U.S. patent application number 10/533750 was filed with the patent office on 2006-06-15 for method of detecting gene mutation.
This patent application is currently assigned to Daiichi Pure Chemicals Co., Ltd.. Invention is credited to Shigeo Kure, Yoichi Matsubara.
Application Number | 20060127907 10/533750 |
Document ID | / |
Family ID | 32310418 |
Filed Date | 2006-06-15 |
United States Patent
Application |
20060127907 |
Kind Code |
A1 |
Matsubara; Yoichi ; et
al. |
June 15, 2006 |
Method of detecting gene mutation
Abstract
DNA amplification and hybridization are successively carried out
in a reaction system containing primers for the DNA amplification
and hybridization probes, followed by detecting the hybrid in the
reaction solution by affinity chromatography, wherein at least one
of the primers to be used in the DNA amplification is labeled with
a first labeling agent so that the amplified DNA will be labeled
with the first labeling agent, a hybridization probe is labeled
with a second labeling agent and contained in a reaction solution
for effecting the DNA amplification, the base sequence of the
hybridization probe is designed not to inhibit the DNA
amplification, and a hybrid is detected by affinity chromatography
with the use of the first and second labeling agents.
Inventors: |
Matsubara; Yoichi;
(Sendai-shi, JP) ; Kure; Shigeo; (Sendai-shi,
JP) |
Correspondence
Address: |
SONNENSCHEIN NATH & ROSENTHAL LLP;FOR PAULA EVANS
P.O. BOX 061080
WACKER DRIVE STATION, SEARS TOWER
CHICAGO
IL
60606-1080
US
|
Assignee: |
Daiichi Pure Chemicals Co.,
Ltd.
|
Family ID: |
32310418 |
Appl. No.: |
10/533750 |
Filed: |
November 7, 2003 |
PCT Filed: |
November 7, 2003 |
PCT NO: |
PCT/JP03/14204 |
371 Date: |
December 5, 2005 |
Current U.S.
Class: |
435/6.18 ;
435/6.1; 435/91.2; 536/25.32 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 1/6827 20130101; C12Q 2535/131 20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 536/025.32 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34; C07H 21/04 20060101
C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 7, 2002 |
JP |
323419/2002 |
Claims
1. A method of detecting a base sequence, comprising the steps of:
amplifying DNA containing a target base sequence to be detected
having a mutation site using DNA polymerase; hybridizing the
amplified DNA to a hybridization probe having a base sequence
complementary to the target base sequence to be detected; and
detecting a hybrid formed by the hybridization, wherein at least
one of primers to be used in the DNA amplification is labeled with
a first labeling agent so that the amplified DNA will be labeled
with the first labeling agent, the hybridization probe is labeled
with a second labeling agent and contained in a reaction solution
for effecting the DNA amplification, the base sequence of the
hybridization probe is designed not to inhibit the DNA
amplification, and the hybrid is detected by affinity
chromatography with the use of the first and second labeling
agents.
2. The method according to claim 1, wherein the mutation site is a
point mutation, and the reaction solution for effecting the DNA
amplification further contains an unlabeled oligonucleotide having
a base sequence different in a single base at the position of the
point mutation from the base sequence of the labeled hybridization
probe, in an amount sufficient to enhance the specificity of
hybridization of the amplified DNA to the hybridization probe.
3. The method according to claim 1 or 2, wherein the DNA
amplification is carried out by PCR.
4. A kit comprising: primers for amplifying DNA containing a target
base sequence to be detected having a mutation site using DNA
polymerase; a hybridization probe having a base sequence
complementary to the target base sequence to be detected; and a
test strip for affinity chromatography, wherein at least one of the
primers to be used in the DNA amplification is labeled with a first
labeling agent so that the amplified DNA will be labeled with the
first labeling agent, the hybridization probe is labeled with a
second labeling agent, the base sequence of the hybridization probe
is designed not to inhibit the DNA amplification, and the test
strip allows of detection of a hybrid of the amplified DNA and the
hybridization probe with the use of the first and second labeling
agents.
5. The kit according to claim 4, wherein the mutation site is a
point mutation and the kit further comprises an unlabeled
oligonucleotide having a base sequence different in a single base
at the position of the point mutation from the base sequence of the
labeled hybridization probe.
6. The kit according to claim 4 or 5, wherein the primers are
primers for PCR.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of detecting a
base sequence, and more particularly to a method of detecting a
base sequence containing a mutation site such as a point mutation,
thereby detecting a gene mutation.
BACKGROUND ART
[0002] There exist a number of gene polymorphisms on the genome,
which have been considered to be deeply associated with
susceptibility to diseases, individual variations in drug
metabolism, and the like. The detection of the gene polymorphism is
indispensable for so-called tailor-made medicine and becomes one of
the most important subjects on the clinical applications of genomic
science. Among others, much interest is lately focused on SNP
(single nucleotide polymorphism; gene polymorphism caused by
substitution of a single base) as a marker of the gene
polymorphism, on which huge research funds have been spent on a
global basis. On the other hand, data on gene mutations associated
with various genetic diseases has been accumulated into databases
by virtue of progress on molecular genetics research. Accordingly,
it has become reality to make the diagnosis of genetic diseases or
the prediction of clinical categories by screening for known gene
mutations already found to be pathogenic on the basis on these
databases. In particular, a gene mutation that occurs with high
frequency within a certain population or interracially is of great
diagnostic value.
[0003] The gene polymorphism and gene mutation include, for
example, a base substitution, deletion, insertion, and variations
in the number of repetitive sequences, and among them, a point
mutation caused by substitution of a single base makes up the
overwhelming majority. A method of simply and quickly detecting a
point mutation is indispensable for applying the outcomes of human
genome research to clinical purposes.
[0004] Until now, a variety of methods have been devised for
detecting a point mutation (see Cotton RGH. Mutation Detection. pp.
1-198, Oxford University Press, Oxford, 1997). Typical methods
include the allele specific oligonucleotide hybridization (ASO)
method, allele specific amplification method, restriction enzyme
digestion method, ligase chain reaction, and minisequencing method.
These methods require complicated procedures including
hybridization or electrophoresis after DNA amplification. On the
other hand, the TaqMan method, invader assay, DNA microarray (DNA
chip) assay, TOF-MASS method with the use of a mass spectrometer,
and the like, which have been recently developed for promoting the
human genome analysis and research, are suited to deal with a large
number of samples. However, these methods require high-priced,
specialized instruments and cannot be easily performed at clinical
laboratories. Alternatively, the SSCP method, chemical cleavage
method, and DHPLC method are widely used for screening of gene
mutations, and are highly effective for broad screening of unknown
gene mutations; but are inadequate to reliable detection of a known
mutation. In addition, the detection of a point mutation by the use
of the sequencing method requires complicated procedures and high
expenses, and is of undeniably too much quality for the detection
of a known mutation. At present, all of these methods described
above involve special examinations performed at gene research
laboratories and find a great difficulty in quick performance in
clinical settings (or at bedside).
[0005] Probes used in the ASO method has been conventionally 15 to
25 mer (see Saiki R K, Erlich H A. Detection of mutations by
hybridization with sequence-specific oligonucleotide probes. In:
Mutation Detection: A Practical Approach. pp. 113-129, IRL Press,
Oxford, 1998). Moreover, it is known that the specificity of a
labeled probe for hybridization is enhanced using an
oligonucleotide that competes with the probe (see Nozari G, Rahbar
S, Wallace R B. Discrimination among the transcripts of the allelic
human .beta.-globin genes .beta..sup.A, .beta..sup.S and
.beta..sup.C using oligodeoxynucleotide hybridization probes. Gene
43: 23-28, 1986).
DISCLOSURE OF THE INVENTION
[0006] An object of the present invention is to provide a method of
simply and quickly detecting a gene mutation.
[0007] The present inventors have used to achieve the present
invention the findings that the use of certain primers and probes
under a certain condition enables both amplification and
hybridization of nucleic acids in one reaction system, and also
enables a easy detection of a hybrid formed by the
hybridization.
[0008] The present invention provides the following:
[0009] (1) A method of detecting a base sequence, comprising the
steps of: amplifying DNA containing a target base sequence to be
detected having a mutation site using DNA polymerase; hybridizing
the amplified DNA to a hybridization probe having a base sequence
complementary to the target base sequence to be detected; and
detecting a hybrid formed by the hybridization,
[0010] wherein at least one of primers to be used in the DNA
amplification is labeled with a first labeling agent so that the
amplified DNA will be labeled with the first labeling agent, the
hybridization probe is labeled with a second labeling agent and
contained in a reaction solution for effecting the DNA
amplification, the base sequence of the hybridization probe is
designed not to inhibit the DNA amplification, and the hybrid is
detected by affinity chromatography with the use of the first and
second labeling agents.
[0011] (2) The method according to item (1), wherein the mutation
site is a point mutation, and the reaction solution for effecting
the DNA amplification further contains an unlabeled oligonucleotide
having a base sequence different in a single base at the position
of the point mutation from the base sequence of the labeled
hybridization probe, in an amount sufficient to enhance the
specificity of hybridization of the amplified DNA to the
hybridization probe.
[0012] (3) The method according to item (1) or (2), wherein the DNA
amplification is carried out by PCR.
[0013] (4) A kit comprising: primers for amplifying DNA containing
a target base sequence to be detected having a mutation site using
DNA polymerase; a hybridization probe having a base sequence
complementary to the target base sequence to be detected; and a
test strip for affinity chromatography,
[0014] wherein at least one of the primers to be used in the DNA
amplification is labeled with a first labeling agent so that the
amplified DNA will be labeled with the first labeling agent, the
hybridization probe is labeled with a second labeling agent, the
base sequence of the hybridization probe is designed not to inhibit
the DNA amplification, and the test strip allows of detection of a
hybrid of the amplified DNA and the hybridization probe with the
use of the first and second labeling agents.
[0015] (5) The kit according to item (4), wherein the mutation site
is a point mutation and the kit further comprises an unlabeled
oligonucleotide having a base sequence different in a single base
at the position of the point mutation from the base sequence of the
labeled hybridization probe.
[0016] (6) The kit according to item (4) or (5), wherein the
primers are primers for PCR.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the principle of the detection method according
to the present invention (when normal DNA is used as a sample).
[0018] FIG. 2 shows the principle of the detection method according
to the present invention (when mutant DNA is used as a sample).
[0019] FIG. 3 shows an example of procedures used in the detection
method according to the present invention.
[0020] FIG. 4 shows the result of detection (chromatogram images)
in the case of using a 17-mer hybridization probe.
[0021] FIG. 5 shows the result of detection (chromatogram images)
in the case of using a 17-mer hybridization probe and adding a
competing probe.
[0022] FIG. 6 shows the result of detection (chromatogram images)
in the case of using hybridization probes of various lengths and
adding a competing probe.
[0023] FIG. 7 shows the result of detection (chromatogram images)
in the case of using a 12-mer hybridization probe and adding a
competing probe.
[0024] FIG. 8 shows the result of the detection (chromatogram
images) of a variety of mutations.
[0025] FIG. 9 shows the result of the detection (chromatogram
images) of a variety of mutations.
BEST MODE FOR CARRYING OUT THE INVENTION
<1> Detection Method of the Present Invention
[0026] In the present invention, there is provided a method of
detecting a base sequence, which comprises the steps of: amplifying
DNA containing a target base sequence to be detected having a
mutation site using DNA polymerase; hybridizing the amplified DNA
to a hybridization probe having a base sequence complementary to
the target base sequence to be detected; and detecting a hybrid
formed by the hybridization;
[0027] characterized in that at least one of primers to be used in
the DNA amplification is labeled with a first labeling agent so
that the amplified DNA will be labeled with the first labeling
agent, the hybridization probe is labeled with a second labeling
agent and contained in a reaction solution for effecting the DNA
amplification, the base sequence of the hybridization probe is
designed not to inhibit the DNA amplification, and the hybrid is
detected by affinity chromatography with the use of the first and
second labeling agents. Hereinafter, each of the steps will be
described.
(1) DNA Amplification
[0028] The DNA amplification is carried out, if using DNA
polymerase, without any particular limitation. Any amplification
methods comprising the step of synthesizing DNA with the use of DNA
polymerase can be employed. Examples of the DNA amplification
method include PCR, TMA, NASBA, and LAMP methods.
[0029] The synthesis of DNA with DNA polymerase requires primers.
The primers are designed by a method known in the art depending on
an amplification method to be used and a target base sequence to be
detected. In the present invention, at least one of primers to be
used in DNA amplification is labeled with a first labeling agent so
that the amplified DNA will be labeled with the first labeling
agent.
[0030] For example, when DNA amplification is effected by the PCR
method, a pair of primers are used and at least one thereof is
labeled so that the amplified DNA can be labeled. Alternatively, a
primer that functions at the stage of DNA synthesis in DNA
amplification by the NASBA and TMA methods or at least one of inner
primers in DNA amplification by the LAMP method is labeled, thereby
allowing the labeling of the amplified DNA.
[0031] The labeling of primers is carried out so as not to inhibit
DNA synthesis reaction. Such labeling can be carried out according
to a method known in the art, and a primer is usually labeled at
its 5' end.
[0032] A labeling agent to be used in the labeling may be those to
which a corresponding substance can be biospecifically bound. A
pair of the labeling agent and the substance biospecifically bound
thereto includes an antigen and an antibody, an enzyme and an
inhibitor, a sugar chain and lectin, a hormone and a receptor, and
a metal-binding protein and a metal element. Specifically, a pair
of digoxigenin and an anti-digoxigenin antibody and a pair of
biotin and streptavidin may be used. In these pairs, either of the
two may be given as a labeling agent. However, the smaller
molecular weight partner is generally used as a labeling agent.
[0033] Primers and DNA amplification conditions to be used are
appropriately adjusted on the basis of type of an amplification
method and a target base sequence to be detected. For example, see:
Molecular Cloning: A Laboratory Manual (3rd ed.), Volume 2, Chapter
8, pp. 8.1-8.126, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, 2001 on the PCR method; PCR Methods and Applications, 1,
25-33 (1991) on the NASBA method; and Nucleic Acids Research, Vol.
28, No. 12, pp. i-vii (2000) on the LAMP method.
[0034] Test DNA that functions as a template in DNA amplification
can be prepared from a test sample by a conventional method.
[0035] The target base sequence to be detected is appropriately
selected depending on the type of an amplification method so that
the target base sequence having a mutation site can be specifically
amplified. In general, the mutation site contained in the target
base sequence to be detected is known as a site of gene mutation or
gene polymorphism. The mutation of the site may be a point
mutation, an insertion mutation or a deletion mutation.
[0036] Common examples of the gene mutation and gene polymorphism
to be analyzed by the detection method of the present invention
include, but not limited to: g727t mutation observed with high
frequency in Japanese patients with glycogen storage disease type
Ia; a985g mutation (Lys329Glu mutation) observed with high
frequency in Caucasian patients with medium-chain acyl-CoA
dehydrogenase deficiency; g1691t mutation (Ser564Ile mutation) of
GLDC gene observed with high frequency in Finnish patients with
hyperglycinemia; gene polymorphism (CYP2C19*2, g681a) in drug
metabolizing enzyme gene CYP2C19; gene polymorphism (E487K) of an
aldehyde dehydrogenase 2 determining individual variations in
alcohol metabolism; deltaF508 deletion mutation in the gene of
cystic fibrosis transmembrane regulator protein; 1277insTATC
insertion mutation in HEXA gene associated with Tay-Sachs disease;
5382insC insertion mutation in BRCA1 gene associated with breast
cancer; 6174delT deletion mutation in BRCA2 gene associated with
breast cancer; and G1691A point mutation in Blood Coagulation
Factor V gene associated with thrombosis.
[0037] Glycogen storage disease type Ia is a congenital disorder of
carbohydrate metabolism, inherited in an autosomal recessive
manner, caused by deficiency of glucose-6-phosphatase in the
glycogen metabolic pathway, and leads to an excess accumulation of
glycogen mainly in the liver. Patients with glycogen storage
disease type Ia are found to have hypoglycemia, hepatomegaly, short
statue, renal damage, hyperlipidemia, hyperuricemia, or the like.
Mutation g727t in the gene of this enzyme is a highly frequent
mutation making up approximately 90% of pathogenic mutations in
Japanese cases, and generates aberrant splicing of its mRNA.
Although the diagnosis of this disease has been usually performed
until very recently by measuring enzyme activity of liver tissues,
the emergence of genetic diagnosis has eliminated the need of liver
biopsy. The number of carriers having this mutation in Japanese
population is one in about 200 people.
[0038] Non-ketotic hyperglycinemia is a congenital disorder of
amino acid metabolism (autosomal recessive inheritance) caused by
deficiency of an enzyme of the glycine cleavage system, and
exhibits severe neurological symptoms including neonatal convulsion
during neonatal period. Mutation g1691t in GLDC gene of the enzymes
in the glycine cleavage system is observed with high frequency
(approximately 70% of mutated genes) in Finnish patients. This
mutation causes an amino acid substitution of Ser564Ile.
[0039] Medium-chain acyl-CoA dehydrogenase deficiency is a
congenital disorder of organic acid metabolism (autosomal recessive
inheritance) caused by deficiency of the enzyme (medium-chain
acyl-CoA dehydrogenase, MCAD) playing a key role in fatty acid
.beta. oxidation pathway, and brings about hypoglycemia and
consciousness disturbance at fasting and infection. It is known
that medium-chain acyl-CoA dehydrogenase deficiency is often
misdiagnosed as sudden infant death syndrome or acute
encephalopathy (Reye syndrome). Mutation a985g in the gene of this
enzyme is a highly frequent mutation making up approximately 90% of
pathogenic mutations in Caucasian cases, and produces an amino acid
substitution of Lys329Glu. Moreover, carriers having this gene
mutation are found with high frequency in Caucasian population (one
in 40 people in the U.K.). In U.S.A. and European countries, the
genetic diagnosis of detecting this a985g mutation is widely used
for diagnosis of this disease.
[0040] CYP2C19 gene plays a key role in the metabolism of
omeprazole (inhibitor of gastric acid secretion) or the like.
CYP2C19*2, a SNP in the gene, shows 681A>G mutation in exon 5,
leading to aberrant splicing, and thus finally decreases in the
metabolic activity to this drug. An individual having such a
polymorphism (poor metabolizer) needs a decreased amount of the
drug to be administrated to the subject. Therefore, it is
clinically advantageous to determine the genotype of a patient
before the medication. This gene polymorphism is found in
approximately 23% of the gene in Japanese population.
[0041] The gene polymorphism (Glu487Lys) of aldehyde dehydrogenase
2 is a SNP, observed largely in oriental population, to determine
individual variations in alcohol metabolism. Because the enzyme
having the gene polymorphism is less active to slow down the
metabolism of acetaldehyde generated from alcohol, an individual
having this polymorphism shows a constitutional "low tolerance for
alcohol". Approximately 30% in Japanese population have a
heterozygote of this gene polymorphism and approximately 5% have a
homozygote thereof.
(2) Hybridization
[0042] The hybridization of the amplified DNA to a hybridization
probe having a base sequence complementary to the target base
sequence to be detected may be carried out in the same manner as
general hybridization except that a particular hybridization probe
is used.
[0043] The hybridization probe used in the present invention is
labeled with a second labeling agent and contained in a reaction
solution for effecting the DNA amplification, and the base sequence
of the hybridization probe is designed not to inhibit the DNA
amplification.
[0044] The second labeling agent is defined as described in the
first labeling agent; provided that the substance used for it must
be different from the first labeling agent. The labeling of the
hybridization probe can be carried out by a method known in the art
so as not to inhibit the hybridization. The labeling of the
hybridization probe is preferably carried out at its 3' end. This
is because such labeling prevents the oligonucleotide chain from
being extended during the DNA amplification reaction. If the chain
is extended in length, the Tm value thereof is increased and thus
hybridization may occur even though there are mismatches.
[0045] The design of the base sequence of the hybridization probe
so as not to inhibit the DNA amplification can be generally
selected by adjusting the chain length or the like of the
hybridization probe so that the hybridization of the hybridization
probe will not occur under the DNA amplification condition.
[0046] The hybridization probe used in the present invention has a
base sequence designed so as not to inhibit the DNA amplification,
and can be thus previously contained in a reaction solution for
effecting the DNA amplification. Therefore, the reaction solution
after the DNA amplification is completed is placed directly under
such a condition that the amplified DNA can be hybridized to the
hybridization probe, thereby allowing the hybridization
thereof.
[0047] The chain length of the hybridization probe and the
condition of hybridization thereof are appropriately selected
depending on a method used in the DNA amplification. In DNA
amplification with the use of DNA polymerase, because the
amplification is effected under a temperature condition suitable to
for the DNA polymerase exhibit its activity, the chain length is
selected so that the hybridization will not occur at this
temperature. In addition, the temperature at which the
hybridization occurs is not particularly limited as long as DNA
amplification is not inhibited, but is preferably selected so that
the generated hybrid may not be dissociated even at room
temperature.
[0048] A specific condition under which the base sequence of the
probe does not inhibit the DNA amplification includes a condition
that the Tm of the probe is designed to be 25 to 40.degree. C.
(preferably 30 to 35.degree. C.) lower than the Tm of primers.
[0049] For example, with consideration given to general conditions
of the PCR method, the probe should be typically 10-mers to
13-mers. This is much shorter than 15-mer to 25-mer probes (see the
above-mentioned non-patent reference 2) that have been
conventionally used as a probe for allele specific oligonucleotide
hybridization. In the context of that longer probes have been
extensively used heretofore, there has been the theory that a
sequence of at least about the 15th power of 4 is required for
constructing a probe having specificity by combinations of four
different bases in the whole genome sequence (3 billion base
pairs). However, this holds true for the case where hybridization
is directed toward the whole genome sequences. When hybridization
is directed to a PCR-amplified DNA fragment having several hundreds
of bases, such length or specificity is not considered necessary
for probes that the specificity of hybridization is sufficiently
maintained with the former probe as shown above.
[0050] When using the detection method of the present invention for
the detection of any gene mutation or polymorphism, the
hybridization probe is required to be adjusted to an optimum chain
length. The optimum length can be determined by a standard
experimentation, as described in Examples below. Because an
extremely short length of a probe is usually used in the detection
method of the present invention, formation of a diagnostic line is
found to dramatically vary depending on the length variations by a
single base. When the emergence of false positive or weak positive
reaction is observed, it is preferred that a probe having a shorter
or longer length than that of the probe designed based on its Tm
value may be constructed to choose the most suitable one. In this
respect, because a probe having a normal base sequence and a probe
having a mutant base sequence are different in Tm value due to the
base substitution even though they have the same chain length, each
optimum chain length should be designed independently.
[0051] It is preferred to design the base sequence of the
hybridization probe so that the mutation site will be positioned in
approximately middle of the base sequence.
[0052] Hybridization is usually carried out by increasing a
temperature until double-stranded DNA is denatured, followed by
gradually lowering the temperature. Thus, the hybridization can be
carried out by only a procedure of changing the temperature of a
reaction solution in which DNA amplification is completed, without
the need of any other procedures. In the case of using a
programmable thermal cycler in DNA amplification, a temperature
condition for hybridization can be programmed in addition to a
temperature condition requisite to the DNA amplification, thereby
effecting the amplification and the hybridization as a series of
reactions, after a sample is loaded in the thermal cycler.
[0053] The use of a short length of the probe designed as described
above provides the following three advantages: 1) the difference in
Tm values between the case where there is a mismatch of a single
base and the case where there is no mismatch can be rendered larger
than that of a longer length of a probe, and thus the specificity
of the probe can be relatively increased; 2) the hybridization
temperature of the probe can be given as low as 25.degree. C. in
the detection method of the present invention, although
conventionally 37 to 65.degree. C., and thus a subsequent series of
procedures can be carried out at room temperature; and 3) a short
length of the probe has a reduced Tm value and does not hybridize
during PCR reaction, and thus the probe does not affect the PCR
reaction even though it is previously mixed in the PCR reaction
solution. This probe enables the procedures PCR.fwdarw.heat
denaturation.fwdarw.hybridization to be carried out as a series of
reactions, without performance of additional procedures such as the
addition of a reagent during the reactions. These advantages can be
similarly obtained in other DNA amplification methods with the use
of an extension reaction by DNA polymerase, as in the PCR
method.
(3) Detection of Hybrid
[0054] A hybrid formed by the hybridization has both the first
labeling agent and the second labeling agent. The hybrid is
detected by affinity chromatography with the use of the first and
second labeling agents.
[0055] The affinity chromatography can be carried out with a test
strip constructed for this purpose. The detection of a hybrid by
affinity chromatography with the use of two different labeling
agents can be carried out according to a method known in the art,
and a test strip used in this method can be constructed according
to a general method.
[0056] An example of such a test strip is constructed so that a
hybrid will be reacted with a substance which is capable to be
specifically bound to the first labeling agent and is coupled with
a labeling agent (e.g., gold colloid) to become visible when
accumulated; and transferred onto a chromatography support on which
a substance capable to be specifically bound to the second labeling
agent is immobilized, to allow of the observation of the visible
labeling agent when accumulated on that immobilization site. Such a
test strip itself has been also used so far in a method of simply
detecting a certain gene (J. Clin. Microbiol. 38: 2525-2529,
2000).
[0057] Hereinafter, an illustration will be provided in a specific
case where the first labeling agent is digoxigenin, the second
labeling agent is biotin, and the labeling agent that is visible
when accumulated is gold colloid. The following sites are placed in
the order named in the migration direction of a chromatography
solvent (generally, a buffer solution): an immersion site that is
immersed in a chromatography solvent to provide the chromatography
solvent to the strip of the chromatography support; a
complex-carrying site that has a pad carrying an anti-digoxigenin
antibody conjugated with gold colloid (a complex) in a manner that
this antibody can be released into the chromatography solvent; a
sample-applying site to which the reaction solution containing a
hybrid is applied; a streptavidin-immobilized site on which a band
of streptavidin is immobilized perpendicularly to the migration
direction of the chromatography solvent; an antibody-immobilized
site on which an antibody against the anti-digoxigenin antibody is
immobilized; and an absorption site that has a pad absorbing the
chromatography solvent.
[0058] This test strip is used as described below. After the
reaction solution containing the hybrid is applied to the
sample-applying site and the immersion site is immersed in the
chromatography solvent, the test strip is removed from the
chromatography solvent and left to stand. When the chromatography
solvent migrates through the chromatography support by capillary
action and reaches the complex-carrying site, from which the
chromatography solvent containing the complex will migrate forward.
When this chromatography solvent reaches the sample-applied site,
the digoxigenin of the hybrid in the applied reaction solution will
bind to the anti-digoxigenin antibody of the complex to form the
hybrid having the gold colloid, which further migrates forward
through the chromatography support by the chromatography solvent.
When the hybrid reaches the streptavidin-immobilized site, this
hybrid will be accumulated on the streptavidin-immobilized site
through the binding of biotin and streptavidin; consequently a
visible signal shall be seen when the hybrid is present. The
complex that has passed through the streptavidin-immobilized site
is accumulated on the antibody-immobilized site to generate a
visible signal showing that the chromatogram has proceeded
normally. The chromatography solvent further migrating will be
absorbed and held in the absorption site.
[0059] In the detection method of the present invention, if the
mutation site is a point mutation, preferably, the reaction
solution for effecting DNA amplification further contains, along
with the hybridization probe, an unlabeled oligonucleotide
(hereinafter, also referred to as a "competing probe") having a
base sequence different in a single base at the position of the
point mutation from the base sequence of the labeled hybridization
probe, in an amount sufficient to enhance the specificity of the
hybridization of the amplified DNA to the labeled hybridization
probe.
[0060] The competing probe is designed in the same way as the
hybridization probe except that it differs from the hybridization
probe in a single base at the position of the point mutation. The
length of the competing probe may be different from that of the
hybridization probe.
[0061] The amount of the competing probe sufficient to enhance the
specificity of the hybridization of the amplified DNA to the
labeled hybridization probe varies depending on conditions such as
the target base sequence to be detected and the base sequence of
the hybridization probe, whereas in principle, the competing probe
may be usually contained in the range from an equal amount to
5-fold amount (molar ratio) with respect to the amount of the
hybridization probe. Nevertheless, when positive reaction is
significantly reduced, the omission of the competing probe, if it
is confirmed not to cause false positives, may sometimes produce
the best result. Because the formation of a diagnostic line is
significantly affected by the chain length of the hybridization
probe and the presence or absence of the competing probe, an
optimum reaction condition will be relatively easily found.
[0062] The specificity of the hybridization probe can be enhanced
and non-specific hybridization can be suppressed by adding an
unlabeled competing oligonucleotide in the hybridization.
[0063] In the detection method of the present invention, different
labeling agents may be used for labeling a hybridization probe for
detecting a normal base sequence and a hybridization probe for
detecting a mutant base sequence, to integrate two reaction systems
for detecting a normal base sequence and for detecting a mutant
base sequence into one reaction system. That is, the hybridization
probes for detecting a normal base sequence and for detecting a
mutant base sequence can be differently labeled and mixed together
in the ratio of 1:1 to integrate the reaction systems into one
while these hybridization probes are allowed to compete with each
other. After the reaction, using the complexes of substances which
are capable of specifically binding to the labeling agents,
respectively, and a labeling agent that becomes visible when
accumulated, affinity chromatography is carried out to determine a
genotype.
[0064] The detection method of the present invention has the
following advantages: (1) versatility: the method is based on
allele specific oligonucleotide hybridization that has been widely
used as a detection method for a long time, and therefore adaptable
to detection of a wide range of base sequence mutations such as an
insertion mutation, a substitution mutation, and a point mutation;
(2) rapidity: the determination of a genotype can be carried out
within 10 minutes after the amplification and hybridization
reactions have been completed in a thermal cycler, and the use of a
capillary-type PCR amplification device in the nucleic acid
amplification also enables all steps to be completed within 1 hour,
if a DNA sample is ready; and (3) simplicity: after PCR reaction, a
genotype can be macroscopically determined, thereby eliminating the
need for an instrument such as a gel electrophoresis device or a
fluorescence detector. A thermal cycler for effecting PCR reaction
is a general-purpose instrument for clinical examination, for
example, an examination for infectious diseases, and has been
already placed in many hospitals. Moreover, the reaction procedure
is simple without the need for special technical skills. The
above-described advantages can be also obtained when nucleic acid
reactions (TMA, NASBA, LAMP, etc.) other than PCR are used.
[0065] The principle of the detection method of the present
invention will be more fully illustrated in the case using PCR with
reference to FIGS. 1 to 3.
[0066] FIG. 1 shows a reaction with the use of normal DNA as a
sample. Reaction system 1 is a system to which is added a
hybridization probe for detecting a normal base sequence and
reaction system 2 is a system to which is added a hybridization
probe for detecting a mutant base sequence. In this figure, the
black circle represents a normal base, the black triangle
represents a mutant base, Dig represents a digoxigenin label, B
represents a biotin label, and GP represents a gold particle.
[0067] At first, a gene site having a point mutation (the target
base sequence to be detected) is amplified by PCR. One primer of
the PCR primer pair used in this case has been previously labeled
at its 5' end with digoxigenin. In a PCR reaction solution, two
oligonucleotides (hybridization probe and competing probe) have
been mixed with typical components. In this combination of the
oligonucleotides, there exist two combinations for detecting a
normal base sequence and for detecting a mutant base sequence. In
the combination for detecting a normal base sequence, one is an
oligonucleotide (normal probe) having a normal base sequence with
the point mutation site located in the middle portion thereof and
labeled with biotin at its 3' end; and the other is an unlabeled
competing oligonucleotide (mutant probe) having a mutant base
sequence with the point mutation site located in the middle portion
thereof. In the combination for detecting a mutant base sequence,
one is an oligonucleotide (mutant probe) having a mutant base
sequence with the point mutation site located in the middle portion
thereof and labeled with biotin at its 3' end and the other is an
unlabeled competing oligonucleotide (normal probe) having a normal
base sequence with the point mutation site located in the middle
portion thereof. Any of these oligonucleotides are designed to be a
reverse strand relative to the PCR primer labeled with
digoxigenin.
[0068] The composition of the PCR reaction solution is, for
example, 50 to 100 ng of sample DNA, 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl.sub.2, 250 .mu.M each dNTPs, 1 .mu.M PCR forward
primer (labeled with digoxigenin at its 5' end), 1 .mu.M PCR
reverse primer, 600 nM hybridization probe (labeled with biotin at
its 3' end), 3 .mu.M unlabeled competing oligonucleotide, and 1.25
U Taq DNA polymerase, and the amount of the PCR reaction solution
is 20 .mu.l. The PCR condition is: for example, heating at
94.degree. C. for 2 minutes; 35 cycles of 98.degree. C. for 10
seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 30
seconds; followed by 72.degree. C. for 3 minutes; 98.degree. C. for
3 minutes; 65.degree. C. for 1 minute; 55.degree. C. for 1 minute;
45.degree. C. for 1 minute; 35.degree. C. for 1 minute; and
25.degree. C. for 1 minute. In this process after the cycle
reactions are repeated, a PCR product labeled with digoxigenin is
hybridized with oligonucleotide having a base sequence completely
complementary to the base sequence of the PCR product. For example,
when oligonucleotides for detecting a normal base sequence is used
with the DNA having a normal base sequence in combination, a PCR
product labeled with digoxigenin and an oligonucleotide labeled
with biotin form a hybrid (FIG. 1, reaction system 1). An aliquot
(5 .mu.l) of this solution is spotted onto the sample-applying site
in a test strip of affinity chromatography, such as DNA detection
test strip (Roche, #1-965-484), on which streptavidin is
immobilized and in which an anti-digoxigenin antibody labeled with
gold colloid is held in a manner that the antibody can migrate, and
the bottom end of the test strip is immersed in a buffer for 5
seconds. As the strip is left to stand at room temperature for 5
minutes while the buffer is developed, the anti-digoxigenin
antibody labeled with gold colloid binds to the hybrid of the PCR
product labeled with digoxigenin and the oligonucleotide labeled
with biotin. This hybrid is further captured by streptavidin
immobilized on the test strip to form a red line that can be
macroscopically observed. On the other hand, when oligonucleotides
for detecting a mutant base sequence is used with the DNA having a
normal base sequence in combination, a PCR product labeled with
digoxigenin and an unlabeled oligonucleotide form a hybrid. After
this solution is spotted to the sample-applying site of the test
strip and subjected to a development with a buffer, an
anti-digoxigenin antibody labeled with gold colloid binds to the
hybrid of the PCR product labeled with digoxigenin and the
unlabeled oligonucleotide. However, because this hybrid is not
captured by streptavidin on the test strip, a red line is not
formed (FIG. 1, reaction system 2). As described above,
macroscopically observing a formation of red line in each of the
two different reaction systems will make it possible to make a
determination of the genotype of DNA given as a sample. The
principle of the reaction of DNA having a mutant base sequence is
the same as above (FIG. 2).
[0069] The operation procedures in this aspect are shown in FIG. 3.
At first, a DNA as a sample is mixed with a reaction reagent in a
PCR tube and heated/cooled with a thermal cycler according to the
program to effect the DNA amplification and the formation of a
hybrid (Step 1). An aliquot (5 .mu.l) of the reaction solution is
spotted to the sample-applying site of the test strip and the
bottom end of the test strip is immersed in a buffer, followed by
standing at room temperature (Step 2). After 5 minutes, the
diagnosis is conducted on the basis of the presence or absence of
the diagnostic line to determine a genotype (Step 3). Whether the
affinity chromatography is normally completed or not can be
confirmed by examining the presence or absence of a control
line.
[0070] The detection method of the present invention is a method
capable of quickly and simply determining the presence or absence
of a gene mutation with accuracy and without the use of a special
device, and is suitable to conduct a genetic test in a hospital
outpatient clinic or at bed side. That is, the detection method
allows of a genetic diagnosis as a Point of Care. More
particularly, the gene polymorphism of drug-metabolizing enzymes
including CYP2C19 will be decided, which made it possible to
determine on the spot whether or not a certain drug is suitable for
a patient and to assist the adjustment of the dosage. In this case,
an important advantage is that a test result can be obtained in a
short time.
<2> Kit of the Present Invention
[0071] The kit of the present invention comprises: primers for
amplifying DNA containing a target base sequence to be detected
having a mutation site using DNA polymerase; a hybridization probe
having a base sequence complementary to the target base sequence to
be detected; and a test strip for affinity chromatography;
[0072] characterized in that at least one of the primers to be used
in the DNA amplification is labeled with a first labeling agent so
that the amplified DNA will be labeled with the first labeling
agent, the hybridization probe is labeled with a second labeling
agent, the base sequence of the hybridization probe is designed not
to inhibit the DNA amplification, and the test strip allows of
detection of a hybrid of the amplified DNA and the hybridization
probe with the use of the first and second labeling agents. The kit
of the present invention can be used for carrying out the detection
method of the present invention.
[0073] The primers, the hybridization probe, and the test strip for
affinity chromatography are as described above in the detection
method of the present invention.
[0074] If the mutation site is a point mutation, preferably, the
kit of the present invention further comprises an unlabeled
oligonucleotide (competing probe) having a base sequence different
in a single base at the position of the point mutation from the
base sequence of the labeled hybridization probe. This
oligonucleotide is as described above in the detection method of
the present invention.
EXAMPLES
[0075] The present invention will be described in detail with
reference to the following examples, which are only intended to
concretely illustrate the present invention, but not intend to
restrict the scope of the present invention in any way.
Example 1
Detection of Mutation g727t in Glycogenosis Type Ia
(1) Reaction System and Experimental Procedure
[0076] For detecting g727t mutation in glycogenosis Type Ia,
primers listed in Table 1 were prepared on the basis of known base
sequences around the mutation site. TABLE-US-00001 TABLE 1 Primers
and probes for detection of g727t mutation in glycogenosis type Ia
PCR forward primer (G6P-E5-1F-Dig):
5'-Dig-CCCAAATCCTTCCTATCTCTCACAG-3' (SEQ ID NO: 1) PCR reverse
primer (G6P-E5-1R(20)): 5'-TGCTGGAGTTGAGAGCCAGC-3' (SEQ ID NO:
2)
[0077] For examining the effect of chain lengths of probes,
oligonucleotides listed in Table 2 were prepared as hybridization
probes and competing probes. TABLE-US-00002 TABLE 2 (I)
Biotin-labeled oligonucleotide for detection of normal base
sequence: 17 mer: 5'-AAGCTGAACAGGAAGAA-Biotin-3' (SEQ ID NO: 3) 15
mer: 5'-AGCTGAACAGGAAGA-Biotin-3' (SEQ ID NO: 4) 13 mer:
5'-GCTGAACAGGAAG-Biotin-3' (SEQ ID NO: 5) 11 mer:
5'-CTGAACAGGAA-Biotin-3' (SEQ ID NO: 6) (II) Unlabeled competing
oligonucleotide for de- tection of normal base sequence: 17 mer:
5'-AAGCTGAAAAGGAAGAA-3' (SEQ ID NO: 7) 15 mer:
5'-AGCTGAAAAGGAAGA-3' (SEQ ID NO: 8) 13 mer: 5'-GCTGAAAAGGAAG-3'
(SEQ ID NO: 9) 11 mer: 5'-CTGAAAAGGAA-3' (SEQ ID NO: 10) (III)
Biotin-labeled oligonucleotide for detection of mutant base
sequence: 17 mer: 5'-AAGCTGAAAAGGAAGAA-Biotin-3' (SEQ ID NO: 11) 15
mer: 5'-AGCTGAAAAGGAAGA-Biotin-3' (SEQ ID NO: 12) 13 mer:
5'-GCTGAAAAGGAAG-Biotin-3' (SEQ ID NO: 13) 11 mer:
5'-CTGAAAAGGAA-Biotin-3' (SEQ ID NO: 14) (IV) Unlabeled competing
oligonucleotide for de- tection of mutant base sequence: 17 mer:
5'-AAGCTGAACAGGAAGAA-3' (SEQ ID NO: 15) 15 mer:
5'-AGCTGAACAGGAAGA-3' (SEQ ID NO: 16) 13 mer: 5'-GCTGAACAGGAAG-3'
(SEQ ID NO: 17) 11 mer: 5'-CTGAACAGGAA-3' (SEQ ID NO: 18)
[0078] The PCR reaction solution consists of 50-100 ng of sample
DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, 250
.mu.M each dNTPs, 1 .mu.M of PCR forward primer, 1 .mu.M of PCR
reverse primer (labeled with digoxigenin at the 5' end), 600 nM of
hybridization probe (labeled with biotin at the 3' end), unlabeled
competing oligonucleotide at a predetermined concentration, and
1.25 U Taq DNA polymerase in a final volume of 20 1.mu.l. The PCR
was carried out by heating at 94.degree. C. for 2 minutes and
repeating 35 times a cycle of 98.degree. C. for 10 seconds,
55.degree. C. for 30 seconds and 72.degree. C. for 30 seconds,
followed by 72.degree. C. for 3 minutes, 98.degree. C. for 3
minutes, 65.degree. C. for 1 minute, 55.degree. C. for 1 minute,
45.degree. C. for 1 minute, 35.degree. C. for 1 minute, and
25.degree. C. for 1 minute.
[0079] An aliquot (5 .mu.l) of the solution was spotted on a
sample-applying site of a test strip (DNA Detection Test Strip,
Roche Co., Ltd., #1-965-484, an affinity chromatographic test strip
on which streptavidin is immobilized and in which an
anti-digoxigenin antibody labeled with gold colloid is held in a
movable manner) and then the bottom end of the strip was immersed
in a buffer for 5 seconds. Then the test strip was left to stand
for 5 minutes at room temperature to allow the buffer to move
through the strip. After the keeping, the presence or absence of a
genotype diagnostic line was macroscopically determined.
(2) Examination of Competition with Unlabeled Oligonucleotide
[0080] The labeled hybridization probe used was of 17 mers and the
detection was then carried out without the addition of a competing
probe in a reaction solution. The DNA samples used were a
homozygote for g727 allele (normal DNA) and a homozygote for t727
allele (mutant DNA), and the hybridization probes used were those
for the detection of a normal base sequence and for the detection
of a mutant base sequence. The results are shown in FIG. 4. In this
figure, the indications "Wt" and "Mut" with respect to "DNA"
represent the normal DNA and the mutant DNA, respectively, while
the indications "Wt" and "Mut" with respect to "hybridization
probe" represent the hybridization probes for the detection of a
normal base sequence and for the detection of a mutant base
sequence, respectively (the same holds for FIG. 5 to FIG. 7
described below).
[0081] In any of the combinations tested, a false positive red
reaction line was recognized. Thus, the genotyping was unsuccessful
(FIG. 4, lanes 1-4).
[0082] A similar experiment was carried out by adding 5-50 times
more amount (molar concentrations) of the competing probe (17 mers)
than that of the hybridization probe to the reaction solution. The
results are shown in FIG. 5.
[0083] The addition of the competing probe resulted in a
substantial decrease in false positive reactions. In other words,
only very slight red reaction lines were observed in reaction
systems of probes for detecting a mutant base sequences with a
normal DNA (FIG. 5, lanes 6-8) and in reaction systems of probes
for detecting a normal base sequences with a mutant DNA (FIG. 5,
lanes 10-12). No difference was found in inhibitory effect on a
false positive reaction in any amounts of the competing probe
added, and even the addition of 50-fold amount could not completely
inhibit the false positive reaction. On the contrary, it was found
that the addition of 25-50 fold amounts inhibited normal positive
reactions so that the reaction lines would tend to become fairly
pale (FIG. 5, lanes 3, 4, 15, and 16).
(3) Examination of Chain Length of Hybridization Probe
[0084] The hybridization probes and competing probes used in this
study were of 17 mers, 15 mers, 13 mers, and 11 mers. The amount of
the competing probes added to the reaction solution was fixed to 30
times more than that of the hybridization probe. The results are
shown in FIG. 6.
[0085] In the reaction system of probes for detecting a mutant base
sequence to a normal DNA, a faint false positive line appeared in
case of 15 mers (FIG. 6, lane 4), but not appeared in cases of 13
mers and 11 mers (FIG. 6, lanes 5 and 6). In the reaction system of
probes for detecting a normal base sequence to a mutant DNA, no
false positive appeared in any cases of 15 mers, 13 mers and 11
mers (FIG. 6, lanes 7 to 9). Nevertheless, it was found that the
normal positive reactions tended to become decreased in case of 11
mer (FIG. 6, lanes 3 to 12).
[0086] In consideration of the results of examinations as described
above, using the hybridization and competing probes of 12 mers in
chain length (Table 3) with the five-fold amount of the competing
probe added, the detection was carried out in a similar manner for
samples of normal DNA (homozygote of g727 allele), carrier's DNA
(heterozygote of g727 allele and t727 allele), and patient's DNA
(homozygote of t727 allele). The results are shown in FIG. 7.
[0087] The results obtained completely corresponds to the genotype
with distinct positive reaction lines observed (FIG. 7, lanes 1 and
4 and lanes 5 and 6). On the contrary, no false positive reaction
were found (lanes 2 and 3). TABLE-US-00003 TABLE 3 Biotin-labeled
oligonucleotide for detection of normal base sequence
(GSD727-ASO-W12-Bio): 5'-GCTGAACAGGAA-Biotin-3' (SEQ ID NO: 19)
Unlabeled competing oligonucleotide for detection of normal base
sequence (GSD727-ASO-M12): 5'-GCTGAAAAGGAA-3' (SEQ ID NO: 20)
Biotin-labeled oligonucleotide for detection of mutant base
sequence (GSD727-ASO-M12-Bio): 5'-GCTGAAAAGGAA-Biotin-3' (SEQ ID
NO: 21) Unlabeled competing oligonucleotide for detection of mutant
base sequence (GSD727-ASO-W12): 5'-GCTGAACAGGAA-3' (SEQ ID NO:
22)
Example 2
[0088] Detection of Mutation a985g of Medium-Chain Acyl-CoA
Dehydrogenase Deficiency, Mutation g1691t of GLDC Gene in
Hyperglycinemia, Mutation g681a of Drug-Metabolizing Enzyme Gene
CYP2C19, and Point Mutation of Glu487Lys of Aldehyde Dehydrogenase
2 Polymorphism
[0089] The detection method of the present invention was carried
out to detect a point mutation, including mutation a985g of
medium-chain acyl-CoA dehydrogenase deficiency, mutation g1691t of
GLDC gene in hyperglycinemia, mutation g681a of drug-metabolizing
enzyme gene CYP2C19, and point mutation of Glu487Lys of aldehyde
dehydrogenase 2 polymorphism.
[0090] The PCR primers for amplifying base sequences containing the
respective point mutation sites were adjusted in chain length so as
to carry out PCR reactions with setting of an annealing temperature
of 55.degree. C. In addition, the hybridization probes were
designed to have Tm values in the range of 35 to 40.degree. C. As a
result, the chain lengths thereof were 10 mers to 15 mers. The base
sequences of primers, hybridization probes, and competing probes
are listed in Table 4. TABLE-US-00004 TABLE 4 (I) Primers and
probes for detection of a985g mutation of gene of medium-chain
acyl-CoA dehydrogenase deficiency PCR forward primer (Dig-MCAD-F1):
5'-Dig- CTTTTTAATTCTAGCACCAAGCAATATC-3' (SEQ ID NO: 23) PCR reverse
primer (Dig-MCAD-R1): 5'-Dig-TCCAAGTATCTGCACAGCAT-3' (SEQ ID NO:
24) Biotin-labeled oligonucleotide for detection of normal base
sequence (Bio-MCAD985-W13): 5'-GCAATGAAAGTTG-Biotin-3' (SEQ ID NO:
25) Unlabeled competing oligonucleotide for detection of normal
base sequence (MCAD985-M13): 5'-GCAATGGAAGTTG-3' (SEQ ID NO: 26)
Biotin-labeled oligonucleotide for detection of mutant base
sequence (Bio-MCAD985-M12): 5'-AACTTCCATTGC-Biotin-3' (SEQ ID NO:
27) Unlabeled competing oligonucleotide for detection of mutant
base sequence (MCAD985-W12): 5'-AACTTTCATTGC-3' (SEQ ID NO: 28)
(II) Primers and probes for detection of g1691t mutation of GLDC
gene PCR forward primer (Dig-GLDC-F):
5'-Dig-GTCTCTTGGTCCTACCTAATA-3' (SEQ ID NO: 29) PCR reverse primer
(GLDC-R): 5'-TTAGTGAAGCTAGAACACTG-3' (SEQ ID NO: 30) Biotin-labeled
oligonucleotide for detection of normal base sequence
(Bio-S564I-W13): 5'-GACCAACTGTTCA-Biotin-3' (SEQ ID NO: 31)
Unlabeled competing oligonucleotide for detection of normal base
sequence (S564I-M13): 5'-GACGAAATGTTCA-3' (SEQ ID NO: 32)
Biotin-labeled oligonucleotide for detection of mutant base
sequence (Bio-S564I-M): 5'-GACGAAATGTTCA-Biotin-3' (SEQ ID NO: 33)
Unlabeled competing oligonucleotide for detection of mutant base
sequence (S564I-W): 5'-GACGAACTGTTCA-3' (SEQ ID NO: 34) (III)
Primers and probes for detection of gene polymorphism CYP2C19*2 of
CYP2C19 gene PCR forward primer (CYP2C19-P1):
5'-AATTACAACCAGAGCTTGGC-3' (SEQ ID NO: 35) PCR reverse primer
(Dig-CYP2C19- P2): 5'-Dig-AATATCACTTTCCATAAAAGCAAG-3' (SEQ ID NO:
36) Biotin-labeled oligonucleotide for detection of normal base
sequence (Bio-CYP2C19-W): 5'-TCCCGGGAAC-Biotin-3' (SEQ ID NO: 37)
Unlabeled competing oligonucleotide for detection of normal base
sequence (CYP2C19-M): 5'-TTCCCAGGAAC-3' (SEQ ID NO: 38)
Biotin-labeled oligonucleotide for detection of polymorphic base
sequence (Bio-CYP2C19-M): 5'-TTCCCAGGAAC-Biotin-3' (SEQ ID NO: 39)
Unlabeled competing oligonucleotide for detection of polymorphic
base sequence (CYP2C19-W): 5'-TCCCGGGAAC-3' (SEQ ID NO: 40) (IV)
Primers and probes for detection of polymor- phism of aldehyde
dehydrogenase 2 gene PCR forward primer (Dig-ALDH2-AF): 5'-
Dig-CAAATTACAGGGTCAACTGCTATGA-3' (SEQ ID NO: 41) PCR reverse primer
(Dig-ALDH2- AR2): 5'-Dig-AGCAGGTCCTGAACTTCCAGCAG-3' (SEQ ID NO: 42)
Biotin-labeled oligonucleotide for detection of normal base
sequence (Bio-ALDH2-PW2): 5'-Biotin-ATACACTGAAGTGA-Biotin-3' (SEQ
ID NO: 43) Unlabeled competing oligonucleotide for detection of
normal base sequence (ALDH2-CM2): 5'-ATACACTAAAGTGA-3' (SEQ ID NO:
44) Biotin-labeled oligonucleotide for detection of polymorphic
base sequence (Bio-ALDH2-PM2): 5'- Biotin-ATACACTAAAGTGAA-Biotin-3'
(SEQ ID NO: 45) Unlabeled competing oligonucleotide for detection
of polymorphic base sequence (ALDH2-CW2): 5'-ATACACTGAAGTGAA-3'
(SEQ ID NO: 46)
[0091] All the PCR conditions or conditions including the
concentrations of probes were the same as those of Example 1 in any
cases of detecting the mutations described above, except that the
above primers and probes must be used.
[0092] The detections, as carried out under these conditions,
showed the correct determination of genotypes in all of the
detection systems (FIG. 8, a, b, c, and d). In the detection of
Glu487Lys of aldehyde dehydrogenase 2 gene polymorphism, the
reaction line obtained was weak in the reaction system for
detecting a mutant base sequence. However, when unlabeled competing
oligonucleotide was not mixed in this reaction system, the
formation of distinct reaction line was observed. In each of the
reactions, no false positive was observed.
[0093] The determination of genotype was completed within 10
minutes after the completion of reaction in the thermal cycler.
Even at least two years after the test strip was dried without any
treatment and stored at room temperature, a macroscopic
determination thereof was possible.
[0094] The above-mentioned results show that the detection method
of the invention allows of the simple and quick detection of each
mutation or polymorphism of the five genes to determine the
genotype of sample DNA, even though the design of primers and
hybridization probes and competing probes, and the reaction
conditions are required to be adjusted slightly for and depending
on the respective gene mutations. Therefore, it is concluded that
the detection method of the present invention can be used for many
purposes.
Example 3
[0095] Detection of Delta F508 Deletion Mutation in Cystic Fibrosis
Transmembrane Regulator Protein Gene, 1277insTATC Insertion
Mutation in HEXA Gene of Tay-Sachs Disease, 5382insC Insertion
Mutation in BRCA1 Gene of Breast Cancer, 6174delT Deletion Mutation
in BRCA2 Gene of Breast Cancer, and G1691A Point Mutation in Blood
Coagulation Factor V Gene of Thrombosis
[0096] The detection method of the present invention was carried
out to detect a mutation, including deltaF508 deletion mutation in
the gene of cystic fibrosis transmembrane regulator protein;
1277insTATC insertion mutation in HEXA gene of Tay-Sachs disease;
5382insC insertion mutation in BRCA1 gene of breast cancer;
6174delT deletion mutation in BRCA2 gene of breast cancer; and
G1691A point mutation in Blood Coagulation Factor V gene of
thrombosis.
[0097] The PCR primers for amplifying base sequences containing the
respective mutation sites were adjusted in chain length so as to
carry out PCR reactions with setting of an annealing temperature of
55.degree. C. In addition, the hybridization probes were designed
to have Tm values in the range of 35 to 40.degree. C. As a result,
the chain lengths thereof were 10 mers to 15 mers. The base
sequences of primers, hybridization probes, and competing probes
are listed in Table 5. In (I) to (IV), the target mutations to be
detected were base deletions or insertions and thus no competing
probe was used. In addition, in (III) to (V) no probe for detecting
the normal base sequence was used because even a patient of
heterozygote of the mutation in question shows the symptom,
indicating no clinical need of investigating the presence or
absence of the gene having the normal base sequence. TABLE-US-00005
TABLE 5 (I) Primers and probes for detection of delta F508 deletion
mutation in the gene of cystic fibrosis transmembrane regulator
protein PCR forward primer: 5'-ATTATGCCTGGCACCATTAAAG-3' (SEQ ID
NO: 47) PCR reverse primer: 5'-Dig-CATTCACAGTAGCTTACCCA-3' (SEQ ID
NO: 48) Biotin-labeled oligonucleotide for detection of normal base
sequence: 5'-AATATCATTGGTGTT-Biotin-3' (SEQ ID NO: 49)
Biotin-labeled oligonucleotide for detection of mutant base
sequence: 5'-TATCATCTTTGGTG-Biotin-3' (SEQ ID NO: 50) (II) Primers
and probes for detection of 1277insTATC insertion mutation in HEXA
gene of Tay-Sachs disease PCR forward primer:
5'-CCAGGAATCTCCTCAGCTTTGTGT-3' (SEQ ID NO: 51) PCR reverse primer:
5'-Dig-AGCCTCCTTTGGTTAGCAAGG-3' (SEQ ID NO: 52) Biotin-labeled
oligonucleotide for detection of normal base sequence:
5'-TATATCTATCCTATG-Biotin-3' (SEQ ID NO: 53) Biotin-labeled
oligonucleotide for detection of mutant base sequence:
5'-GTATATCCTATGG-Biotin-3' (SEQ ID NO: 54) (III) Primers and probes
for detection of 5382insC insertion mutation in BRCA1 gene of
breast cancer PCR forward primer: 5'-CTTTCAGCATGATTTTGAAGTC-3' (SEQ
ID NO: 55) PCR reverse primer: 5'-Dig-GGGAGTGGAATACAGAGTGG-3' (SEQ
ID NO: 56) Biotin-labeled oligonucleotide for detection of mutant
base sequence: 5'-AGAATCCCCAGGA-Biotin-3' (SEQ ID NO: 57) (IV)
Primers and probes for detection of 6174delT deletion mutation in
BRCA2 gene of breast cancer PCR forward primer:
5'-GATGAATGTAGCACGCATTC-3' (SEQ ID NO: 58) PCR reverse primer:
5'-Dig-TCTTGTGAGCTGGTCTGAA-3' (SEQ ID NO: 59) Biotin-labeled
oligonucleotide for detection of mutant base sequence:
5'-ACAGCAAGGGAAAAT-Biotin-3' (SEQ ID NO: 60) (V) Primers and probes
for detection of G1691A mutation in blood coagulation factor V gene
of thrombosis PCR forward primer: 5'-GGTTCCAAGTAGAATATTTAAAGAA-3'
(SEQ ID NO: 61) PCR reverse primer:
5'-Dig-CCATTATTTAGCCAGGAGACCT-3' (SEQ ID NO: 62) Biotin-labeled
oligonucleotide for detection of mutant base sequence:
5'-ACAGGCAAGGAA-Biotin-3' (SEQ ID NO: 63) Unlabeled competing
oligonucleotide for detection of mutant base sequence:
5'-ACAGGCGAGGAA-3' (SEQ ID NO: 64)
[0098] All the PCR conditions or conditions including the
concentrations of probes were the same as those of Example 1 in any
cases of detecting the mutations described above, except that the
above primers and probes must be used.
[0099] The detections, as carried out under those conditions,
showed the correct determination of genotypes in all of the
detection systems (FIG. 9). In each of the reactions, no false
positive was observed.
[0100] The determination of genotype was completed within 10
minutes after the completion of reaction in the thermal cycler.
Even at least two years after the test strip was dried without any
treatment and stored at room temperature, a macroscopic
determination thereof was possible.
[0101] The above-mentioned results show that the detection method
of the invention enables the simple and quick detection of
mutations including insertion and deletion mutations to determine
the genotype of sample DNA, even though the design of primers and
hybridization probes and competing probes and the reaction
conditions are required to be adjusted slightly for and depending
on the respective gene mutations. Therefore, it is concluded that
the detection method of the present invention can be used for many
purposes.
INDUSTRIAL APPLICABILITY
[0102] According to the present invention, the identification of
pathogenic gene mutation and the detection of polymorphisms of
disease-related genes and drug metabolism enzyme genes can be
carried out in a simple, quick and accurate manner without use of
other special devices and equipments than a conventional thermal
cycler. The detection method of the present invention allows of the
detection at bed side and is thus considered to facilitate the
tailor-made medicine.
Sequence CWU 1
1
64 1 25 DNA Artificial Sequence primer 1 cccaaatcct tcctatctct
cacag 25 2 20 DNA Artificial Sequence primer 2 tgctggagtt
gagagccagc 20 3 17 DNA Artificial Sequence probe 3 aagctgaaca
ggaagaa 17 4 15 DNA Artificial Sequence probe 4 agctgaacag gaaga 15
5 13 DNA Artificial Sequence probe 5 gctgaacagg aag 13 6 11 DNA
Artificial Sequence probe 6 ctgaacagga a 11 7 17 DNA Artificial
Sequence probe 7 aagctgaaaa ggaagaa 17 8 15 DNA Artificial Sequence
probe 8 agctgaaaag gaaga 15 9 13 DNA Artificial Sequence probe 9
gctgaaaagg aag 13 10 11 DNA Artificial Sequence probe 10 ctgaaaagga
a 11 11 17 DNA Artificial Sequence probe 11 aagctgaaaa ggaagaa 17
12 15 DNA Artificial Sequence probe 12 agctgaaaag gaaga 15 13 13
DNA Artificial Sequence probe 13 gctgaaaagg aag 13 14 11 DNA
Artificial Sequence probe 14 ctgaaaagga a 11 15 17 DNA Artificial
Sequence probe 15 aagctgaaca ggaagaa 17 16 15 DNA Artificial
Sequence probe 16 agctgaacag gaaga 15 17 13 DNA Artificial Sequence
probe 17 gctgaacagg aag 13 18 11 DNA Artificial Sequence probe 18
ctgaacagga a 11 19 12 DNA Artificial Sequence probe 19 gctgaacagg
aa 12 20 12 DNA Artificial Sequence probe 20 gctgaaaagg aa 12 21 12
DNA Artificial Sequence probe 21 gctgaaaagg aa 12 22 12 DNA
Artificial Sequence probe 22 gctgaacagg aa 12 23 28 DNA Artificial
Sequence primer 23 ctttttaatt ctagcaccaa gcaatatc 28 24 20 DNA
Artificial Sequence primer 24 tccaagtatc tgcacagcat 20 25 13 DNA
Artificial Sequence probe 25 gcaatgaaag ttg 13 26 13 DNA Artificial
Sequence probe 26 gcaatggaag ttg 13 27 12 DNA Artificial Sequence
probe 27 aacttccatt gc 12 28 12 DNA Artificial Sequence probe 28
aactttcatt gc 12 29 21 DNA Artificial Sequence primer 29 gtctcttggt
cctacctaat a 21 30 20 DNA Artificial Sequence primer 30 ttagtgaagc
tagaacactg 20 31 13 DNA Artificial Sequence probe 31 gacgaactgt tca
13 32 13 DNA Artificial Sequence probe 32 gacgaaatgt tca 13 33 13
DNA Artificial Sequence probe 33 gacgaaattg tca 13 34 13 DNA
Artificial Sequence probe 34 gacgaactgt tca 13 35 20 DNA Artificial
Sequence primer 35 aattacaacc agagcttggc 20 36 24 DNA Artificial
Sequence primer 36 aatatcactt tccataaaag caag 24 37 10 DNA
Artificial Sequence probe 37 tcccgggaac 10 38 11 DNA Artificial
Sequence probe 38 ttcccaggaa c 11 39 11 DNA Artificial Sequence
probe 39 ttcccaggaa c 11 40 10 DNA Artificial Sequence probe 40
tcccgggaac 10 41 25 DNA Artificial Sequence primer 41 caaattacag
ggtcaactgc tatga 25 42 23 DNA Artificial Sequence primer 42
agcaggtcct gaacttccag cag 23 43 14 DNA Artificial Sequence probe 43
atacactgaa gtga 14 44 14 DNA Artificial Sequence probe 44
atacactaaa gtga 14 45 15 DNA Artificial Sequence probe 45
atacactaaa gtgaa 15 46 15 DNA Artificial Sequence probe 46
atacactgaa gtgaa 15 47 22 DNA Artificial Sequence primer 47
attatgcctg gcaccattaa ag 22 48 20 DNA Artificial Sequence primer 48
cattcacagt agcttaccca 20 49 15 DNA Artificial Sequence probe 49
aatatcattg gtgtt 15 50 14 DNA Artificial Sequence probe 50
tatcatcttt ggtg 14 51 24 DNA Artificial Sequence primer 51
ccaggaatct cctcagcttt gtgt 24 52 21 DNA Artificial Sequence primer
52 agcctccttt ggttagcaag g 21 53 15 DNA Artificial Sequence probe
53 tatatctatc ctatg 15 54 13 DNA Artificial Sequence probe 54
gtatatccta tgg 13 55 22 DNA Artificial Sequence primer 55
ctttcagcat gattttgaag tc 22 56 20 DNA Artificial Sequence primer 56
gggagtggaa tacagagtgg 20 57 13 DNA Artificial Sequence probe 57
agaatcccca gga 13 58 20 DNA Artificial Sequence primer 58
gatgaatgta gcacgcattc 20 59 19 DNA Artificial Sequence primer 59
tcttgtgagc tggtctgaa 19 60 15 DNA Artificial Sequence probe 60
acagcaaggg aaaat 15 61 25 DNA Artificial Sequence primer 61
ggttccaagt agaatattta aagaa 25 62 22 DNA Artificial Sequence primer
62 ccattattta gccaggagac ct 22 63 12 DNA Artificial Sequence probe
63 acaggcaagg aa 12 64 12 DNA Artificial Sequence probe 64
acaggcgagg aa 12
* * * * *