U.S. patent application number 14/883043 was filed with the patent office on 2016-02-04 for method for detection of target nucleic acid.
This patent application is currently assigned to NGK INSULATORS, LTD.. The applicant listed for this patent is NGK INSULATORS, LTD.. Invention is credited to Toshikazu Hirota, Mitsuo Kawase, Kousuke Niwa.
Application Number | 20160032367 14/883043 |
Document ID | / |
Family ID | 43922011 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160032367 |
Kind Code |
A1 |
Kawase; Mitsuo ; et
al. |
February 4, 2016 |
Method for Detection of Target Nucleic Acid
Abstract
An object of the disclosure of the present specification is to
provide a method for detection of a target nucleic acid which
allows construction of an effective detection system of a target
nucleic acid. For this purpose, in the disclosure of the present
specification, a first primer comprising an identification sequence
complementary to a target sequence in a target nucleic acid and a
tag addition sequence, and a second primer having a label are
prepared. The first primer and the second primer are used for the
target nucleic acid in a sample to amplify a chimeric DNA having a
tag sequence and the label. The chimeric DNA is hybridized with a
detection probe on a solid phase to obtain signal intensity
information based on the label, and the target nucleic acid is
detected based on the signal intensity information.
Inventors: |
Kawase; Mitsuo; (Chita-shi,
JP) ; Hirota; Toshikazu; (Nagoya-shi, JP) ;
Niwa; Kousuke; (Niwa-gun, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NGK INSULATORS, LTD. |
Aichi-ken |
|
JP |
|
|
Assignee: |
NGK INSULATORS, LTD.
Aichi-ken
JP
|
Family ID: |
43922011 |
Appl. No.: |
14/883043 |
Filed: |
October 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13497000 |
Mar 19, 2012 |
9175339 |
|
|
14883043 |
|
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Current U.S.
Class: |
506/16 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 1/6858 20130101; C12Q 1/6858 20130101; C12Q 2565/514 20130101;
C12Q 2563/107 20130101; C12Q 2537/143 20130101; C12Q 2531/107
20130101; C12Q 1/689 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 29, 2009 |
JP |
2009-249122 |
Claims
1. A set of primers for use in a method of detection of a target
nucleic acid, wherein the set of priers comprises: a first primer
having an identification sequence complementary to a target
sequence in a target nucleic acid and a tag addition sequence
complementary to a tag sequence, wherein the tag sequence is
complementary to a detection probe correlated to the target nucleic
acid; and a second primer having a partial sequence that has the
same sequence as a partial sequence adjacent to the target
sequence, wherein the set of primers does not include a universal
primer.
2. The set of primers of claim 1, wherein the set of primers
consists of a first primer having an identification sequence
complementary to a target sequence in a target nucleic acid and a
tag addition sequence complementary to a tag sequence, and wherein
the tag sequence is complementary to a detection probe correlated
to the target nucleic acid, and a second primer having a partial
sequence that has the same sequence as a partial sequence adjacent
to the target sequence.
3. A kit for carrying out the detection of a target nucleic acid,
said kit comprising; a set of detection probes bound to a solid
support, wherein the detection probes have different base
sequences; a set of primers a first primer having an identification
sequence complementary to a target sequence in a target nucleic
acid and a tag addition sequence complementary to a tag sequence,
wherein the tag sequence is complementary to a detection probe
correlated to the target nucleic acid, and a second primer having a
partial sequence that has the same sequence as a partial sequence
adjacent to the target sequence, wherein the set of primers does
not include a universal primer.
4. A kit for the detection of a target nucleic acid, comprising; an
array comprising a set of detection probes respectively having
different base sequences, said probes bound to a solid support; and
a set of primers which does not include a universal primer, wherein
the set of primers consists of a first primer having an
identification sequence complementary to a target sequence in a
target nucleic acid and a tag addition sequence complementary to a
tag sequence, and wherein the tag sequence is complementary to a
detection probe correlated to the target nucleic acid, and a second
primer having a partial sequence that has the same sequence as a
partial sequence adjacent to the target sequence and the label a
detection probe hybridizable to the tag sequence having been
correlated to the target nucleic acid.
Description
[0001] This application is a Divisional of, and claims priority
under 35 U.S.C. .sctn.120 to, U.S. patent application Ser. No.
13/497,000, filed on Mar. 19, 2012, which was a national phase
entry under 35 U.S.C. .sctn.371 of PCT Patent Application No.
PCT/JP2010/068964, filed on Oct. 26, 2010, which claimed priority
under 35 U.S.C. .sctn.119 to Japanese Patent Application No.
2009-249122, filed Oct. 29, 2009, all of which are incorporated by
reference. Also, the Sequence Listing filed electronically herewith
is hereby incorporated by reference (File name:
1027-0022DIV_Seq_List_as-filed; File size: 18 KB; Date recorded:
Oct. 14, 2015).
TECHNICAL FIELD
[0002] The present application claims priority to Japanese Patent
Application No. 2009-249122 filed on Oct. 29, 2009, which is
incorporated herein by reference in its entirety. The present
invention relates to a technology for detecting target nucleic
acids.
BACKGROUND ART
[0003] It has been conventionally proposed to exhaustively detect
or quantitate nucleic acid sequences in order to carry out genetic
analyses of individual organisms and to test an infection of
biological samples with viruses or bacteria. For example,
microarrays (hereinafter merely referred to as "arrays") are used
for detecting an expression level of nucleic acid sequences to be
detected (target nucleic acids) in samples (e.g., see Non-patent
documents 1 to 4). Arrays are carriers on which multiple nucleic
acid fragments (detection probes) having known base sequences are
independently fixed. As shown in FIG. 8, in conventional any
methods, a forward primer (F primer) and a reverse primer (R
primer) designed so as to flank the target sequence are used to
amplify a DNA fragment (target nucleic acid) containing the target
sequence. The amplified nucleic acid is then separated to a single
strand. The target nucleic acid then binds on the carrier by
hybridization of the target sequence with a portion complementary
to a partial sequence characteristic to the target nucleic acid
(target sequence). The hybridized target nucleic acid is detected
by any suitable method to determine a presence or absence of the
nucleic acid in the sample.
[0004] Arrays specific for detection of single nucleotide
polymorphisms (SNPs) have been developed (e.g., see Patent
documents 1 and 2). By this method, a type of SNPs of the target
nucleic acid in the sample can be detected by using DNA computer
technology.
CITATION LIST
Patent Literature
[0005] Patent document 1: Japanese Patent Application Laid-open No.
2006-211982 Patent document 2: Japanese Patent Application
Laid-open No. 2006-101844
Non Patent Literature
[0005] [0006] Non-patent document 1: Baio Jikken de Shippai Shinai!
Kenshutsu to Teiryo no Kotsu (Successful Biotechnological
Experiments: Tips for Detection and Quantification), Supplementary
volume of Jikken Igaku (Medical Experiments), Yodosha, Chapter 3,
10. Maikuroarei no Kotsu (Tips for Microarrays) [0007] Non-patent
document 2: Bioview, No. 45, pp 14-18, 2004, Takara-Bio [0008]
Non-patent document 3: Biotechnology series: DNA chip application
technology, CMC Publishing, Chapter 5, Practice and application of
DNA microarrays [0009] Non-patent document 4: Ministry of Health,
Labour and Welfare Grant-in-Aid for Scientific Research, Research
project on securement of food safety and reliability (Annual Report
of Ministry of Health, Labour and Welfare, 2006)
SUMMARY
[0010] In the methods disclosed in Non-patent documents 1 to 4, the
detection probe fixed on the array is hybridized with the target
sequence, which hybridization requires a prolonged period of time.
In addition, the detection probe may bind non-specifically to other
nucleic acid sequences having similarity (homology) with the target
sequence. Namely, upon detection of multiple target sequences in
the sample, the presence or absence thereof may not be accurately
detected.
[0011] Regarding the non-specific binding problems, Non-patent
document 2 discloses that homology of the detection probe can be
minimized by reducing a length thereof. However, the reduction in
the length of the detection probe may decrease an intensity of
signal of a label upon detection. Non-patent document 4 discloses
that non-specific binding may be decreased by increasing
hybridization temperature. However, when the problem is not solved
by these methods, the sequence of the detection probe needs to be
re-designed and the array needs to be re-prepared. Thus, users of
arrays need to consider an influence of homology, making process
steps for obtaining an appropriate detection system for the target
nucleic acid significantly intricate.
[0012] On the other hand, the methods disclosed in Patent documents
1 and 2 are specific for detection of SNPs, which allow accurate
detection of SNPs. However, seven different probes are required for
the detection of one SNP and procedures are further intricate. In
addition, to design probes is troublesome because it is required
for the user to ligate amplification byproducts of the target
nucleic acid before hybridization of the target nucleic acid to the
array.
[0013] As described above, a lot of effort is required in
conventional methods for constituting the detection system of
intended target nucleic acids. It has been also difficult to
accurately detect the target nucleic acid in a short time.
Accordingly, an object of the disclosure of the present
specification is to provide a method for detection of the target
nucleic acid allowing effective construction of the detection
system of the target nucleic acid.
[0014] The present inventors have studied in order to effectively
construct the detection system on various methods which allow
effective hybridization of the detection probe fixed on the carrier
with the target nucleic acid while maintaining selectivity. As a
result, they have reached to a conclusion that it is difficult to
effectively construct the detection system based on a hybridization
reaction due to sequence specificity of the target nucleic acid on
a solid carrier. They have also found that consideration on
hybridization conditions may be omitted and non-specific binding
may be excluded and high selectivity can be achieved by using
multiple sets of detection probes and tag sequences which have been
designed so as to be able to specifically hybridize and attaching
the tag sequences to the target nucleic acid. In addition, without
requiring ligation of such a chimeric target nucleic acid using a
probe specific to the target sequence, non-specific binding between
the labeled target nucleic acid and the detection probe can be
reduced by amplifying the labeled target nucleic acid using primers
specifically hybridizable to a partial sequence having low
homology, i.e. a sequence characteristic to the target nucleic
acid. The following method is provided based on these findings.
[0015] The disclosure of the present specification relates to a
method for detection of the target nucleic acid in the sample. The
present method for detection comprises steps of preparing a solid
phase comprising detection probes respectively having certain
different base sequences, cam/Mg out PCR on the sample to obtain
chimeric DNAs each having a label and a tag sequence complementary
to each of the detection probes having been correlated to the
target nucleic acid, bringing the chimeric DNAs into contact with
the detection probes such that the chimeric DNAs and the detection
probes can hybridize through the tag sequences, obtaining signal
intensity information based on the label on the solid phase, and
detecting the target nucleic acid based on the signal intensity
information.
[0016] The step of PCR comprises preparing a first primer having an
identification sequence complementary to the target sequence in the
target nucleic acid and a tag addition sequence complementary to
the tag sequence, and a second primer having a partial sequence
identical to a partial sequence adjacent to the target sequence and
the label, and carrying out PCR on the sample using the first
primer and the second primer to synthesize the chimeric DNA having
the target sequence, the tag sequence and the label.
[0017] In the step of PCR, two or more first primers and one second
primer common to the two or more target nucleic acids may be used
for two or more target nucleic acids.
[0018] The step of PCR may be the step of amplifying the chimeric
DNAs by asymmetric PCR.
[0019] The target nucleic acid can be detected by using the array
comprising the detection probe hybridizable to the tag sequence
having been correlated to the target nucleic acid.
BRIEF DESCRIPTION OF DRAWINGS
[0020] FIG. 1 is a schematic view of an example of the method for
detection of the present invention;
[0021] FIG. 2 is a view depicting relationship between the solid
phase carrier, and the detection probes and the chimeric DNA on the
solid phase carrier according to the present invention;
[0022] FIG. 3 is a view showing the step of amplification of the
labeled target nucleic acid according to the present invention;
[0023] FIG. 4 is a flow chart for preparation of the array and the
target;
[0024] FIG. 5 is a table showing base sequences of detection
probes;
[0025] FIG. 6 is a view showing detection results obtained in
examples of the present invention;
[0026] FIG. 7 is a view showing detection results obtained in
examples of the conventional method; and
[0027] FIG. 8 is a view showing an exemplary conventional detection
method of target nucleic acids.
DESCRIPTION OF EMBODIMENTS
[0028] The present invention relates to the array for detection of
the target sequence in the target nucleic acid which is to be
detected. According to the method for detection of the target
nucleic acid of the present invention, a procedure can be avoided
for constructing the detection system by designing the detection
probes having different unique base sequences respectively for all
target nucleic acids and fixing them on the solid phase carrier. By
carrying out PCR on the sample so as to obtain the chimeric DNA
having a detection sequence complementary to the detection probe
having been correlated to the target nucleic acid and the label,
the target nucleic acid can be identified and the chimeric DNA
which has the detection sequence having been correlated to the
detection probe, is specific to the target nucleic acid and is
labeled can be obtained by PCR for preparation of DNA for
hybridization. By hybridizing the chimeric DNA and the detection
probe via the detection sequence, the chimeric DNA hybridizes to
the detection probe based on the detection probe and the detection
sequence which have been correlated to each other, effectively
suppressing or avoiding non-specific binding upon
hybridization.
[0029] According to the disclosure of the present specification,
the first primer having the identification sequence complementary
to the target sequence in the target nucleic acid and the tag
addition sequence and the second primer having the partial sequence
adjacent to the target sequence and the label are used when PCR is
carried out, thereby suppressing or avoiding complicated design of
probes or primers. The set of primers allows easy preparation of
the chimeric DNAs for hybridization with the detection probes which
directly identify the target sequences and are specific to the
target nucleic acids.
[0030] This method ensures detection of each of multiple target
nucleic acids and can be used for detection of single nucleotide
polymorphisms (SNPs) or modified sites in genetically modified
nucleic acids or for detection of expression genes such as RNAs.
Namely, SNPs, modified sites, expression genes, polymorphisms or
mutations in the target nucleic acids can be detected by obtaining
chimeric DNAs based on the same concept as the detection of the
mutation to be detected.
[0031] FIG. 4 shows an outline of the procedures of the preparation
of the array and the target for detection of the target nucleic
acid. FIG. 4 also shows the flow chart for the method disclosed in
the present specification as well as the conventional detection
method. The steps shown with the solid line are the steps common
for the method disclosed in the present specification and the
conventional method, and the steps shown with the dashed line are
the ones necessary only for the conventional method. In the
conventional method, multiple steps are carried out for preparation
of arrays for respective target nucleic acids. Information on the
sequence of the target nucleic acid to be detected is first
obtained and the detection probe is designed according to the
sequence information. The detection probe is then synthesized
according to the design and prepared in a spot solution for the
array in order to fix the probe on the array. Meanwhile the target
nucleic acid to be detected (a target such as RNA or DNA) is
extracted and purified from the sample. Primers for amplifying the
target nucleic acid are designed and synthesized with a labeling.
The target nucleic acid is then amplified with the synthesized
primers. The amplified target nucleic acid is then hybridized with
the detection probe on the array prepared (hybridization). The
occurrence of hybridization is examined according to detection of
signal of the label on the array and the obtained signal of the
label is converted to the numeral value. When the obtained results
are not the ones expected such as abnormal or absence of
fluorescence signal etc., the array has to be designed again or the
sample has to be prepared again, as shown with the solid and dashed
lines in FIG. 4.
[0032] The conventional methods disclosed in Non-patent documents 1
to 4 require many reviews on hybridization conditions, primers, and
even sequences of the detection probes on the array. It takes a
prolonged period of time to re-design and synthesize oligo DNAs for
the detection probes and query probe sequences. According to the
present method, the detection probes and query probes may be merely
selected from 100 different sequences (see Sequence Listing). The
methods disclosed in Patent documents 5 and 6 require seven
different primers and probes for detection of one target sequence.
However, the present invention requires only two kinds of primers
for detection of one target sequence.
[0033] On the other hand, the method disclosed in the present
specification merely requires preparation of the array comprising
multiple detection probes respectively having the unique detection
sequence preliminarily determined regardless of the target nucleic
acid. As the array can be applied regardless of the target nucleic
acid, design, synthesis and fixation of probes for respective
target nucleic acids and review of hybridization conditions may all
be avoided, unlike the conventional method. The detection system
may be constructed according to the method disclosed in the present
specification by mainly considering only the design of the primers
upon preparation of the target.
[0034] According to the method disclosed in the present
specification, the detection probes can be prepared for which
hybridization conditions are optimized, thus the target nucleic
acid can be accurately detected in a short time.
[0035] As used herein, the "nucleic acid" includes all DNAs and
RNAs including cDNA, genomic DNA, synthetic DNA, mRNA, total RNA,
hnRNA and synthetic RNA as well as artificial synthetic nucleic
acids such as peptide nucleic acid, morpholino-nucleic acid,
methylphosphonate-nucleic acid and S-oligo nucleic acid. The
nucleic acid may be single-stranded or double-stranded. As used
herein, the "target nucleic acid" is any nucleic acid having any
sequence. Typically, the target nucleic acid includes nucleic acids
which may have base sequences genetically indicative for
constitution or disease incidence, disease diagnosis, disease
prognosis, drug or treatment selection of specific diseases such as
genetic diseases or cancer in human or non-human animals. The
genetically indicative base sequences include polymorphisms such as
SNPs and inherent or acquired mutations. The target nucleic acid
also includes nucleic acids derived from microorganisms such as
pathogens and viruses.
[0036] The target nucleic acid may be the sample described below or
a nucleic acid fraction thereof and is preferably an amplified
product in which all of the multiple target nucleic acids have been
amplified by preferably amplification reaction with PCR, more
preferably multiplex PCR.
[0037] As used herein, the "sample" refers to the sample which may
contain the target nucleic acid. The sample may be any sample
containing a nucleic acid including cells, tissues, blood, urine,
saliva and the like. A person skilled in the art may appropriately
obtain a fraction containing the nucleic acid from such various
samples according to the conventional art.
[0038] As used herein, the "target sequence" refers to a sequence
formed by one or more bases characteristic to the target nucleic
acid to be detected. The target sequence may be a partial sequence
having low homology among the target nucleic acids or a sequence
having low complementarity or homology to other nucleic acids which
may be contained in the sample. The target sequence may be a
sequence characteristic to the target nucleic acid. The target
sequence may have a sequence artificially modified.
[0039] Representative and non-limiting specific examples of the
disclosure of the specification are described herein after with
referring to the drawings. The detailed description merely intends
to illustrate the details to a person skilled in the art for
carrying out the preferred examples of the disclosure of the
present specification, while it does not intend to limit the scope
of the disclosure of the present specification. Additional features
and disclosures hereinafter may be used separately or in
conjunction with other features or inventions in order to provide a
further improved method for detection of the target nucleic acid
and the like.
[0040] Combinations of the features and steps disclosed hereinafter
in the detailed description are not requisite for carrying out the
disclosure of the present specification in its broadest meaning,
but are particularly described merely for illustrating
representative specific examples of the disclosure of the present
specification. Various features of the above- and below-described
representative specific examples as well as various features of
those described in independent and dependent claims are not the
ones which have to be combined as the specific examples or in the
same order as described herein in order to provide additional and
useful modes of the disclosure of the present specification.
[0041] All features described in the present specification and/or
claims intend to be disclosed, individually and independently each
other, as limitations for specific items described in the
disclosure and claims at the time of filing the present
application, separately from a structure of the features described
in examples and/or claims. Descriptions on all numerical ranges and
groups or sets intend to disclose intermediate aspects thereof as
limitations for specific items described in the disclosure and
claims at the time of filing the present application.
[0042] FIG. 1 is a schematic view showing a principle of the method
for detection of the present invention. FIG. 2 shows an example of
the solid phase 100 used for the present invention and FIG. 3 shows
details for the step of amplification in FIG. 1. FIGS. 1 and 3 show
an example for the method for detection and the primer for
detecting one target nucleic acid contained in the sample. In the
descriptions hereinafter, a base sequence designated with a number
and (-) means a complementary base sequence of a base sequence
designated with the same number.
[0043] [Method for Detection of the Target Sequence in the Target
Nucleic Acid]
[0044] The method for detection disclosed in the present
specification comprises steps of preparing the solid phase
comprising multiple detection probes respectively having different
unique base sequences, carrying out PCR on the sample so as to
obtain chimeric DNAs respectively having tag sequence complementary
to the detection sequence of the detection probe having been
correlated to the target nucleic acid and the label, hybridizing
the chimeric DNAs and the detection probes through the detection
sequence and the tag sequence, obtaining signal intensity
information based on the label on the carrier, and detecting the
target nucleic acid based on the signal intensity information. The
method for detection according to the disclosure of the present
specification is applied to one or more target nucleic acids and
more specifically, aims to detect the target sequence(s)
characteristic in the target nucleic acid(s). A series of the steps
for detection of one target nucleic acid is mainly illustrated
hereinafter. However, the steps described below may also be applied
for simultaneous detection of several or many target nucleic
acids.
[0045] (Step of Preparation of Solid Phase Carrier)
[0046] The method for detection disclosed in the present
specification (hereinafter merely referred to as the present method
for detection) may comprise the step of preparing the solid phase
100 as shown in FIG. 1. The solid phase 100 may be preliminary
prepared prior to carrying out the method for detection, may be
commercially obtained or may be prepared every time when carrying
out the method for detection.
[0047] As shown in FIG. 1, the solid phase 100 may comprise
multiple detection probes 104 respectively comprising the detection
sequences 106 which are different unique base sequences on the
carrier 102. Preparation of such a solid phase 100 may avoid design
and synthesis of probes, preparation of arrays and consideration on
hybridization conditions.
[0048] FIG. 2 shows an example of the solid phase 100. The
detection probes 104 contain the detection sequences 106 which are
respectively unique base sequences for probing. Such detection
sequences 106 may be established irrespectively of the sequence
characteristic to the target nucleic acid 10, i.e. the target
sequence 12. The detection sequences 106 in the detection probes
104 are irrespective of the target sequence 12 and may be
established so as to suppress or avoid non-specific binding between
multiple detection probes 104 and obtain suitable hybridization
conditions such as temperature and time. In addition, same
detection probes 104 may be used all the time irrespective of the
variation of the target nucleic acid 10.
[0049] The detection sequence 106 in the detection probe 104 may be
base sequences of SEQ ID NO: 1 to SEQ ID NO: 100 or their
complementary base sequences. These base sequences have the same
base length and have a melting temperature (Tm) of 40.degree. C. or
higher and 80.degree. C. or lower, more preferably 50.degree. C. or
higher and 70.degree. C. or lower, thereby giving homogeneous
hybridization results under the same hybridization conditions.
[0050] The detection sequence 106 in the detection probe 104 may be
appropriately selected from such candidate base sequences. Two or
more detection probes 104 to be used preferably have melting
temperatures as close as possible to each other. When multiple
target nucleic acids 10 are exhaustively and simultaneously
detected, multiple detection probes 104 for respective multiple
target nucleic acids 10 are preferably combined so as to have
melting temperatures closest to each other. For example, when
detection probes 104 are arranged in order of their melting
temperatures, two or more detection probes 104 for respective two
or more target nucleic acids 10 to be distinguished may be selected
from two base sequences adjacent in the arrangement by melting
temperatures. The detection sequence 106 in the detection probe 104
for another target nucleic acid 10 may be selected from base
sequences immediately consecutive to or apart from the base
sequence which has already been selected. It is also preferable to
use the base sequences which have consecutive melting temperatures
in the arrangement by the melting temperatures for all detection
probes for multiple target nucleic acids 10 to be detected
simultaneously.
[0051] The melting temperature may be the one calculated according
to a GC % method, a Wallace method, a method according to Current
Protocols in Molecular Biology (described in Biotechnology
Experiments Illustrated 3, Honto ni fueru PCR (Truly amplifiable
PCR), Shujunsha, p. 25); however, it is preferably calculated by a
Nearest-Neighbor method to which impacts of a range of the melting
temperature and a concentration of the base sequence in the present
invention may be included. The melting temperature by the
Nearest-Neighbor method can be easily obtained by using, for
example, software equipped with Visual OMP (Tomy Digital Biology
Co., Ltd.) or software provided by Nihon Gene Research Laboratories
Inc. (http://www.ngrl.co.jp/) (OligoCalculator;
http://www.ngrl.cojp/tool/ngrl_tool.html). SEQ ID NO: 1 to SEQ ID
NO: 100 are arranged in descending order of the melting
temperatures calculated with Visual OMP (0.1 M probe concentration,
50 mM Na.sup.+ ion and 1.5 mM Mg.sup.+ ion).
[0052] The detection sequence 106 in the detection probe 104 is
called as a orthonormalization sequence and is designed based on
the calculations on a consecutive identical length, melting
temperature prediction by the Nearest-Neighbor method, a Hamming
distance, secondary structure prediction on DNA sequences having
certain base lengths obtained from random numbers. The
orthonormalization sequences are base sequences of nucleic acids
which have homogeneous melting temperatures and thus are designed
so as to have the melting temperatures in a constant range, which
do not inhibit hybridization with the complementary sequences
because nucleic acids are structured intramolecularly, and which do
not stably hybridize with base sequences other than complementary
base sequences. Sequences contained in one orthonormalization
sequence group hardly react or do not react to sequences other than
a desired combination or within their sequences. When
orthonormalization sequences are amplified by PCR, the amount of
the nucleic acids quantitatively amplified correspond to an initial
amount of the nucleic acids having the orthonormalization sequences
without influenced by a problem such as cross-hybridization as
mentioned above. Such orthonormalization sequences are reviewed in
H. Yoshida and A. Suyama, "Solution to 3-SAT by breadth first
search", DIMACS Vol. 54, 9-20 (2000) and Japanese Patent
Application No. 2003-108126. The orthonormalization sequences can
be designed by using the methods described in these documents.
[0053] The detection probes 104 are fixed on the carrier 102. The
carrier 102 may be the solid phase carrier. The carrier 102 may be,
for example, plastics, glass or any other material without
limitation. A shape of the carrier 102 may be a plate as shown in
FIG. 1 or may be a bead without limitation. The solid phase 100 is
preferably the any (particularly microarray) in which the support
102 is a solid phase plate and multiple detection probes 104 are
fixed with a regular sequence. The array can be fixed with many
detection probes 104 and is suitable for detecting various target
nucleic acids 10 simultaneously and exhaustively. The solid phase
100 may comprise multiple defined array regions on the carrier 102.
On the multiple array regions, the same sets of detection probes
104 or different sets of detection probes 104 may be fixed. When
different combinations of the sets of detection probes 104 are
fixed on multiple array regions, individual array regions may be
assigned for detection of target nucleic acids 10 in different
genes.
[0054] The preferred solid phase 100 may comprise two or more
detection probes 104 arranged in order of their melting
temperatures. For example, by using such a solid phase 100 in which
two or more detection probes 104 for two or more target nucleic
acids 10 corresponding to two or more target sequences 12 which may
exist at certain sites in certain genes are arranged in such order,
variation in hybridization due to the difference in melting
temperatures of detection sequences 106 in detection probes 104 or
to positions to where detection probes 104 are fixed is suppressed,
thereby allowing accurate detection of target nucleic acids 10 in
the sample.
[0055] The detection probes 104 may be fixed by any mode without
limitation, which may be covalent or non-covalent. The detection
probes 104 may be fixed on the surface of the carrier 102 by any
various well-known methods in the art. The surface of the carrier
102 may comprise appropriate linker sequences. The linker sequences
preferably have the same base length and same sequence for the
respective detection probes 104.
[0056] (Step of Obtaining Chimeric DNA: Step of PCR)
[0057] As shown in FIG. 1, the step of PCR may comprise carrying
out PCR on the sample so as to obtain the chimeric DNA 60 having
the label 42 and the tag sequence 66 which is able to hybridize
with the detection sequence 106 in the specific detection probe 104
having been correlated to the target nucleic acid 10. By obtaining
such a chimeric DNA 60, the detection probe 104 can be employed
having the unique detection sequence 106 which has been determined
in advance regardless of the base sequence of the target sequence
12 in the target nucleic acid 10. The tag sequence 66 is preferably
complementary such that it can specifically hybridize to the unique
detection sequence 106 in the detection probe 104, and more
preferably completely complementary to the detection sequence 106.
The label is described hereinafter.
[0058] Primers used in the step of PCR are not specifically limited
as long as the above chimeric DNA 60 can be obtained. Exemplary
preferred step of PCR in the present method for detection is now
described with referring to FIG. 1. The upper right of FIG. 1 shows
a step of carrying out PCR on the target nucleic acid 10 and its
complementary strand 20 in the sample with the first primer 30 and
the second primer 40 to obtain an amplification product, chimeric
DNA 60.
[0059] (First Primer)
[0060] As shown in FIG. 1, the first primer 30 contains the
identification sequence 32 and the tag addition sequence 36. The
first primer 30 is prepared as many as the target nucleic acids 10.
When two kinds of mutations are expected at a certain part in a
genomic DNA of a certain kind of an animal, which are, for example,
single nucleotide substitutions with A for a wild type and T for a
mutation, there are two target nucleic acids 10 for this part.
Thus, one target nucleic acid 10 for this part contains the target
sequence 12 having the wild type base and the other target nucleic
acid 10 contains the target sequence 12 having the mutated base.
Accordingly, when there are two target nucleic acids 10 for a
certain site of a gene, two first primers 30 are prepared each
having the identification sequence 32 complementary to the target
sequence 12 in each of the target nucleic acids 10
[0061] (Identification Sequence)
[0062] The identification sequence 32 can specifically hybridize to
the target sequence 12 which is a characteristic sequence in the
target nucleic acid 10, in order to identify the target nucleic
acid 10. The identification sequence 32 is established to be
complementary such that it can hybridize to the target sequence 12
in the target nucleic acid 10 with high selectivity, and preferably
is established to be completely complementary (specific). The
preferred length of the identification sequence 32 may vary
according to mutations and is not specifically limited, but is
preferably 15 bases or more, for example. The identification
sequence 32 having 15 bases or more in length can hybridize to the
target sequence 12 with high selectivity. The identification
sequence 32 having 60 bases or less in length is preferable due to
reduced non-specific hybridization.
[0063] (Tag Addition Sequence)
[0064] The first primer 30 may comprise the tag addition sequence
36 for adding the tag sequence 66 to the amplified product,
chimeric DNA 60, so as to allow the chimeric DNA 60 being able to
hybridize to the detection sequence 106 in the detection probe 104.
The tag sequence 66 in the chimeric DNA 60 is for detecting the
target nucleic acid 10, thus is established to be able to hybridize
to the detection sequence 106 in the detection probe 104 for every
target nucleic acid 10. Thus, one chimeric DNA 60 corresponding to
one target nucleic acid 10 is correlated to one detection probe
104. The tag sequence 66 is preferably completely complementary to
the unique detection sequence 106 in the detection probe 104. Thus,
the tag addition sequence 36 preferably has the same base sequence
as the unique detection sequence 106 in the detection probe 104 for
detection.
[0065] As described above, the first primer 30 is prepared so as to
specifically bind to the target sequence 12 in the target nucleic
acid 10 and is prepared as many as the target nucleic acids 10,
thereby specifically amplifying the target nucleic acids 10 while
detecting the same. The first primer 30 is also formed to allow
specific binding of the PCR amplified product, chimeric DNA 60, to
the particular detection probe 104 which has been correlated to the
target nucleic acid 10.
[0066] (Second Primer)
[0067] As shown in FIG. 1, the second primer 40 may contain the
label 42 and the partial sequence 44 which is identical to the base
sequence adjacent to the target sequence 12 in the target nucleic
acid 10. The label 42 may be at the 5'-side of the second
primer.
[0068] (Label)
[0069] The label 42 is for detecting the PCR amplified product,
chimeric DNA 60. The label 42 may be appropriately selected from
well-known labels. The label may be any of various dyes emitting
fluorescent signal after excitation such as fluorescent substances,
or a substance emitting any of various signal after combining it
with a secondary component by enzyme reaction or antigen-antibody
reaction. The label may be typically fluorescent labeling
substances such as Cy3, Alexa 555, Cy5, Alexa 647. The detection by
color development may be used by combining biotin and
streptoavidin-HRP and processing them with a substrate.
[0070] (Partial Sequence)
[0071] The partial sequence 44 has the same base sequence as the
partial sequence 14 adjacent to the target sequence 12 in the
target nucleic acid 10. The partial sequence 14 adjacent to the
target sequence 12 does not mean that the partial sequence 14 is
immediately at the 5'-side of the target sequence 12 without
interposing one base (nucleotide) therebetween, but may be the
sequence interposing appropriate number of bases (nucleotides). The
partial sequence 44 in the second primer 40 is the sequence
allowing annealing of the second primer 40 to the complementary
sequence 20 of the target nucleic acid 10.
[0072] When a mutation on DNA is detected, the first primer 30 and
the second primer 40 are designed for the target nucleic acids 10
respectively of the wild type and the mutant. In this case, the
partial sequence 44 of the second primer 40 may be common to these
target nucleic acids 10. Namely, the partial sequence 44 may be a
common partial sequence adjacent to the target sequence 12 in these
target nucleic acids 10. The common partial sequence is a base
sequence which is common regardless of the mutation. Due to this,
amplification efficiency of the target nucleic acids 10 can be
averaged and the amount of the first primer 40 to be used may be
decreased. The partial sequence 44 may be the sequence having
homology to multiple target nucleic acids 10 corresponding to
multiple target sequences 12 constituting mutations.
[0073] As described above, the second primer 40 contains the label
42 and the partial sequence 44, and is formed so as to synthesize
the chimeric DNA 60 containing the target sequence 12 due to the
partial sequence 44. When the present method is to detect multiple
target nucleic acids 10 having multiple target sequences 12
constituting mutations, the second primer 40 may have the common
partial sequence 44 which allows amplification of multiple target
nucleic acids 10 having multiple target sequences 12 constituting
mutations under the same condition.
[0074] The step of obtaining the chimeric DNA 60 with the first
primer 30 and the second primer 40 is now described with referring
to FIGS. 1 and 3. In the following description, only PCR reaction
which may give the desired chimeric DNA 60 is explained.
[0075] As shown in FIG. 3, the first primer 30 anneals to the
target sequence 12 in the target nucleic acid 10 through the
identification sequence 32. As a result, a new DNA strand is
extended from the first primer 30 with the target nucleic acid 10
as a template, thereby synthesizing a DNA strand 50 comprising a
newly synthesized partial sequence 14 (-). The obtained DNA strand
50 has the tag addition sequence 36, the identification sequence 32
and the partial sequence 14 (-).
[0076] To the partial sequence 14 (-) in the thus obtained DNA
strand 50 then anneals the second primer 40 through its partial
sequence 44. As a result, a new DNA strand is extended from the
second primer 40 with the DNA strand 50 as a template, thereby
synthesizing a DNA strand 60 comprising a base sequence
complementary to the identification sequence 32 and a base sequence
complementary to the tag addition sequence 36. As the
identification sequence 32 has identical base sequence as a target
sequence 12 (-), a base sequence complementary to the
identification sequence 32 has the same sequence as the target
sequence 12. As the tag addition sequence 36 is identical to the
unique detection sequence 106 in the detection probe 104, a base
sequence complementary to the tag addition sequence is the tag
sequence 66 which is complementary to the detection sequence 106 in
the detection probe 104. The thus obtained DNA strand 60 is the
chimeric DNA 60 comprising the label 42 and has been correlated to
the target sequence 12 and the detection probe 104. The chimeric
DNA 60 is used as a template in further amplification reaction.
[0077] The step of PCR for obtaining the chimeric DNA 60 is
preferably the step of asymmetric PCR. Asymmetric PCR can be
carried out by varying the concentrations of the first and second
primers, for example.
[0078] As the chimeric DNA 60 is obtained as a double-stranded DNA,
it is dissociated to single strands for subjecting them to the step
of hybridization. The dissociation in this context can be achieved
by a denaturing treatment comprising chemical denaturation and
thermal denaturation. When oligonucleotides linked are dissociated
by chemical denaturation, a treatment known to a person skilled in
the art such as alkaline denaturation may be carried out. When
oligonucleotides linked are dissociated by thermal denaturation,
they may be placed under a temperature of 85.degree. C. or more,
preferably 90.degree. C. or more under physiological conditions;
however, a person skilled in the art can select appropriate
dissociation method.
[0079] According to the step of PCR in which the sample which may
possibly contain the target nucleic acid 10 is subjected to the
step of PCR, chimeric DNAs 60 can be obtained at once which can
specifically detect the target nucleic acids 10 via the detection
probes 104 having been correlated to the target nucleic acid
10.
[0080] A PCR reaction product may be subjected to a next step
without collecting chimeric DNAs 60, because only chimeric DNAs 60
can bind to the detection probes 104 which are then detected
through the label 42. Chimeric DNAs 60 may be collected by a
well-known method. For example, the chimeric DNAs 50 may be
separated and collected by a well-known method such as using an
appropriate solid phase carrier after being dissociated into single
strands.
[0081] (Step of Hybridization)
[0082] The step of hybridization is the step in which the detection
probes 104 having the detection sequences 106 complementary to the
tag sequences 66 in the chimeric DNAs 60 on the solid phase 100
fixed on the carrier 102 and the chimeric DNAs 60 are brought into
contact so as to allow hybridization. As shown in FIGS. 1 and 2
(c), when the chimeric DNA 60 is complementary to the detection
sequence 106 in the detection probe 104 such that they can
specifically hybridize each other under certain conditions, they
hybridize each other to form a double-strand at a certain detection
probe 104 on the carrier 102 in this step. A washing step may
further be appropriately contained following to the step of
hybridization.
[0083] To the step of hybridization is provided the chimeric DNA 60
which has been synthesized in the step of PCR only when the target
nucleic acid 10 is present in the sample and which hybridizes only
to the detection probe 104 having been correlated. The detection
sequence 106 in the detection probe 104 and the tag sequence 66 in
the chimeric DNA 60 are selected with high selectivity so that
mishybridization is highly suppressed, thereby highly suppressing
non-specific hybridization of the chimeric DNA 60 to the detection
probe 104 in the step of hybridization.
[0084] (Step of Obtaining Signal Intensity Information)
[0085] The step of obtaining signal intensity information is the
step in which signal intensity information about the target nucleic
acid 10 based in the label 42 on the carrier 102 is obtained after
hybridization. According to the present step of obtaining signal
intensity information, the chimeric DNA 60 hybridizes to the
detection probe 104 to provide signal intensity information based
on the label 42.
[0086] As shown in FIG. 1, in the step of obtaining signal
intensity information, signal 48 derived from the label 42
associated with the detection probe 104 on the solid phase 100 may
be detected. As the position of the detection probe 104 correlated
has been already known on the solid phase 100, the presence or
absence or ratio of the target nucleic acid 10 can be determined by
detecting the signal 48 in the nest step of detection.
[0087] The step of obtaining signal intensity information may be
carried out by selecting a conventional well-known method according
to the form of the carrier 102 or the label 42. Typically, after
removing non-hybridized oligonucleotides and the like from the
carrier 102 by washing, fluorescent signal of the added labeling
substance may be detected with an array scanner and the like or the
labeling substance may be subjected to chemical luminescence
reaction. When the carrier is a bead, a detection method using a
flow cytometer may be employed.
[0088] (Step of Detection)
[0089] The step of detection is the step in which the presence or
absence or ratio of the target nucleic acid 10 in the sample is
detected based on signal intensity information of the label 42
obtained for the detection probe 104. According to the present
method, even when multiple target nucleic acids 10 are detected
simultaneously, the target sequences can be surely detected.
According to the present method, as non-specific binding to the
detection probe 104 is highly suppressed in the step of
hybridization, the target nucleic acid 10 can be accurately
detected with high detection sensitivity and the presence or
absence or ratio thereof can be obtained.
[0090] (Primer Set)
[0091] The primer set of the present invention comprises the first
and second primers described hereinabove. The primer set is used in
combination with the solid phase on which the detection probes 104
have been fixed, and is suitable for obtaining the chimeric DNA
described hereinabove. The first primer comprises the
identification sequence 32 which is specific to the particular
target nucleic acid for detecting a mutation among individuals
regarding the same gene and the like or a difference between
species or genera and the tag addition sequence 36 having been
correlated to the detection sequence 106. The second primer
comprises the label. The primer set may be for detecting two or
more target nucleic acids. In this case, the primer set may
comprise the first primers specific to respective target nucleic
acids and the single second primer common to two or more target
nucleic acids. The primer set of the present invention may be
comprised in a kit together with the carrier such as the array to
which the detection probes described hereinabove have been
fixed.
Example 1
[0092] The present invention is specifically described with the
following examples, which do not limit the present invention.
Example 2
[0093] The target nucleic acid was detected with the method for
detection of the present invention in the present example according
to the following procedures, which are now described step by
step.
(1) Preparation of DNA microarray (2) Preparation and amplification
of target nucleic acids and primers
(3) Hybridization
[0094] (4) Detection with scanner (5) Data analysis
[0095] (1) Preparation of DNA Microarray
[0096] On a plastic plate, aqueous solutions of synthetic oligo
DNAs (Nihon Gene Research Laboratories Inc.) modified at a 3'-end
with an amino group were spotted as the detection probes using a
GENESHOT.RTM. spotter at NGK Insulators, Ltd. As shown in Table 1,
100 synthetic oligo DNAs were used which were D1.sub.--001 to
D1.sub.--100 shown in Supplementary Table 1 in a document
(Analytical Biochemistry 364 (2007) 78-85) (see FIG. 5). After
spotting, the plate was baked at 80.degree. C. for an hour. These
probes are arranged in descending order of melting temperatures
corresponding to Tm calculated with Visual OMP (0.1 M probe
concentration, 50 mM Na+ ion and 1.5 mM Mg+ ion).
[0097] The synthetic oligo DNAs were fixed according to the
following procedures. Namely, the plate was washed with
2.times.SSC/0.2% SDS for 15 minutes, with 2.times.SSC/0.2% SDS at
95.degree. C. for 5 minutes, before three times of washing with
sterilized water (mixing by turning vertically for 10 times). The
plate was then dried by centrifugation (1000 rpm.times.3
minutes).
[0098] (2) Preparation of Target Nucleic Acids and Primers and
Amplification
[0099] Sample genes to be detected were derived from two types of
oral microorganisms, Enterococcus faecalis (sample 1) and
Pseudorambibacter alactolyticus (sample 2). The length of these
samples was about 150 bp, which surrounded characteristic sequences
of microorganisms, and artificial genes having these base sequences
were used as target nucleic acids. Primers for amplifying these
target nucleic acids were artificially synthesized as follows. The
second primer, i.e. the forward primer (F primer) was
5'-AGGTTAAAACTCAAAGGAATTGACG-3' (SEQ ID NO: 101), which was labeled
with Cy3 at the 5'-side. The first primers, i.e. the reverse
primers (R primers) were prepared according to the target sequences
of the samples. The reverse primer for the sample 1 was
5'-GCAGATTCATTGGTCAGAGAACATATCTCTAGAGTGGT-3' (SEQ ID NO: 102) and
the reverse primer for the sample 2 was
5'-CATCTAAAGCGTTCCCAGTTCCATATCTCTATTGCGCT-3' (SEQ ID NO: 103).
[0100] These samples were amplified as follows. A reagent used for
amplifying the samples was a multiple PCR kit from QIAGEN. A
thermal cycler used was GeneAmp PCR System 9700 from Applied
Biosystems.
[0101] The following reagents were prepared for each sample. The F
primer and R primers used were respectively adjusted to 10
pmol/.mu.l.
(Reagent Preparation)
[0102] dH.sub.2O 15.0 .mu.l multiple PCR kit 25.0 .mu.l F primer
3.75 .mu.l R primer 3.75 .mu.l
Sample 2.5 .mu.l
Total 50.0 .mu.l
[0103] The prepared reagents were transferred to a thermal cycle
plate and thermal cycle reaction (95.degree. C. for 15 min; then 50
cycles of 94.degree. C. for 30 sec, 62.degree. C. for 30 sec and
72.degree. C. for 30 min; 72.degree. C. for 10 min, and decreased
to 4.degree. C.) was carried out. The amplified labeled samples
were purified with MinElute PCR Purification Kit from QIAGEN,
before verifying that amplified products had a desired length.
[0104] (3) Hybridization
[0105] In order to hybridize the amplified samples obtained in (2)
with the detection probes fixed on the microarray, the following
Hybri control and Hybri solution were prepared, which were used for
preparation of a hybridization reagent. An Alexa 555-labeled oligo
DNA sequence used for Hybri control was Alexa555-rD1.sub.--100
which was obtained by labeling the 5'-end of a complementary
sequence of D1.sub.--100, among those probes described in FIG. 5,
with Alexa 555.
[0106] (Hybri Control)
Alexa555-rD1.sub.--100 10 .mu.l
TE (pH 8.0) 390 .mu.l
Total 400 .mu.l
[0107] (Hybri Solution)
20.times.SSC 2.0 ml
10% SDS 0.8 ml
100% Formamide 12.0 ml
100 mM EDTA 0.8 ml
[0108] milliQ 24.4 ml
Total 40.0 ml
[0109] (Reagent for Hybridization)
Hybri control 1.5 .mu.l Hybri solution 9.0 .mu.l
Subtotal 10.5 .mu.l
[0110] Labeled sample 7.5 .mu.l
Total 18.0 .mu.l
[0111] A prepared labeled sample solution was heated in GeneAmp PCR
system 9700 from Applied Biosystems at 90.degree. C. for 1 minute
prior to heating in a heat block (TAITEC, DTU-N) at 80.degree. C.
for 1 minute. The sample solutions (9 .mu.l each) were deposited on
a spotted area of the microarray and left to stand at 37.degree. C.
for 30 minutes for hybridization reaction while preventing
evaporation with Thermoblock Slide for Comfort/plus
(Eppendorf).
[0112] (Washing)
[0113] After hybridization, the microarray substrate after
hybridization reaction was soaked in a glass staining vat filled
with washing solution having the following composition, incubated
with vertical shaking for 5 minutes, and the glass substrate was
transferred to a glass staining vat filled with sterilized water,
incubated with vertical shaking for 1 minute, and dried by
centrifugation at 2000 rpm for 1 minute to remove remaining water
on the surface of the microarray substrate.
(Composition of Washing Solution)
[0114] milliQ 188.0 ml
20.times.SSC 10.0 ml
10% SDS 2.0 ml
Total 200.0 ml
[0115] (4) Detection with Scanner
[0116] Fluorescent images were obtained with ArrayWoRx from Applied
Precision, Inc. by appropriately adjusting time of exposure.
Fluorescent signal from the obtained images were converted to
numerical values with GenePix Pro.
[0117] (5) Data Analysis
[0118] Fluorescent signal from the obtained images were converted
to numerical values with GenePix Pro, which is software for
numerical conversion of images. FIG. 6 shows the results of
verification on whether or not the samples 1 and 2 respectively
bound non-specifically to the detection probes.
[0119] As shown in FIG. 6 (a), the reaction with the mixture of the
samples 1 and 2 gave fluorescent signal for both probes, indicating
that the samples were detected. As shown in FIGS. 6(b) and 6(c) in
which either of the sample 1 or sample 2 was subjected to the
reaction without mixing, it was found that non-specific binding to
an undesired probe was significantly decreased. It was also found
that each sample specifically bound to the respective detection
probes designed to identify the respective samples.
[0120] Next, a conventional detection method of target nucleic
acids (method described in Non-patent document 4) was verified as a
comparative example. In the following comparative example, the
target nucleic acid was detected with the conventional detection
method according to the following procedures, which are now
described step by step.
[0121] On a plastic plate, aqueous solutions of synthetic oligo
DNAs (Nihon Gene Research Laboratories Inc.) modified at the 3'-end
with an amino group were spotted as the detection probes using a
GENESHOT.RTM. spotter at NGK Insulators, Ltd. The used synthetic
oligo DNA sequences were 5'-ACCACTCTAGAGATA-3' (SEQ ID NO: 104) for
a sample 1, and 5'-AGCGCAATAGAGATA-3' (SEQ ID NO: 105) for a sample
2. After spotting, the plate was baked at 80.degree. C. for an
hour, and the DNAs were arranged in descending order of Tm.
[0122] Sample genes to be detected were the same samples 1 and 2
used in the example. Common primers for amplifying the target
nucleic acids were artificially synthesized as follows. The F
primer was 5'-AGGTTAAAACTCAAAGGAATTGACG-3' (SEQ ID NO: 106), which
was labeled with Cy3 at the 5'-side. The R primer was
5'-ATGGTGTGACGGGCGGTGTGT-3' (SEQ ID NO: 107).
[0123] The samples 1 and 2 were amplified by the thermal cycle
reaction as described in the above (3) to (5), and hybridized with
the detection probes prepared in Example 6 before washing and
signal detection. FIG. 7 shows the results of verification on
whether or not the samples 1 and 2 respectively bound
non-specifically to the detection probes.
[0124] As shown in FIGS. 7(a) to 7(c), weak signal was detected
with the detection probe for the sample 1 even when the sample did
not contain the sample 1, and weak signal was detected against the
detection probe for the sample 2 even when the sample did not
contain the sample 2, which were thus showing non-specific binding
of the samples.
[0125] The time required for hybridization in the conventional
method was about two hours. Non-specific reaction to the detection
probes was observed (about 10% in fluorescent intensity). On the
other hand, the time required for hybridization in the present
invention was decreased to about 30 minutes and non-specific
reaction of the samples to undesired detection probes on the DNA
microarray could be significantly reduced (less than 1% in
fluorescent intensity). Thus, according to the present invention,
hybridization can always be carried out at a constant temperature
(about 37.degree. C.) in about 30 minutes of time (one-fourth of
the conventional method). The present invention can also provide
results with more intense signal than the conventional method, and
allows more accurate detection of bases in a particular nucleic
acid and more accurate determination of sequence than the
conventional method. The conventional method sometimes requires
optimization of hybridization conditions, re-design of probe
sequences or re-preparation of arrays until desired result are
obtained. On the other hand, the present invention does not require
re-design of probe sequences or re-production of arrays and allows
examination with arrays having the same specification all the
time.
[0126] [Sequence Listing Free Text]
SEQ ID NOs: 1 to 100: probes, SEQ ID NOs: 101 to 103: primers, SEQ
ID NOs: 104 and 105: probes, SEQ ID NOs: 106 and 107: primers
[0127] [Sequence Listing]
Sequence CWU 1
1
107123DNAArtificialProbe 1gcctatatga accaagccac tgc
23223DNAArtificialProbe 2gagacaggta aaccctcaga gca
23323DNAArtificialProbe 3gtcccaaaag cttcttacgg acg
23423DNAArtificialProbe 4cgatcagctc tatttccctc cca
23523DNAArtificialProbe 5gcattgaggt attgttgctc cca
23623DNAArtificialProbe 6gcctcacttg taataagcgg gac
23723DNAArtificialProbe 7ggggtgtgag agctttttag acg
23823DNAArtificialProbe 8cgcgataatt gatacctacg ggc
23923DNAArtificialProbe 9cgatcacgga ttaatgtcac ccc
231023DNAArtificialProbe 10cgcagtttgc aagaacgaac aaa
231123DNAArtificialProbe 11cgcgacattt agtccaggag atg
231223DNAArtificialProbe 12accactatga ttgaggaaac gcg
231323DNAArtificialProbe 13cgctgttggt attaccttcc tcg
231423DNAArtificialProbe 14gagtcgaaga cctcctccta ctc
231523DNAArtificialProbe 15tggaactggg aacgctttag atg
231623DNAArtificialProbe 16cgtctttagt atcaaccctc cgc
231723DNAArtificialProbe 17ggggggtact tcatacaaga tgc
231823DNAArtificialProbe 18tgccgtcatt taaacgtaag ggt
231923DNAArtificialProbe 19catctccaag aattgaccca cca
232023DNAArtificialProbe 20atgccgttgt caagagttat ggt
232123DNAArtificialProbe 21cgagagtctg taatagccga tgc
232223DNAArtificialProbe 22cacgcttagt tcctacctta ggc
232323DNAArtificialProbe 23gcccgggaat agattataac gca
232423DNAArtificialProbe 24gcagccctta tagataacgg gac
232523DNAArtificialProbe 25cgctctggtt actattggac gtt
232623DNAArtificialProbe 26gcatttttag taatccgagc gcc
232723DNAArtificialProbe 27cgccattata caacggttca tgc
232823DNAArtificialProbe 28ggctggttaa atgtaaatcc gcg
232923DNAArtificialProbe 29gtcggtatcg aaaaggtact gca
233023DNAArtificialProbe 30cgccaatgac aataagttga ggc
233123DNAArtificialProbe 31ggtcgtaaca ttgagaggag acg
233223DNAArtificialProbe 32gaagccatga tactgttcag ggt
233323DNAArtificialProbe 33aggcagttca acctatatct gcg
233423DNAArtificialProbe 34gcctcacata actggagaaa cct
233523DNAArtificialProbe 35gcatatagtg acggtaaggc gaa
233623DNAArtificialProbe 36tgccggttat acctttaagg acg
233723DNAArtificialProbe 37gcctatagtg tcgattgtcc tcg
233823DNAArtificialProbe 38ggctcgtagt actccttaca tgc
233923DNAArtificialProbe 39ctagtccatt gtaacgaagg cca
234023DNAArtificialProbe 40ccgtcgtgtt attaaagacc cct
234123DNAArtificialProbe 41ccgtgtgtat gagtatgaca gca
234223DNAArtificialProbe 42tgccggctat cgtaagtata tgc
234323DNAArtificialProbe 43gggataggta ttatgctcca gcc
234423DNAArtificialProbe 44ccatcagtta ttcggaggga ctc
234523DNAArtificialProbe 45agtcgcttaa ttactccgga tgg
234623DNAArtificialProbe 46gcagctgaat tgctatgatc acc
234723DNAArtificialProbe 47gcacctcata ccttcataga gca
234823DNAArtificialProbe 48agtcagtcca aatctcagga tgg
234923DNAArtificialProbe 49aggtccggta gtaatttagg tgc
235023DNAArtificialProbe 50cgcctaaatg aaactcactc tgc
235123DNAArtificialProbe 51gcccacactc ttacttatcg act
235223DNAArtificialProbe 52ttcgcttcgt tgtaatttcg gac
235323DNAArtificialProbe 53agacaattag aatcagtgcc cct
235423DNAArtificialProbe 54agtcagttaa tcagacgtga gca
235523DNAArtificialProbe 55cgcggtacta ttagaaaggg cta
235623DNAArtificialProbe 56ggctctacaa acttgtgtcc atg
235723DNAArtificialProbe 57cgatcatgta aagctaactc gcg
235823DNAArtificialProbe 58tagcacccgt taaaacggaa atg
235923DNAArtificialProbe 59tttgttgttc gatatcaggc gtg
236023DNAArtificialProbe 60gcactaccgc taactatacg cta
236123DNAArtificialProbe 61tatgtttagt tgttgaaccg gcg
236223DNAArtificialProbe 62tggcaattac agttgttaac gca
236323DNAArtificialProbe 63cgcgatataa cattaaccga ggc
236423DNAArtificialProbe 64ggggtcaaac caacaattga tct
236523DNAArtificialProbe 65tggcaataca ataacgtatc gcg
236623DNAArtificialProbe 66aggcatccta agaaatcgct act
236723DNAArtificialProbe 67gagtagcagg caaataccct aga
236823DNAArtificialProbe 68cgcgattcct attgattgat ccc
236923DNAArtificialProbe 69gcccattgat agaattacga ggc
237023DNAArtificialProbe 70gagtccgcaa aaatatagga ggc
237123DNAArtificialProbe 71tgccgtgata cttaactacg cta
237223DNAArtificialProbe 72ttcggttgtc gatatgagga tct
237323DNAArtificialProbe 73cgcgtcgaat tacttaatca cca
237423DNAArtificialProbe 74gaaggatcgc ttttatctgg cat
237523DNAArtificialProbe 75ggcgatttat tgctaactgg cta
237623DNAArtificialProbe 76ggtggagtga atctcactag act
237723DNAArtificialProbe 77gcatacgaac ttctatatcg gcg
237823DNAArtificialProbe 78tgcactctga tatatacagg cca
237923DNAArtificialProbe 79ccgtctgggt taaagattgc tag
238023DNAArtificialProbe 80aagagattta acttgagctc gcc
238123DNAArtificialProbe 81tgttctctga ccaatgaatc tgc
238223DNAArtificialProbe 82gggatccgta acaagtgtgt tag
238323DNAArtificialProbe 83tagcccagtg atttatgaca tgc
238423DNAArtificialProbe 84ccatatccga ttattagcga cgg
238523DNAArtificialProbe 85tgctcactta cattacgtcc atg
238623DNAArtificialProbe 86catttgtcag gtacagtcca ctt
238723DNAArtificialProbe 87catggataag ttttcaagct gcg
238823DNAArtificialProbe 88cgctgttact gtaagcgtac tag
238923DNAArtificialProbe 89tgctgtcttc gtgttttacc tag
239023DNAArtificialProbe 90tacacctatc aactcgtaga gca
239123DNAArtificialProbe 91cgccgtcagt acttgtatag atg
239223DNAArtificialProbe 92tattctacca acgacatcac tgc
239323DNAArtificialProbe 93cattcgacat aagctgttga tgc
239423DNAArtificialProbe 94tgcagtgtaa gcaactattg tct
239523DNAArtificialProbe 95ctaggtacaa caccaactgt ctc
239623DNAArtificialProbe 96gcctattaag gtctacgtca tcg
239723DNAArtificialProbe 97atgccaatat gtactcgtga ctc
239823DNAArtificialProbe 98agtcatacag tgaggaccaa atg
239923DNAArtificialProbe 99tagccaactc taaataacgg acg
2310023DNAArtificialProbe 100ctagcacaat taatcaatcc gcc
2310125DNAArtificial SequencePrimer 101aggttaaaac tcaaaggaat tgacg
2510238DNAArtificial SequencePrimer 102gcagattcat tggtcagaga
acatatctct agagtggt 3810338DNAArtificial SequencePrimer
103catctaaagc gttcccagtt ccatatctct attgcgct 3810415DNAArtificial
SequenceProbe 104accactctag agata 1510515DNAArtificial
SequenceProbe 105agcgcaatag agata 1510625DNAArtificial
SequencePrimer 106aggttaaaac tcaaaggaat tgacg 2510721DNAArtificial
SequencePrimer 107atggtgtgac gggcggtgtg t 21
* * * * *
References