 5 pmol/.mu.l) 20 mut1 primer
(mutagenic primer; 5 pmol/.mu.l) 5 pKF19kM (5 ng/.mu.l) 5 pKF18k
and pKF19k (100 D/ml) 10 each Escherichia coli MV1184 100 (stored
in 10% glycerol solution) Remark: pKF18k and pKF19k being
manufactured by Takara Shuzo Co., Ltd..
INDUSTRIAL APPLICABILITY
[0097] According to the present invention, there can be provided a
method for performing site-directed mutagenesis and a kit, which is
useful for genetic engineering and protein engineering, more simply
and rapidly. By using the method and kit of the present invention,
it is possible to efficiently obtain a mutation-introduced gene at
a desired position by simply transforming host with a PCR product
obtained by PCR.
Sequence CWU 1
1
9 1 20 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 1 gggttttccc agtcacgacg 20 2 20 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 2
gattgcgcct gagcgagacg 20 3 23 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 3 cggtacccgg ggctctagag tcg 23
4 23 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 4 cggtacccgg ggtagagtcg acc 23 5 20 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 5
gacggccagt gtaatcatgg 20 6 24 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 6 gctggcgtga attcagcgaa gagg
24 7 30 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 7 ccgaaaatga cccagggatc cgccggcacc 30 8 60 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 8 gccaagcttg catgcctgca ggtcgactct agaggatccc cgggtaccga
gctcgaattc 60 9 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 9 caacgtcgtg actaggaaaa ccctggc
27
* * * * *