U.S. patent application number 11/712307 was filed with the patent office on 2007-07-05 for method for detecting target nucleic acid.
Invention is credited to Ken Inose.
Application Number | 20070154940 11/712307 |
Document ID | / |
Family ID | 29561162 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070154940 |
Kind Code |
A1 |
Inose; Ken |
July 5, 2007 |
Method for detecting target nucleic acid
Abstract
A target nucleic acid having a target sequence in a sample is
detected according to the steps of: (a) mixing a first probe
including a nucleic acid which has a specific region having a
sequence complementary to the target sequence and a nonspecific
region having a sequence that is not complementary to the target
sequence of the target nucleic acid; a second probe including a
nucleic acid which has a first region that is complementary to at
least a portion of the nonspecific region of the first probe, a
loop region that does not have a sequence complementary to the
first probe, and a second region that is complementary to at least
a portion of the specific region of the first probe, the loop
region being capable of forming a loop when it is annealed with the
first probe, wherein the nucleic acid is labeled with a labeling
material generating a signal by which formation of the
aforementioned loop can be detected; and a sample under conditions
in which the first probe and the second probe are annealed and the
first probe and the target nucleic acid are annealed; and (b)
detecting a signal of the labeling material.
Inventors: |
Inose; Ken; (Kyoto-shi,
JP) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
29561162 |
Appl. No.: |
11/712307 |
Filed: |
February 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10511458 |
Oct 13, 2004 |
7220544 |
|
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PCT/JP03/05773 |
May 8, 2003 |
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11712307 |
Feb 27, 2007 |
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Current U.S.
Class: |
435/6.11 ;
536/25.32 |
Current CPC
Class: |
C12Q 2565/101 20130101;
C12Q 2525/301 20130101; C12Q 2525/161 20130101; C12Q 1/6818
20130101; C12Q 1/6818 20130101; C12Q 2565/1015 20130101; C12Q
1/6818 20130101; C12Q 2537/1373 20130101; C12Q 2525/161
20130101 |
Class at
Publication: |
435/006 ;
536/025.32 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 8, 2002 |
JP |
2002-132995 |
Claims
1-4. (canceled)
5. A kit for detecting a target nucleic acid having a target
sequence in a sample, comprising a first probe comprising a nucleic
acid which has a specific region having a sequence complementary to
the target sequence and a nonspecific region having a sequence that
is not complementary to the target sequence of the target nucleic
acid; and a second probe comprising a nucleic acid having a first
region that is complementary to at least a portion of the
nonspecific region of the first probe, a loop region that does not
have a sequence complementary to the first probe, and a second
region that is complementary to at least a portion of the specific
region of the first probe, the loop region being capable of forming
a loop when it is annealed with the first probe, wherein the
nucleic acid is labeled with a labeling material generating a
signal by which formation of the loop can be detected.
6. The kit according to claim 5, wherein the second region of the
second probe is shorter than the specific region of the first
probe.
7. The kit according to claim 5, wherein the labeling material
comprises a fluorescent material and a quencher that quenches the
fluorescence of the fluorescent material when the quencher is near
the fluorescent material, arranged so as to sandwich the loop
region, with the fluorescence of the fluorescent material being
quenched by the quencher when the first probe and the second probe
are annealed to form the loop and not quenched when the first probe
and the second probe are not annealed as compared when the probes
are not annealed.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method and kit for
detecting a target nucleic acid having a target sequence in a
sample. The method and kit of the present invention permit the
target nucleic acid to be detected in real time and are useful in
fields of biochemistry and so forth.
BACKGROUND ART
[0002] A method for detecting a target nucleic acid having a target
sequence in a sample, which has been used, includes a hybridization
method in which a probe is used, a PCR method in which
oligonucleotide primers are used, and other methods. Further, the
PCR method is generally used in various fields including the
detection and cloning of target nucleic acid, and various improved
methods have been developed.
[0003] A so-called real time PCR has been known, which is a PCR
method that performs amplification of a target sequence and
analysis of the amplified product simultaneously. Means for
analyzing the amplified products that has been known include, for
example, a Taq-Man probe method (U.S. Pat. No. 5,210,015 A, JP
06-500021 A, and Holland et al., Pro. Natl. Aca. Sci. USA., 88,
7276-7280, 1991), a molecular beacon method (JP 05-123195 A, Sanjay
Tyagi et al., Nature Biotechnology, vol 14, March 1996), an
intercalator method (Bio/Technology, 10, 413-417; 1992,
Bio/Technology, 11, 1026-1030, 1993, and JP05-237000 A), and the
like.
[0004] In the Taq-Man probe method, a fluorescent material and a
probe labeled with a quencher that quenches fluorescence emitted by
the fluorescent material are used. When the probe is hybridized
with a target nucleic acid, the quencher quenches the fluorescence
while the probe is cleaved by the 5'.fwdarw.3' exonuclease activity
of the polymerase used in PCR at the time of amplification
reaction. As a result, the fluorescent material is released from
the quencher to emit fluorescence. The amount of the double
stranded DNA molecule can be known from this fluorescence.
[0005] Further, the molecular beacon method is a method that uses a
probe including a sequence complementary to a target sequence and
an arm having sequences complementary to each other at both sides
thereof as well as a fluorescent material and a quencher bonded to
both the ends. When the probe is annealed to the target nucleic
acid, the fluorescent material emits fluorescence while when the
probe is dissociated from the target nucleic acid, the probe forms
an arm resulting in that the fluorescent material and the quencher
become closer to each other to cause quenching.
[0006] On the other hand, the intercalator method is a method that
detects a double stranded DNA using an intercalator such as
ethidium bromide.
[0007] Although the methods for quantifying PCR amplified products
in real time have been known as described above, they have
problems; the Taq-Man probe method cannot be applied in the case of
amplification methods that use polymerases having no 5'.fwdarw.3'
exonuclease activity, the molecular beacon method is difficult to
design a probe and suffers poor detection efficiency due to the
intermolecular bond, and the intercalator method has no sequence
specificity.
DISCLOSURE OF THE INVENTION
[0008] The present invention has been made from the aforementioned
viewpoint, and it is an object of the present invention to provide
a method and kit for quantifying a target nucleic acid in real time
and in a simple manner without using polymerases having
5'.fwdarw.3' exonuclease activity.
[0009] The inventors of the present invention have made extensive
studies in order to achieve the aforementioned object and as a
result they have found that use of two types of probes differing in
length enables quantification of a target nucleic acid in a simple
manner, thereby accomplishing the present invention.
[0010] That is, the present invention relates to:
[0011] (1) A method for detecting a target nucleic acid having a
target sequence in a sample, comprising the steps of:
[0012] (a) mixing a first probe including a nucleic acid which has
a specific region having a sequence complementary to the target
sequence and a nonspecific region having a sequence that is not
complementary to the target sequence of the target nucleic acid; a
second probe including a nucleic acid which has a first region that
is complementary to at least a portion of the nonspecific region of
the first probe, a loop region that does not have a sequence
complementary to the first probe, and a second region that is
complementary to at least a portion of the specific region of the
first probe, the loop region being capable of forming a loop when
it is annealed with the first probe, wherein the nucleic acid is
labeled with a labeling material generating a signal by which
formation of the aforementioned loop can be detected; and a sample
under conditions in which the first probe and the second probe are
annealed and the first probe and the target nucleic acid are
annealed; and
[0013] (b) detecting a signal of the labeling material.
[0014] (2) A method according to item (1), wherein the second
region of the second probe is shorter than the specific region of
the first probe.
[0015] (3) A method according to item (1) or (2), wherein the
labeling material comprises a fluorescent material and a quencher
that quenches the fluorescence of the fluorescent material when the
quencher is near the fluorescent material, arranged so as to
sandwich the loop region, with the fluorescence of the fluorescent
material being quenched by the quencher when the first probe and
the second probe are annealed to form the loop and not quenched
when the first probe and the second probe are not annealed as
compared when the probes are annealed. (4) A method according to
any one of items (1) to (3), wherein the detection of the signal is
performed quantitatively, thereby quantifying the target nucleic
acid. (5) A kit for detecting a target nucleic acid having a target
sequence in a sample, comprising a first probe including a nucleic
acid which has a specific region having a sequence complementary to
the target sequence and a nonspecific region having a sequence that
is not complementary to the target sequence of the target nucleic
acid; a second probe including a nucleic acid which has a first
region that is complementary to at least a portion of the
nonspecific region of the first probe, a loop region that does not
have a sequence complementary to the first probe, and a second
region that is complementary to at least a portion of the specific
region of the first probe, the loop region being capable of forming
a loop when it is annealed with the first probe, wherein the
nucleic acid is labeled with a labeling material generating a
signal by which formation of the aforementioned loop can be
detected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is schematic diagrams illustrating the concept of the
present invention, with F indicating a fluorescent material and Q
indicating a quencher.
[0017] FIG. 2 is a graph showing the time-course changes of the
intensities of fluorescence of the reaction mixtures (not subjected
to heat treatment), where
[0018] solid line: Probe 1+Probe 2+target oligonucleotide
[0019] dotted line: Probe 1+Probe 2
[0020] alternate longer and shorter dashed lines: Probe 2.
[0021] FIG. 3 is a graph showing the time-course changes of
intensities of fluorescence of the reaction mixtures(subjected to
heat treatment).
BEST MODE FOR CARRYING OUT THE INVENTION
[0022] Hereinafter, the present invention will be described in
detail.
[0023] The method of the present invention is a method for
detecting a target nucleic acid having a target sequence in a
sample. The target nucleic acid is not particularly limited so far
as it has the target sequence; it may be either a DNA or an RNA,
and it may be either single stranded or double stranded. The
present invention is advantageously applied to detection of
particularly a double stranded DNA. In a preferable mode of the
present invention, the target nucleic acid is detected
quantitatively. Note that quantitative detection includes
measurement of an absolute amount of a nucleic acid and measurement
of a nucleic acid relatively to a certain amount.
[0024] The target sequence is usually a sequence specific to the
target nucleic acid and is not particularly limited in sequence and
length so far as it can form a specific hybrid with a probe having
a sequence complementary to that sequence. The length of the target
sequence is preferably 6 bases or more, more preferably 15 bases or
more.
[0025] Samples that contain the target nucleic acid are not
particularly limited and include nucleic acids or nucleic acid
mixtures extracted from cells or tissues, and PCR nucleic acid
amplification reaction mixtures using such nucleic acids or nucleic
acid mixtures as templates.
[0026] In the present invention, two types of probes are used in
detecting the target nucleic acid (see FIG. 1). A first probe
(hereinafter, also referred to as "Probe 1") includes a nucleic
acid which has a specific region having a sequence complementary to
the target sequence and a nonspecific region having a sequence that
is not complementary to the target sequence of the target nucleic
acid (FIG. 1A). Due to such a structure, when Probe 1 is hybridized
with the target nucleic acid, the nonspecific region remains as a
single strand and forms a flap (FIG. 1B). The length of the
nonspecific region is preferably 10 bases or more, more preferably
10 to 30 bases.
[0027] A second probe (hereinafter, also referred to as "Probe 2")
includes a nucleic acid which has a first region that is
complementary to at least a portion of the aforementioned
nonspecific region of the Probe 1, a loop region that does not have
a sequence complementary to Probe 1, and a second region that is
complementary to at least a portion of the specific region of Probe
1, the loop region being capable of forming a loop when it is
annealed with Probe 1, and the nucleic acid is labeled with a
labeling material generating a signal by which formation of the
aforementioned loop can be detected (FIG. 1A).
[0028] Note that FIG. 1 shows examples of Probe 1 that has the
specific region on the 5' side and the nonspecific region on the 3'
side and of Probe 2 that has the first region, the loop region, and
the second region in order from the 5' side. In the present
invention, Probe 1 may have the specific region on the 3' side and
the nonspecific region on the 5' side and Probe 2 may have the
first region, the loop region, and the second region in order from
the 3' side.
[0029] The first region has a sequence that is complementary to at
least a portion of the nonspecific region of Probe 1 and preferably
has a sequence that is complementary to the whole nonspecific
region. On the other hand, the second region has a sequence that is
complementary to at least a portion of the specific region of Probe
1 and preferably is shorter than the specific region. By making the
second region shorter than the specific region, Probe 1 can be
annealed with the target nucleic acid preferentially than Probe 2.
The length of the second region is preferably 6 bases or more, more
preferably 6 to 20 bases. Alternatively, it is desirable that the
second region is made shorter by preferably one base or more, more
preferably 4 bases or more than the specific region.
[0030] The loop region is a region that does not have a sequence
complementary to Probe 1 and preferably it does not have a sequence
complementary to the target nucleic acid. The loop region forms a
protruded portion in the form of a loop when Probe 2 is bonded to
Probe 1 but when Probe 2 and Probe 1 are dissociated from each
other, the loop structure is eliminated (FIG. 1C). Probe 2 is
labeled with a labeling material generating a signal by which
formation of a loop can be detected. The labeling material
specifically includes, for example, an energy donor and an energy
receptor arranged so as to sandwich the loop region. The energy
donor and energy receptor include, for example, a fluorescent
material and a quencher that quenches the fluorescence generated by
the fluorescent material. The quencher quenches fluorescence when
it is near the fluorescent material but it will no longer quench
fluorescence when the distance between the fluorescent material and
the quencher is equal to or greater than a certain distance. With
such a fluorescent material and quencher, the fluorescence of the
fluorescent material is quenched by the quencher when Probe 1 and
Probe 2 are annealed to form a loop while the fluorescence is not
quenched when Probe 1 and Probe 2 are not annealed. Therefore, by
measuring the fluorescence from the fluorescent material, formation
of a loop, that is the state of hybridization of Probe 1 and Probe
2 can be detected. Examples of the fluorescent material include
fluorescein dyes such as fluorescein and fluorescein isothiocyanate
(FITC) and examples of the quencher include rhodamine dyes such as
tetramethyl rhodamine isothiocyanate (TRITC) and Sulfo Rhodamine
101 chlorosulfonate derivative (trade name: Texas Red). Among
these, a preferable combination is FITC and Texas Red. These
labeling materials can be introduced into any desired portion of
the sequence by performing chemical synthesis of Probe 2 using
oligonucleotides having bonded thereto these labeling materials.
Any of the fluorescent material and quencher may be on the 5'
side.
[0031] The sequence and length of the loop region are not
particularly limited so far as a loop structure is formed when
Probe 1 and Probe 2 are annealed and signals from the labeling
material differ between the case where the loop structure is formed
and the case where it is eliminated. Note that the loop region is
preferably designed such that the loop region forms neither a
partial double strand with the first region and second region of
Probe 2 nor a partial double strain within the loop region. The
length of the loop region is preferably 10 bases or more and more
preferably 20 bases or more. It is desirable that the labeling
material is bonded to a portion usually within 3 bases form the
both terminal bases in the loop region, preferably to the terminal
bases.
[0032] The aforementioned Probe 1, Probe 2, and a sample are mixed
in the conditions under which Probe 1 and Probe 2 are annealed and
Probe 1 and the target nucleic acid are annealed. However, from the
aforementioned structures of the probes, Probe 1 is annealed with
the target nucleic acid preferentially than Probe 2. The order of
mixing in not particularly questioned; for example, Probe 1 and
Probe 2 are mixed and then the sample is added. Further, a
premixture of Probe 1 and Probe 2 may be prepared in advance.
Furthermore, it is preferable that a reaction mixture that does not
contain Probe 1 and/or a sample is used as a control.
[0033] Probe 1 and Probe 2 are mixed in a molar ratio of preferably
1:1. The final concentration of each of Probe 1 and Probe 2 in the
reaction mixture is preferably 0.1 .mu.M or more.
[0034] After Probe 1, Probe 2, and the sample are mixed, either the
mixture as it is may be exposed to the conditions in which Probe 1
and Probe 2 are annealed and Probe 1 and the target nucleic acid
are annealed or the mixture may be subjected to heat denaturation
treatment and then exposed to the aforementioned conditions.
[0035] After leaving the aforementioned reaction mixture to stand a
given time, preferably after it has reached an equilibrium state,
the signal of the labeling material is detected. More preferably,
the detection of the signal is performed with time. The
aforementioned conditions include, for example, a temperature that
is lower by 3 to 10.degree. C. than the (Tm) s of Probe 1 and Probe
2. Optimal conditions can be readily determined by performing
detection of signals with time with varying temperature in several
stages to select those that give the clearest difference from the
control. In the case where FITC is used as a fluorescent material
and Texas is used Red as a quencher, the detection of signals is
performed by measuring intensity of fluorescence at a fluorescent
wavelength of 515 nm attributable to FITC. The measurement of the
intensity of fluorescence is performed using a commercially
available apparatus. The results of the measurements are shown in
FIGS. 2 and 3. Those results will be described in detail in the
example below.
[0036] By quantitatively detecting the signals of the labeling
material, the amount of the target nucleic acid can be
quantified.
[0037] The kit of the present invention is a kit that is used in
order to detect the aforementioned target nucleic acid and includes
Probe 1 and Probe 2. Each probe maybe either a solution or a
freeze-dried preparation. Further, each probe maybe either charged
in a separate container or in the same container as a mixture. The
kit of the present invention may further contain a buffer for
dissolving or diluting each probe or a sample.
[0038] The method and kit of the present invention can be
advantageously used in quantifying the amplified product in the
nucleic acid amplification reaction mixture in a real time. The kit
of the present invention may contain an oligonucleotide primer for
amplifying such a target nucleic acid by a nucleic acid
amplification method. The primer is charged in a separate container
from that in which each probe is contained.
EXAMPLE
[0039] Hereinafter, the present invention will be described in more
detail by examples.
[0040] Probe 1 (SEQ ID NO: 1), Probe 2 (SEQ ID NO: 2), and a target
oligonucleotide (SEQ ID NO: 3) were synthesized. The synthesis of
each oligonucleotide was requested to Japan Bio Service Co., Ltd.
The nucleotide (T) of the base 21 of Probe 2 was labeled with FITC
and the nucleotide (T) of base 52 was labeled with Texas Red. Note
that the target oligonucleotide is a partial sequence of human
amylin gene.
[0041] The bases 1 to 28 of Probe 1 are complementary to the bases
6 to 33 of the target oligonucleotide. The bases 11 to 3.3 and
bases 35 to 55 of Probe 1 are complementary to the bases 52 to 74
and bases 1 to 21 of Probe 2, respectively. Note that the bases 57
to 74 of SEQ ID NO: 2 is homologous to the bases 6 to 23 of SEQ ID
NO: 3.
[0042] Each oligonucleotide was dissolved in TE buffer to 5 .mu.M.
In a 1.5-ml tube were added 2.2 .mu.l of 10.times.Ex Taq buffer
(Takara Shuzo Co., Ltd., Lot. A6501-1), 19.8 .mu.l of sterilized
distilled water, and 1 .mu.l of a Probe 2 solution (5 .mu.M), which
were mixed well and 23 .mu.l of the mixture was dispensed to each
25-.mu.l tube for a reaction machine (Cepheid Co., Smart Cycler).
In each tube, 1 .mu.l of the Probe 1 solution (5 .mu.M) or TE
buffer was added and further 1 .mu.l of the target oligonucleotide
solution (5 .mu.M) or TE buffer to obtain a reaction mixture. This
operation was performed at room temperature.
[0043] The aforementioned reaction mixture or the aforementioned
reaction mixture subjected to heat treatment at 94.degree. C. for 3
minutes was set in the Smart Cycler, which was adjusted to
47.degree. C. and fluorescence at a wavelength of 505 to 537 nm was
measured. The results on the reaction mixture that was not
subjected to the heat treatment are shown in FIG. 1 and the results
on the reaction mixture subjected to the heat treatment are shown
in FIG. 2.
[0044] With Probe 2 only, fluorescence was observed but in the case
where Probe 1 was added, the fluorescence was quenched
considerably. In the case where the target oligonucleotide in
addition to Probe 1 was added to Probe 2, fluorescence with an
intermediate intensity between both the cases was observed. The
results were the same regardless of presence or absence of the heat
treatment.
[0045] As described above, higher intensity of fluorescence
observed in the case where the target sequence was present than the
case where no target sequence was present confirmed that Probe 1
preferentially bonds to the target sequence than Probe 2 and a
portion of Probe 2 was free.
INDUSTRIAL APPLICABILITY
[0046] By the present invention, a target nucleic acid can be
quantified in a real time and in a simple manner. The method of the
present invention does not require a polymerase having a
5'.fwdarw.3' exonuclease activity, so that it can be applied to
various reaction systems.
Sequence CWU 1
1
3 1 56 DNA Artificial Sequence Description of Artificial Sequence
probe 1 1 aaagttgttg ctggaatgaa ctaaaaaatg gcaatattca catgtacagg
actcag 56 2 75 DNA Artificial Sequence Description of Artificial
Sequence probe 2 2 ctgagtccag tacaactgaa taaaaaaaaa aaaaaaaaaa
aaaaaaaaaa attgccattt 60 tttagttcat tccag 75 3 50 DNA Artificial
Sequence Description of Artificial Sequence target oligonucleotide
3 ggcaaatttt ttagttcatt ccagcaacaa ctttggtgcc attctctcat 50
* * * * *