U.S. patent application number 13/499618 was filed with the patent office on 2012-07-26 for method for designing probe in dna microarray, and dna microarray provided with probe designed thereby.
This patent application is currently assigned to Toyota Jidosha Kabushiki Kaisha. Invention is credited to Hiroyuki Enoki, Aya Murakami, Satoru Nishimura.
Application Number | 20120190582 13/499618 |
Document ID | / |
Family ID | 44167263 |
Filed Date | 2012-07-26 |
United States Patent
Application |
20120190582 |
Kind Code |
A1 |
Enoki; Hiroyuki ; et
al. |
July 26, 2012 |
METHOD FOR DESIGNING PROBE IN DNA MICROARRAY, AND DNA MICROARRAY
PROVIDED WITH PROBE DESIGNED THEREBY
Abstract
Provided is a probe to be used in a DNA microarray having an
excellent detection rate of a polymorphism such as SNP contained in
genomic DNA. A method for designing a probe according to the
invention includes the steps of: specifying one or more regions
covering at least a part of fragments flanked by restriction enzyme
recognition sites recognized by a restriction enzyme, contained in
genomic DNA derived from an organism to be tested; and designing a
probe for the specified one or more regions for detecting the
fragment in the organism to be tested.
Inventors: |
Enoki; Hiroyuki;
(Okazaki-shi, JP) ; Nishimura; Satoru;
(Nagoya-shi, JP) ; Murakami; Aya; (Hoi-Gun,
JP) |
Assignee: |
Toyota Jidosha Kabushiki
Kaisha
Toyota-shi, Aichi
JP
|
Family ID: |
44167263 |
Appl. No.: |
13/499618 |
Filed: |
December 13, 2010 |
PCT Filed: |
December 13, 2010 |
PCT NO: |
PCT/JP2010/072322 |
371 Date: |
March 30, 2012 |
Current U.S.
Class: |
506/9 ; 506/16;
506/26 |
Current CPC
Class: |
C12Q 1/683 20130101;
C12Q 1/6837 20130101; C12Q 1/6855 20130101; C12Q 1/6806 20130101;
C12Q 1/6809 20130101; C12Q 1/683 20130101; C12Q 2565/501 20130101;
C12Q 2535/125 20130101; C12Q 2521/307 20130101; C12Q 1/6837
20130101; C12Q 2535/125 20130101; C12Q 2521/307 20130101; C12Q
1/6809 20130101; C12Q 2521/307 20130101; C12Q 2535/125 20130101;
C12Q 2565/501 20130101; C12Q 1/6855 20130101; C12Q 2521/307
20130101; C12Q 2535/125 20130101; C12Q 2565/501 20130101 |
Class at
Publication: |
506/9 ; 506/26;
506/16 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/06 20060101 C40B040/06; C40B 50/06 20060101
C40B050/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 14, 2009 |
JP |
2009-283430 |
Claims
1-20. (canceled)
21. A method for designing a probe, comprising the steps of:
specifying one or more regions having a shorter nucleotide length
than genomic DNA fragment flanked by restriction enzyme recognition
sites, contained in genomic DNA derived from a target organism, and
covering at least one portion of the genomic DNA fragment; and
designing the specified one or more regions as a probe for
detecting a mutation contained in the genomic DNA fragment.
22. The method for designing a probe according to claim 21, wherein
the one or more regions is specified by performing the following
steps: (1a) extracting the genomic DNA; (1b) digesting the
extracted genomic DNA with the restriction enzyme; (1c) connecting
an adaptor to the genomic DNA fragments obtained the step (1b);
(1d) amplifying the genomic DNA fragments using a primer capable of
hybridizing to the adaptor; (1e) sequencing the amplified genomic
DNA fragments; and (1f) determining the one or more regions based
on the nucleotide sequence.
23. The method for designing a probe according to claim 22,
wherein, in the step (1b), the genomic DNA is digested with more
than one restriction enzyme.
24. The method for designing a probe according to claim 23,
wherein, in the step (1c), an adaptor is connected corresponding to
one restriction enzyme selected from the more than one restriction
enzyme or corresponding to a part of the more than one restriction
enzyme used.
25. The method for designing a probe according to claim 22, wherein
the adaptor has a complementary sequence to a protruding end of the
genomic DNA fragments obtained in the step (1b).
26. The method for designing a probe according to claim 21, wherein
the one or more regions are specified using the nucleotide sequence
data on the genomic DNA by performing the following steps: (2a)
searching the nucleotide sequence data on the genomic DNA for the
restriction enzyme recognition sequence to specify the nucleotide
sequence of the genomic DNA fragments obtained by digesting the
genomic DNA with the restriction enzyme; and (2b) determining the
one or more regions based on the specified nucleotide sequence.
27. The method for designing a probe according to claim 26,
wherein, in the step (2a), the genomic DNA fragments obtained by
digesting the genomic DNA with more than one restriction enzyme are
sequenced.
28. The method for designing a probe according to claim 27,
wherein, in the step (2b), the one or more regions are determined
with respect to the genomic DNA fragments flanked by one
restriction enzyme selected from the more than one restriction
enzyme or a part of more than one restriction enzyme used.
29. The method for designing a probe according to claim 21, wherein
the one or more regions are determined by performing the following
steps: (3a) extracting the genomic DNA; (3b) digesting the
extracted genomic DNA with the restriction enzyme; (3c) connecting
an adaptor to the genomic DNA fragments obtained in the step (3b);
(3d) amplifying the genomic DNA fragments using a primer capable of
hybridizing to the adaptor; (3e) digesting the amplified genomic
DNA fragment with another restriction enzyme; and (3f) separating
the DNA fragments obtained by digestion in the step (3e) as
probes.
30. The method for designing a probe according to claim 29,
wherein, in the step (3b), the genomic DNA is digested with more
than one restriction enzyme.
31. The method for designing a probe according to claim 30,
wherein, in the step (3c), an adaptor is connected corresponding to
one restriction enzyme selected from the more than one restriction
enzyme or corresponding to a part of the more than one restriction
enzyme used.
32. The method for designing a probe according to claim 29,
wherein, the adaptor has a complementary sequence to a protruding
end of the genomic DNA fragments obtained in the step (3b).
33. The method for designing a probe according to claim 21, wherein
the designed probe has a 20 to 100 nucleotide length.
34. A DNA microarray comprising a probe designed by the method for
designing a probe according to claim 21 and a carrier on which the
probe to be immobilized.
35. The DNA microarray according to claim 34, wherein the probe is
synthesized on the carrier based on the sequence data.
36. A method for detecting a mutation using a DNA microarray,
comprising the steps of: extracting a genomic DNA derived from an
organism to be tested; digesting the genomic DNA with a restriction
enzyme having the same recognition sequence as the restriction
enzyme used in the designing a probe immobilized on the DNA
microarray according to claim 34; connecting an adaptor to the
genomic DNA fragments obtained by the restriction enzyme treatment;
amplifying the genomic DNA fragments using a primer capable of
hybridizing to the adaptor; and detecting a hybrid of the genomic
DNA fragment with the probe by bringing the amplified genomic DNA
fragment into contact with the DNA microarray according to claim
34.
37. The method for detecting a mutation using the DNA microarray
according to claim 36, wherein, in the step of digesting the
genomic DNA, the genomic DNA is digested with more than one
restriction enzyme.
38. The method for detecting a mutation using a DNA microarray
according to claim 37, wherein, in the step of connecting an
adaptor is connected corresponding to one restriction enzyme
selected from the more than one restriction enzyme or corresponding
to a part of the more than one restriction enzyme are
connected.
39. The method for detecting a mutation using a DNA microarray
according to claim 36, wherein the adaptor has a complementary
sequence to a protruding end of the genomic DNA fragments obtained
in the step of digesting the genomic DNA with a restriction
enzyme.
40. The method for detecting a mutation using a DNA microarray
according to claim 36, wherein the organism to be tested is
different from the organism used in preparing the DNA microarray.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for designing a
probe used in a DNA microarray for detecting, for example, a
mutation in genomic DNA, a DNA microarray having a probe designed
by the method, and a method for detecting a mutation using the DNA
microarray.
BACKGROUND ART
[0002] Of the polymorphisms represented by a single nucleotide
polymorphism (SNP), there is a polymorphism that can be used as a
mutation characterizing a variation in a homogenous organism. More
specifically, a predetermined variation in a homogenous organism
can be distinguished from other variations by detecting and
identifying a specific mutation such as a polymorphism in genomic
DNA. Furthermore, a variation of an organism to be tested can be
specified by detecting and identifying the mutation.
[0003] As a method for detecting such a mutation in genomic DNA, a
method of directly determining a sequence of a mutation site, a
method of using a restriction enzyme fragment length polymorphism
(RFLP), a method of using an amplification fragment length
polymorphism (AFLP) and the like are known. In addition, a method
of analyzing a variation based on identification of a polymorphism
using a DNA microarray called as a DArT (Diversity Array
Technology) method (Nucleic Acids Research, 2001, Vol. 29, No. 4,
e25) is known.
[0004] A method for preparing a DNA microarray for use in the DArT
method is shown in FIG. 9. First, genomic DNA is extracted from a
predetermined organism species and fractionated with restriction
enzyme A and restriction enzyme B. Next, to the both ends of each
of the genomic DNA fragments obtained by the restriction enzyme
treatment, an adaptor is connected and each of the genomic DNA
fragments is cloned into a vector. Next, using a primer capable of
hybridizing with the adaptor, genomic DNA fragments are amplified
by PCR. Then, genomic DNA fragments amplified are spotted on a
substrate as a probe to prepare a DNA microarray.
[0005] Using the DNA microarray thus prepared, a variation of a
organism species to be tested can be analyzed. First, genomic DNA
is extracted from an organism to be tested, and fractionated with
restriction enzyme A and restriction enzyme B that are used for
preparing the DNA microarray. To the genomic DNA fragments, an
adaptor is connected similarly in the preparation of the DNA
microarray and the resultant fragments are amplified by PCR. The
amplified genomic DNA fragments are tagged with a fluorescent label
etc. and hybridized with the probe spotted on the DNA microarray.
Based on the presence or absence of hybridization of the labeled
genomic DNA fragment with the probe detected, a difference between
the predetermined organism species used in preparation of the DNA
microarray and the organism species to be tested can be
analyzed.
SUMMARY OF INVENTION
Technical Problem
[0006] According to the DArT method, the diversity of an organism
species can be determined in a genotype level in the genomic DNA by
using the DNA microarray prepared as mentioned above. However, the
DNA microarray prepared as mentioned above has a problem in that
the detection ability of a probe, which is defined as a region
flanked by restriction enzyme recognition sites, is not sufficient.
More specifically, even if a genomic DNA fragment derived from an
organism species to be tested contains a small mutation such as
SNP, the genomic DNA fragment may often hybridize with the probe of
the DNA microarray. In other words, the DArT method has a detection
limit, that is, detection cannot be made unless a mutation such as
a polymorphism is present in a restriction enzyme recognition site
or deletion of several hundreds of base pairs is present.
[0007] Then, in the aforementioned circumstances, the present
invention is directed to providing a method for designing a probe
of a DNA microarray having an excellent detection rate of a
polymorphism such as SNP contained in genomic DNA, a DNA microarray
having a probe designed by the method and a method for detecting a
mutation using the DNA microarray.
Solution to Problem
[0008] In the aforementioned circumstances, the present inventors
have made intensive studies and conceived a method for designing a
probe capable of detecting even a small mutation such as SNP in the
genomic DNA with an excellent sensitivity, and a method of
detecting a mutation by using a DNA microarray having the probe
immobilized thereto.
[0009] The present invention includes the followings.
[0010] More specifically, the method for designing a probe
according to the present invention including the steps of:
specifying one or more regions having a shorter nucleotide length
than fragments flanked by restriction enzyme recognition sites
recognized by a restriction enzyme, contained in genomic DNA
derived from a target organism, and covering at least one portion
of the genomic DNA fragments; and designing the specified one or
more regions as a probe for detecting the fragment in an organism
to be tested.
[0011] The one or more regions can be specified by performing the
following steps:
[0012] (1a) extracting the genomic DNA;
[0013] (1b) digesting the extracted genomic DNA with the
restriction enzyme;
[0014] (1c) connecting an adaptor to the genomic DNA fragments
obtained the step (1b);
[0015] (1d) amplifying the genomic DNA fragments using a primer
capable of hybridizing to the adaptor;
[0016] (1e) sequencing the amplified genomic DNA fragment; and
[0017] (1f) determining the one or more regions based on the
nucleotide sequence.
[0018] In the step (1b) herein, the genomic DNA may be digested
with one or more restriction enzymes. Furthermore, in the step
(1c), the adaptor used preferably has a complementary sequence to a
protruding end of the genomic DNA fragments obtained the step (1b).
Moreover, the region to be determined in the step (1f) has, for
example, a 20 to 10000 nucleotide length, preferably, a 100 to 8000
nucleotide length and more preferably, a 200 to 6000 nucleotide
length.
[0019] Furthermore, the one or more regions can be specified using
nucleotide sequence data on the genomic DNA by performing the
following steps:
[0020] (2a) searching the nucleotide sequence data on the genomic
DNA for the restriction enzyme recognition sequence to specify the
nucleotide sequence of the genomic DNA fragments obtained by
digesting the genomic DNA with the restriction enzyme; and
[0021] (2b) determining the one or more regions based on the
specified nucleotide sequence.
[0022] Herein, the region determined in the step (2b) has, for
example, a 20 to 10000 nucleotide length, preferably, a 100 to 8000
nucleotide length, and more preferably, a 200 to 6000 nucleotide
length.
[0023] Furthermore, the one or more regions can be determined by
performing the following steps:
[0024] (3a) extracting the genomic DNA;
[0025] (3b) digesting the extracted genomic DNA with the
restriction enzyme;
[0026] (3c) connecting an adaptor to the genomic DNA fragments
obtained in the step (3b);
[0027] (3d) amplifying the genomic DNA fragments using a primer
capable of hybridizing to the adaptor;
[0028] (3e) digesting the amplified genomic DNA fragment with
another restriction enzyme; and
[0029] (3f) separating the DNA fragments obtained by digestion in
the step (3e) as probes.
[0030] Furthermore, in the method for designing a probe according
to the present invention, a fragment flanked by the restriction
enzyme recognition sites may be a fragment flanked by more than one
restriction enzyme having different recognition sequences.
[0031] In the step (3b) herein, the genomic DNA may be digested
with one or more restriction enzymes. Furthermore, in the step
(3c), the adaptor used preferably has a complementary sequence to a
protruding end of the genomic DNA fragment obtained the step
(1b).
[0032] On the other hand, the DNA microarray according to the
present invention is prepared by immobilizing a probe designed by
the aforementioned method for designing a probe according to the
present invention on a carrier. Particularly, in the DNA microarray
according to the present invention, the probe is preferably
synthesized on a carrier based on the sequence data.
[0033] On the other hand, a method for detecting a mutation using
the DNA microarray according to the present invention is a method
of detecting a mutation in a genomic DNA derived from a target
organism to be tested by using the aforementioned DNA microarray
according to the present invention. Particularly, a mutation
detection method using the DNA microarray according to the present
invention includes the following steps:
[0034] extracting a genomic DNA derived from a target organism to
be tested;
[0035] digesting the genomic DNA with a restriction enzyme having
the same recognition sequence as the restriction enzyme used in the
method for designing a probe according to the present
invention;
[0036] connecting an adaptor to the genomic DNA fragments obtained
by the restriction enzyme treatment;
[0037] amplifying the genomic DNA fragments using a primer capable
of hybridizing to the adaptor; and
[0038] detecting a hybrid of the genomic DNA fragment with the
probe by bringing the amplified genomic DNA fragment into contact
with the DNA microarray according to the present invention.
[0039] Herein, in the step of digesting the genomic DNA with the
restriction enzyme, the genomic DNA may be digested with one or
more restriction enzymes similarly to the method for designing a
probe. Furthermore, in the step of connecting the adaptor, as the
adaptor, one having a complementary sequence to a protruding end of
the genomic DNA fragment obtained in the step of digesting the
genomic DNA with the restriction enzyme is preferably used.
Moreover, the step of amplifying the genomic DNA fragment may
further have a step of adding a labeling molecule to an amplified
genomic DNA fragment or may have a step of allowing the genomic DNA
fragment to incorporate a labeling molecule when the genomic DNA
fragment is amplified.
[0040] The specification of the present invention incorporates the
content described in the specification and/or drawings of JP
Application No. 2009-283430 A, based on which the priority of the
present application is claimed.
Advantageous Effects of Invention
[0041] According to the present invention, it is possible to
provide a method for designing a probe having an excellent
detection rate of a polymorphism such as SNP contained in genomic
DNA, for use in a DNA microarray. Furthermore, according to the
present invention, it is possible to provide a DNA microarray
having an excellent detection rate of a polymorphism such as an SNP
contained in a genomic DNA and a method for detecting a mutation by
use of the DNA microarray.
[0042] Application of the present invention enables to analyze,
i.e., determine and identify, an organism species based on a
genotype, although it has been difficult to detect it by a
conventional method.
BRIEF DESCRIPTION OF DRAWINGS
[0043] FIG. 1 is a flow chart schematically showing a method for
designing a probe to which the present invention is applied.
[0044] FIG. 2 is a flow chart schematically showing another method
for designing a probe to which the present invention is
applied.
[0045] FIG. 3 is a flow chart schematically showing further another
method for designing a probe to which the present invention is
applied.
[0046] FIG. 4 is a flow chart schematically showing a step of
detecting a mutation by using a DNA microarray having a probe
designed by applying the present invention.
[0047] FIG. 5 is a characteristic view showing alignment of
A.sub.--1 and A.sub.--2 and the site of the designed probe.
[0048] FIG. 6 is a characteristic view showing alignment of
B.sub.--1 and B.sub.--2 and the site of the designed probe.
[0049] FIG. 7 is a characteristic graph showing the relationship
between the rate of mutation introduced in a probe and the
intensity of a signal detected.
[0050] FIG. 8 is a characteristic graph showing the relationship
between the ratio of mutation site probe prepared in Example 3 and
the ratio of the sequence data in which a mutation was
detected.
[0051] FIG. 9 is a characteristic view schematically showing a step
of preparing a DNA microarray used in a conventional DArT
method.
DESCRIPTION OF EMBODIMENTS
[0052] Now, the method for designing a probe for use in the DNA
microarray according to the present invention, a DNA microarray
having a probe designed by the method for designing a probe, and a
method of detecting a mutation by use of the DNA microarray, will
be more specifically described, referring to the drawings.
Method for Designing a Probe
[0053] The probe to be designed in the present invention is
preferably applied to, particularly, a so-called oligonucleotide
microarray. The oligonucleotide microarray is a microarray, which
is prepared by synthesizing an oligonucleotide having a desired
nucleotide sequence on a carrier and using the oligonucleotide as a
probe. The synthesized oligonucleotide used as a probe has, for
example, a 20 to 100 nucleotide length, preferably a 30 to 90
nucleotide length, and more preferably a 50 to 75 nucleotide
length.
[0054] Note that, the probe designed in the present invention may
be applied to a microarray, which is prepared by spotting a
synthesized oligonucleotide having the aforementioned nucleotide
length onto a carrier, similarly to so-called Stanford-type
microarray.
[0055] More specifically, the probe designed in the present
invention can be applied to any microarray as long as it is
conventionally known. Therefore, the probe designed in the present
invention can be applied to a microarray using a flat substrate
formed of glass and silicone etc., as a carrier and to a beads
array using micro-beads as a carrier.
[0056] More specifically, in the method for designing a probe
according to the present invention, first, genomic DNA is extracted
from a predetermined organism, as shown in FIG. 1 (Step 1a). As the
organism, any one of a microorganism such as a bacterium and a
fungus, an insect, a plant and an animal may be used. Note that,
the method for designing a probe shown in FIG. 1 is preferably
applied to the case of using an organism whose genomic DNA
nucleotide sequence data has not yet been elucidated. Furthermore,
a method of extracting genomic DNA is not particularly limited and
a method known in the art can be used.
[0057] Next, the extracted genomic DNA is digested with one or more
restriction enzymes (Step 1b). In the example shown in FIG. 1,
genomic DNA is digested with two types of restriction enzymes,
restriction enzyme A and restriction enzyme B, which are
sequentially used in this order. The restriction enzymes used
herein are not particularly limited. For example, PstI, EcoRI,
HindIII, BstNI, HpaII and HaeIII can be used. Particularly, the
restriction enzyme can be appropriately selected in consideration
of an appearance frequency of a recognition sequence such that
genomic DNA is completely digested into genomic DNA fragments
having a 20 to 10000 nucleotide length. Furthermore, in the case
where more than one restriction enzyme is used, it is preferred
that after all restriction enzymes are applied, the genomic DNA
fragments of a 200 to 6000 nucleotide length remain. Moreover, when
more than one restriction enzyme is used, the order of supplying
restriction enzymes to treatment is not particularly limited.
Furthermore, when common treatment conditions (solution
composition, temperature etc.,) are used, more than one restriction
enzyme may be used in the same reaction system. To describe more
specifically, in the example shown in FIG. 1, genomic DNA is
digested by using restriction enzyme A and restriction enzyme B in
this order; however, restriction enzyme A and restriction enzyme B
may be simultaneously used in a same reaction system to digest
genomic DNA. Alternatively, restriction enzyme B and restriction
enzyme A may be used in this order to digest genomic DNA.
Furthermore, the number of restriction enzymes to be used may be 3
or more.
[0058] Next, to genomic DNA fragments treated by the restriction
enzyme, an adaptor is connected (Step 1c). The adaptor herein is
not particularly limited as long as it can connect to both ends of
each of the genomic DNA fragments obtained by the aforementioned
restriction enzyme treatment. As the adaptor, for example, one
having a single strand complementary to a protruding end (sticky
end) formed at both ends of genomic DNA fragments by restriction
enzyme treatment, and having a primer binding sequence to which a
primer to be used in the amplification treatment (specifically
described later) can hybridize, can be used. Furthermore, as the
adaptor, one having a single strand complementary to the protruding
end (sticky end) and having a restriction enzyme recognition site
for use in cloning into a vector.
[0059] Furthermore, in the digestion of the genomic DNA with more
than one restriction enzyme, more than one adaptor can be prepared
for use corresponding to the restriction enzymes. More
specifically, in the digestion of the enomic DNA with more than one
restriction enzyme, more than one protruding end generates. To
correspond to the more than one protruding end, more than one
adaptor having a single strand complementary thereto can be used.
At this time, the more than one adaptor corresponding to the more
than one restriction enzyme may have a common primer binding
sequence such that a common primer can hybridize or may have
different primer binding sequences such that different primers can
hybridize.
[0060] Furthermore, in the digestion of genomic DNA with more than
one restriction enzyme, an adaptor can be prepared for use
corresponding to one restriction enzyme selected from the more than
one restriction enzyme used or corresponding to a part of the
restriction enzymes used.
[0061] Next, a genomic DNA fragment having an adaptor added to the
both ends is amplified (Step 1d). When the adaptor having a primer
binding sequence is used, the genomic DNA fragment can be amplified
by use of a primer capable of hybridizing with the primer binding
sequence. Alternatively, the genomic DNA fragment having an adaptor
added thereto is cloned into a vector by use of the adaptor
sequence. In this case, the genomic DNA fragment can be amplified
by use of a primer capable of hybridizing to a predetermined region
of the vector. Note that, as an amplification reaction of a genomic
DNA fragment by use of a primer, for example, PCR can be used.
[0062] Furthermore, in the case where genomic DNA is digested with
more than one restriction enzyme and more than one adaptor
corresponding to the restriction enzymes are connected to the
genomic DNA fragments, the adaptors will be connected to all
genomic DNA fragments obtained by treatment using more than one
restriction enzyme. In this case, all genomic DNA fragments
obtained can be amplified by a nucleic acid amplification reaction
using a primer binding sequence contained in each of the
adaptors.
[0063] Alternatively, in the digestion of the genomic DNA with more
than one restriction enzyme and the connection to the genomic DNA
fragment an adaptor corresponding to one restriction enzyme
selected from the more than one restriction enzyme used or
corresponding to a part of the restriction enzymes used, only a
genomic DNA fragment, of the obtained genomic DNA fragments, having
a recognition sequence of the selected restriction enzyme at both
ends can be amplified.
[0064] Next, the amplified genomic DNA fragment is sequenced (Step
1e), one or more regions having a shorter nucleotide length than
the genomic DNA fragments and covering at least a part of the
genomic DNA fragments are specified; and a probe for the one or
more regions specified are designed for detecting the amplified
genomic DNA fragment of an organism to be tested (Step 1f). A
method for sequencing a genomic DNA fragment is not particularly
limited. A method known in the art employing the Sanger method etc.
and a DNA sequencer can be used.
[0065] In the steps (Steps 1e and 1f), one or more regions having a
shorter nucleotide length than the amplified genomic DNA fragments
are designed as a probe(s) for detecting the genomic DNA fragment.
Herein, in the case where more than one region of a predetermined
genomic DNA fragment are used for designing, detection of the
genomic DNA fragment using more than one probe is intended.
Furthermore, a single region may be selected from a genomic DNA
fragment and designed, whereas a predetermined number (two or more)
of regions may be selected from another genomic DNA fragment and
designed. In short, the number of regions to be designed may differ
from one genomic DNA fragment to another. As the regions to be
designed herein, those having, for example, a 20 to 10000
nucleotide length, preferably, a 100 to 8000 nucleotide length, and
more preferably, a 200 to 6000 nucleotide length are used, as
mentioned above. Furthermore, in the case where more than one
region is designed, the adjacent regions may be overlapped with
each other or may have an interval of several nucleotides between
them.
[0066] Particularly, more than one region is preferably set so as
to cover the entire region of the sequenced genomic DNA fragment.
In this case, more than one probe responds to a genomic DNA
fragment obtained by a restriction enzyme treatment from genomic
DNA derived from a predetermined organism to detect the genomic DNA
fragment by these more than one probe.
[0067] In the meantime, the method for designing a probe according
to the present invention is not limited to a method including a
step of digesting genomic DNA with a restriction enzyme(s) as
mentioned above, and genomic data of a target organism, as shown in
FIG. 2 may be used.
[0068] In the method shown in FIG. 2, first, nucleotide sequence
data on the genome derived from a target organism is obtained (Step
2a). The nucleotide sequence data on the genome can be obtained
from various types of data bases known in the art. The data base is
not particularly limited; however, DDBJ data base provided by the
DNA Data Bank of Japan, EMBL data base provided by the European
Bioinformatics Institute, Genbank data base provided by the
National Center for Biotechnology Information, the KEGG data base
provided by the Kyoto Encyclopedia of Genes and Genomes or data
base integrated of these data bases can be appropriately used.
[0069] In this method, next, the nucleotide sequence data on the
genomic DNA obtained is searched for the recognition sequence of
the restriction enzyme(s) as mentioned above (Step 2a). The
nucleotide sequences of genomic DNA fragments which will be
obtained by digesting the aforementioned genomic DNA with the
aforementioned restriction enzyme(s) are specified. The recognition
sequence to be searched for herein is a restriction enzyme(s)
corresponding to the restriction enzyme(s) used in the method shown
in FIG. 1. More specifically, in this step, recognition sequences
of one or more restriction enzymes are searched for.
[0070] Next, based on the nucleotide sequence of the determined
genomic DNA fragment, one or more regions covering at least a part
of the genomic DNA fragment are determined (Step 2b). In the step
(Step 2b), one or more regions having a shorter nucleotide length
than the sequenced genomic DNA fragments are designed as a probe
for detecting the genomic DNA fragment. Herein, if more than one
region is designed for a predetermined genomic DNA fragment,
detection of the genomic DNA fragment using more than one probe is
intended. Furthermore, a single region may be selected from a
genomic DNA fragment and designed, whereas a predetermined number
(2 or more) of regions may be selected from another genomic DNA
fragment and designed. In short, the number of regions to be
designed may differ from one genomic DNA fragment to another. As
the region to be designed herein, those having, for example, a 20
to 100 nucleotide length, preferably a 30 to 90 nucleotide length,
and more preferably a 50 to 75 nucleotide length are used, as
mentioned above.
[0071] Furthermore, more than one region is preferably set so as to
cover the entire region of the sequenced genomic DNA fragment. In
this case, more than one probe responds to genomic DNA fragments
obtained by a restriction enzyme treatment from genomic DNA derived
from a predetermined organism to detect the genomic DNA fragment by
these more than one probe.
[0072] In the meantime, the method for designing a probe according
to the present invention may be a method having neither a step of
sequencing nor a step of obtaining nucleotide sequence data using a
database and including a step of digesting the genomic DNA fragment
with a further different restriction enzyme, as shown in FIG. 3. To
describe more specifically, in the method shown in FIG. 3, first,
step 1a to step 1d of the method shown in FIG. 1 are carried out to
amplify a genomic DNA fragment having an adaptor attached to the
both ends (Step 3a to 3d). Then, the amplified genomic DNA fragment
is digested with a restriction enzyme (hereinafter, restriction
enzyme C) having a different recognition sequence from the
restriction enzymes used in step 3b (Step 3e). Owing to this step,
the PCR fragment amplified in step 3d is digested into further
shorter fragments.
[0073] In this manner, more than one region covering at least a
part of the genomic DNA fragments obtained by digesting genomic DNA
with restriction enzyme A and restriction enzyme B can be specified
by more than one DNA fragment without sequencing. As the region to
be specified, those having, for example, a 20 to 100 nucleotide
length, preferably a 30 to 90 nucleotide length, and more
preferably, a 50 to 75 nucleotide length are mentioned, as
mentioned above. In other words, as restriction enzyme C, one
capable of cleaving a genomic DNA fragment obtained by digesting
genomic DNA with restriction enzyme A and restriction enzyme B into
DNA fragments having, for example, a 20 to 100 nucleotide length,
preferably a 30 to 90 nucleotide length, and more preferably a 50
to 75 nucleotide length can be used.
[0074] Next, the DNA fragments obtained by digestion with
restriction enzyme C are separated from type to type to obtain
probes (Step 3f). In this step, the DNA fragments obtained by
digesting with restriction enzyme C can be separated by
electrophoresis, followed by cutting out. Furthermore, the
separated DNA fragment may be further cloned into a vector and used
as a probe, or after cloning, may be further amplified and used as
a probe. Also in this method, more than one probe responds to the
genomic DNA fragments obtained in step 3d from genomic DNA derived
from a predetermined organism.
DNA Microarray
[0075] The DNA microarray having a probe designed as mentioned
above can be prepared by a method known in the art. For example, a
DNA microarray having a probe designed by the method shown in FIG.
1 or FIG. 2 can be prepared by synthesizing an oligonucleotide
having a desired nucleotide sequence on a carrier based on the
nucleotide sequence of each probe designed by the method shown in
FIG. 1 or FIG. 2. Herein, the method for synthesizing an
oligonucleotide is not particularly limited and a method known in
the art can be applied. For example, a method of synthesizing an
oligonucleotide on a carrier by a photolithographic technology in
combination with a light irradiation chemosynthesis technique can
be applied. As another method that can be applied, an
oligonucleotide having a linker molecule, which has a high affinity
for a carrier surface, on an end may be separately synthesized
based on the nucleotide sequence data of each probe designed by the
method shown in FIG. 1 or FIG. 2, and thereafter immobilized at a
predetermined position on the carrier surface.
[0076] Furthermore, the probe designed and prepared by the method
shown in FIG. 3 is immobilized on a carrier to prepare a DNA
microarray having a probe designed by the method shown in FIG. 3.
In this case, for example, the probe designed by the method shown
in FIG. 3 is spotted on a carrier by a pin type arrayer and a
nozzle type arrayer to prepare a DNA microarray.
[0077] The DNA microarray prepared as described above has one or
more probes having a nucleotide length shorter than the genomic DNA
fragment, which is obtained by a restriction enzyme treatment of
genomic DNA derived from a predetermined organism. More
specifically, the DNA microarray prepared as described above is
used in detecting a predetermined genomic DNA fragment by one or
more probes having a nucleotide length shorter than the genomic DNA
fragment. Particularly, the DNA microarray preferably has more than
one probe to a predetermined genomic DNA fragment to detect the
genomic DNA fragment by the more than one probe.
[0078] Note that, as the DNA microarray, any type of microarray may
be used such as a microarray using a flat surface substrate formed
of e.g., glass or silicone as a carrier, a beads array using micro
beads as a carrier or a three dimensional microarray having a probe
immobilized on the inner surface of a hollow fiber.
Method for Detecting Mutation
[0079] A mutation present in genomic DNA can be detected by using
the DNA microarray prepared as described above. The mutation herein
refers to a polymorphism such as a single polymorphism present
between homogenous organisms, a variation of a nucleotide sequence
present between related species or a mutation artificially
introduced into a predetermined organism.
[0080] More specifically, first, a genomic DNA is extracted from an
organism to be tested, as shown in FIG. 4. The organism to be
tested herein is an organism to be compared to the organism used in
preparing the DNA microarray. Then, the extracted genomic DNA is
digested with the restriction enzyme used in preparing the DNA
microarray to prepare more than one genomic DNA fragment.
Subsequently, the obtained genomic DNA fragments are connected to
the adaptor used in preparing the DNA microarray. Next, the genomic
DNA fragment having an adaptor attached to the both ends is
amplified by use of a primer used in preparing the DNA microarray.
In this manner, the genomic DNA derived from an organism to be
tested, which corresponds to the genomic DNA fragment amplified in
step 1d for preparing the DNA microarray, the genomic DNA fragment
whose nucleotide sequence is specified in step 2a, and the genomic
DNA fragment amplified in step 3d, can be amplified.
[0081] In this step, of the genomic DNA fragments having an adaptor
added thereto, a predetermined genomic DNA fragment may be
selectively amplified. For example, in the case where more than one
adaptor is used so as to correspond to more than one restriction
enzyme, the genomic DNA fragment having a specific adaptor added
thereto can be selectively amplified. Furthermore, of the genomic
DNA fragments obtained by digesting genomic DNA with more than one
restriction enzyme, only to a genomic DNA fragment having a
protruding end corresponding to a predetermined restriction enzyme,
an adaptor is added. In this manner, a genomic DNA fragment having
the adaptor added thereto can be selectively amplified. Likewise, a
predetermined genomic DNA fragment can be concentrated by
selectively amplifying it.
[0082] Next, a label is added to the amplified genomic DNA
fragment. As the label, any substance may be used as long as it is
known in the art. As the label, for example, a fluorescent
molecule, a pigment molecule and a radioactive molecule can be
used. Note that, this step may be omitted by performing the step of
amplifying a genomic DNA fragment by using nucleotides having a
label. This is because an amplified DNA fragment is labeled by
amplifying the genomic DNA fragment by use of a nucleotide having a
label in above step.
[0083] Next, the genomic DNA fragment having a label is brought
into contact with a DNA microarray under predetermined conditions
to hybridize the probe immobilized to the DNA microarray with the
genomic DNA fragment having a label. At this time, the probe partly
hybridizes with the genomic DNA fragment under highly stringent
conditions under which the probe does not hybridize if a single
nucleotide mismatch is present but only hybridizes if the
nucleotides completely match with each other. Under such highly
stringent conditions thus employed, a small mutation such as a
single polymorphism can be detected.
[0084] Note that, the stringent conditions can be controlled by a
reaction temperature and a salt concentration. More specifically,
further higher stringent conditions can be obtained by increasing
the temperature and further higher stringent conditions can be
obtained by reducing a salt concentration. For example, when a
probe having a 50 to 75 nucleotide length is used, further higher
stringent conditions are prepared if conditions of 40 to 44.degree.
C., 0.21SDS, 6.times.SSC are employed.
[0085] Furthermore, hybridization between the probe and the genomic
DNA fragment having a label can be detected based on the label.
More specifically after a hybridization reaction between the
aforementioned genomic DNA fragment having a label and a probe,
unreacted genomic DNA fragments etc. were washed away. Thereafter,
the label of the genomic DNA fragment specifically hybridized to
the probe is observed. For example, in the case where the label is
a fluorescent substance, the fluorescent wavelength is detected. In
the case where the label is a pigment molecule, the wavelength of
the pigment is detected. More specifically, a fluorescent detection
apparatus and an image analyzer etc., usually used for DNA
microarray analysis, can be used.
[0086] Particularly, if the aforementioned DNA microarray is used,
the genomic DNA fragment derived from an organism to be tested is
detected by one or more probes having a shorter nucleotide length
than the genomic DNA fragments. In a conventional DArT method (FIG.
9), since a genomic DNA fragment derived from a predetermined
organism is amplified by PCR and used as a probe, even if a genomic
DNA fragment derived from an organism to be tested having a
mismatch of several tens of nucleotides, the probe often hybridized
with it (pseudo-positive reaction). However, in the aforementioned
DNA microarray, since detection was made by use of one or more
probes having a shorter nucleotide length than the genomic DNA
fragments, an incident probability of such a pseudo-positive
reaction can be reduced, with the result that a genomic DNA
fragment derived from an organism to be tested can be highly
accurately detected. Particularly, when a genomic DNA fragment
derived from an organism to be tested is detected by more than one
probe, a small mutation contained in a genomic DNA fragment derived
from an organism to be tested can be detected by detecting the
presence or absence of hybridization in more than one probe.
[0087] Furthermore, in the aforementioned DNA microarray, an
unknown mutation can be detected. In a conventional DNA microarray
using an oligonucleotide synthesized on a carrier as a probe for
mutation detection, a detection target is only a known mutation
having known sequence data. However, according to the
aforementioned method for designing a probe, even if a genomic DNA
fragment contains a mutation whose sequence has not yet been found,
such an unknown mutation can be a target of detection. In other
words, an unknown mutation can be found by use of the DNA
microarray having the aforementioned probe.
[0088] As described in the foregoing, according to the DNA
microarray of the present invention, since a mutation contained in
the genomic DNA of an organism to be tested can be detected in
comparison with that of a predetermined organism used in preparing
the DNA microarray, for example, diversity in homogeneous organisms
can be analyzed at a gene level. Furthermore, if the DNA microarray
according to the present invention is prepared with respect to
various types of variants contained in homogenous organism, which
variant an organism to be tested belongs to can be analyzed at a
gene level.
EXAMPLES
[0089] Now, the present invention will be more specifically
described by way of Examples. The technical range of the present
invention is not limited by the following Examples.
Example 1
[0090] In this Example, it was shown that a mutation present in an
allele of each of sugar cane varieties NiF8 and Ni9 can be detected
by designing a probe in accordance with the procedure shown in FIG.
1 without using the whole sequence data or mutation data.
[0091] (1) Material
[0092] Sugar cane varieties NiF8 and Ni9 were used.
[0093] (2) Treatment with Restriction Enzyme
[0094] Genomic DNA was extracted separately from sugar cane
varieties NiF8 and Ni9 in accordance with a customary method.
Genomic DNA (750 ng) was treated with restriction enzyme PstI (NEB
Inc. 25 units) at 37.degree. C. for 2 hours, followed with
restriction enzyme BstNI (NEB Inc., 25 units) at 60.degree. C. for
2 hours.
[0095] (3) Adaptor Ligation
[0096] To the genomic DNA fragment (120 ng) treated in the step
(2), PstI sequence adaptors (5'-CACGATGGATCCAGTGCA-3' (SEQ ID NO:
1), 5'-CTGGATCCATCGTGCA-3' (SEQ ID NO: 2)) and T4 DNA Ligase (NEB
Inc., 800 units) were added and a treatment was performed at
16.degree. C., all night and all day. In this manner, the adaptor
was selectively added to the genomic DNA fragment having a PstI
recognition sequence at the both ends, among those treated in the
step (2).
[0097] (4) PCR Amplification
[0098] To the genomic DNA fragments (15 ng) having an adaptor
obtained in the step (3), a PstI sequence adaptor recognizing
primer (5'-GATGGATCCAGTGCAG-3' (SEQ ID NO: 3)) and Taq polymerase
(company TAKALA, PrimeSTAR, 1.25 units) were added and genomic DNA
fragments were amplified by PCR (a cycle consisting of 10 seconds
at 98.degree. C., 15 seconds at 55.degree. C., and 1 minute at
72.degree. C. was repeated 30 times and the PCR sample was treated
at 72.degree. C. for 3 minutes and stored at 4.degree. C.).
[0099] (5) Acquisition of Genomic Sequence
[0100] The genomic DNA fragment amplified by PCR in the step (4)
was analyzed by the Sanger method for sequencing. As a result, 2
types of genomic sequence data (A.sub.--1 (SEQ ID NO: 4) and
B.sub.--1 (SEQ ID NO: 5)) derived from NiF8 were obtained.
Furthermore, genomic sequence data (A.sub.--2 (SEQ ID NO: 6) and
B.sub.--2 (SEQ ID NO: 7)) of locus region of Ni9 allele were
obtained by use of sequence data of genomic sequence A.sub.--1 and
B.sub.--1.
[0101] (6) Probe Designing
[0102] Based on the genomic sequence data (A.sub.--1, B.sub.--1) of
the step (5), 5 and 6 probes of a 50 to 70 bp were separately
designed. More specifically, in this Example, a probe of sugar cane
variety NiF8 was designed. A.sub.--1 and A.sub.--2 alignments and
the position of the designed probe are shown in FIG. 5.
Furthermore, B.sub.--1 and B.sub.--2 alignments and the position of
the designed probe are shown in FIG. 6.
[0103] (7) Preparation of Array
[0104] Based on the nucleotide sequence data of the designed
probes, the DNA microarrays having these probes were prepared
(outsource to Roche).
[0105] (8) Sample Preparation
[0106] Fragments from sugar cane varieties NiF8 and Ni9 were
separately amplified by PCR in accordance with the aforementioned
methods (2) to (4). PCR amplification fragments were purified by a
column (company, Qiagen), and thereafter, Cy3-labeled 9mers
(TriLink Inc., 1O.D.) was added. The mixture was treated at
98.degree. C. for 10 minutes and allowed to stand still on ice for
10 minutes. Thereafter, Klenow (NEB Inc., 100 units) was added. The
mixture was treated at 37.degree. C. for 2 hours and then
precipitated with ethanol to prepare a labeled sample.
[0107] (9) Detection of Hybridization Signal
[0108] Hybridization was performed by use of the DNA microarray
prepared in the step (7) and using the labeled sample of the step
(8) in accordance with the NimbleGen Array User's Guide to detect a
signal derived from the label.
[0109] (10) Calculation of Mutation Rate
[0110] A mutation rate was calculated based on homology of the
genome sequence of the loci regions of NiF8 and Ni9 alleles within
respective probes.
[0111] (11) Calculation of signal intensity ratio
[0112] The signal intensity ratio is obtained by dividing the
signal intensity of array using NiF8 as a sample by the signal
intensity of the array using Ni9 as a sample.
[0113] (12) Results and Discussion
[0114] The measurement results of signal intensity and the signal
intensity ratio calculated from the results are shown in Table 1
and Table 2.
TABLE-US-00001 TABLE 1 probe signal length Mutation rate Signal
intensity intensity (bp) (%) NiF8 Ni9 ratio PA_1 60 91.7% 2,304 225
10.2 PA_2 50 54.0% 1,318 249 5.3 PA_3 65 0.0% 4,837 4,554 1.1 PA_4
58 3.4% 1,738 894 1.9 PA_5 60 0.0% 4,240 3,075 1.4
TABLE-US-00002 TABLE 2 Signal Probe length Mutation rate Signal
intensity intensity (bp) (%) NiF8 Ni9 ratio PB_1 69 4.3% 1,921 298
6.4 PB_2 69 10.1% 3,398 272 12.5 PB_3 70 5.7% 541 247 2.2 PB_4 50
30.0% 608 209 2.9 PB_5 52 1.9% 1,463 902 1.6 PB_6 70 1.4% 2,807
2,665 1.1
[0115] From FIG. 5, it was found that a single insertion/deletion
mutation of 101 bp and three mutations of 1 to several-bases
mutation are present between A.sub.--1 and A.sub.--2. From Table 1,
it was found that, in a probe (PA.sub.--3 and PA.sub.--5, mutation
rate 0%) having no mutation between NiF8 and Ni9, high signal
intensity was detected in each of NiF8 and Ni9. This means that
A.sub.--1 and A.sub.--2 sequences corresponding to the sequence
data are present respectively in the samples of NiF8 and Ni9.
Furthermore, since the signal intensity ratio of both samples is as
low as 1.1 to 1.4, signal intensity ratio of a probe having no
mutation was low.
[0116] On the other hand, as a mutation rate increases, the signal
intensity ratio of both samples increased (1.9 (PA.sub.--4) to 10.2
(PA.sub.--1)). This is because, A.sub.--2 sequence is present in
the Ni9 sample but a mutation is present in A.sub.--2 sequence,
which corresponds to PA.sub.--1, PA.sub.--2, and PA.sub.--4 probes,
with the result that hybridization strength decreases and the
signal of Ni9 decreases.
[0117] Similarly, from FIG. 6, it is found that three
insertion/deletion mutations and 14 SNPs are present between
B.sub.--1 and B.sub.--2. Also with respect to B.sub.--1 and
B.sub.--2, a signal intensity ratio increases as a mutation rate
increases (1.1 (PB.sub.--6) to 12.5 (PB.sub.--2)) as is apparent
from Table 2.
[0118] From the above results, it was demonstrated that DNA
mutation of a several-bp level can be detected and the site of a
mutation of several-tens of nucleotides can be specified by using a
probe having a nucleotide length shorter than a genomic DNA
fragment serving as a sample.
Example 2
[0119] In this Example, to the probe derived from NiF8 prepared in
Example 1, a mutation was artificially introduced. Based on the
mutation introduction rate and the signal intensity ratio thereof
to an original probe, the mutation detection ability was
evaluated.
[0120] (1) Material
[0121] Sugar cane variety NiF8 was used.
[0122] (2) Acquisition of Basic Probe Sequence Data
[0123] A PCR amplification fragment of NiF8 was prepared in
accordance with the steps (2) to (4) of Example 1 and the genomic
sequence was determined by the Sanger method. Based on independent
genomic sequence data, 6 basic probes having a 50 to 75 bp were
prepared (Table 3).
TABLE-US-00003 TABLE 3 Sequence Probe Sequence length PC_1
gccgtcgctcacaaggaccaacgaacggaaaggcatgcatgcagag 64 agtt (SEQ ID NO:
8) PC_2 tatgagctatatgtaatgtaagtgtactactctcctgtcaccttgc 71
acttgacagca (SEQ ID NO: 9) PC_3
cctctctttgctccgaaattggtcatgtactcatgttatatgcaat 78 atatacggagtagtact
(SEQ ID NO: 10) PC_4 tcagaaacgcaacattctgcactctgattttactatatgcatcgct
tctcattttactgacttg (SEQ ID NO: 11) 79 PC_5
aagtaatgttatcaatcggcaaatcaaatatggccagaatcaacat 88
aagaaactgagatttggcacagaaatg (SEQ ID NO: 12) PC_6
ttcatctacatttagtactccatgcatatatcgcaagtttgatgtg 90
acggaaatcttttgtttgcacaatacttt (SEQ ID NO: 13)
[0124] (3) Preparation of Mutation Probe
[0125] Probes were prepared by separately inserting, deleting and
substituting with 1, 2, 3, 4, 5, 10, 15, 20 and 25 nucleotides
into, from and for the basic probes of the step (2).
[0126] (4) Array Preparation, Labeling, Hybridization-Signal
Detection
[0127] A DNA microarray was prepared in the same manner as in the
steps (7) to (9) of Example 1. A sample was prepared and a
hybridization reaction and the following signal detection were
performed.
[0128] (5) Calculation of Signal Intensity Ratio
[0129] The value of the signal intensity ratio was obtained by
dividing the signal intensity of a mutation probe by the basic
probe signal intensity. A graph and an approximation curve were
prepared by Excel 2007.
[0130] (6) Results and Discussion
[0131] The relationship between the mutation rate introduced into a
probe and the signal intensity detected is shown in FIG. 7. As
shown in FIG. 7, the mutation rate of a probe and the signal
intensity ratio are highly correlated (y=0.0804x-0.518, R2=0.8068).
From the correlation, it was found that a signal intensity ratio
tends to reduce to 50% or less at a mutation rate of 3% or more.
Even if there is a 1 bp mutation, the signal intensity ratio
decreases up to less than 50% depending upon the probe. From the
above results, it was demonstrated that mutation of a single to
several nucleotides or more can be highly accurately detected by a
probe having a nucleotide length shorter than the genomic DNA
fragment.
Example 3
[0132] In this Example, using sugar cane varieties NiF8 and Ni9
genomic sequence data (5,848 nucleotides), 5 to 15 probes
consisting of several tens of bps were prepared for each genomic
sequence datum and detection of a mutation between both samples was
carried out.
[0133] (1) Material
[0134] Sugar cane varieties NiF8 and Ni9 were used.
[0135] (2) Acquisition of Genomic Sequence Data
[0136] Fragments of NiF8 and Ni9 were amplified by PCR according to
the steps (2) to (4) of Example 1 and analyzed by the Sanger method
to obtain the genomic sequence data. More specifically genomic
sequence data of 5,848 PCR amplification fragments were
obtained.
[0137] (3) Preparation of Probe
[0138] Five to fifteen probes each having 50 to 75 bp were designed
based on the genomic sequence data obtained in the step (2). More
specifically, based on the genomic sequence data (5,848 data),
59,462 probes were designed.
[0139] (4) Array Preparation, Labeling, Hybridization-Signal
Detection
[0140] A DNA microarray was prepared in accordance with the steps
(7) to (9) of Example 1. A sample was prepared and a hybridization
reaction and the following signal detection were performed
[0141] (5) Detection of Mutation-Site Probe
[0142] When a signal intensity ratio of an array using Ni9 as a
sample to an array using NiF8 as a sample is twice or more or 1/2
or less, the probe was determined as a mutation-site probe.
[0143] (6) Ratio of Mutation-Site Probe Per Sequence Data
[0144] A value obtained by dividing the number of mutation-site
probes per sequence datum by the number of probes prepared per
sequence datum, was used.
[0145] (7) Results and Discussion
[0146] 59,462 probes were designed from 5,848 genomic sequence
data. Of them, the number of probes in the case where a signal
intensity ratio was beyond 2, was 5,596. Sequence data having at
least one of such a probe was 1,497. Of these sequence data, the
number of data providing a signal intensity ratio of 2 or more in
all probes were 189, which was 12.6% of the total (FIG. 8). It was
considered that mutation within the sequence data is caused by a
large insertion/deletion of several kbp within a restriction enzyme
recognition sequence. On the other hand, the sequence data in which
a mutation was detected in a part of probes was 87.4% of all data.
This is because an ability to detect a mutation is improved by
designing more than one probe of several tens of bps in the
interior. From the above results, in all probes, the sequence data
in which a mutation of this time was detected is 7.9 fold as large
as the sequence data providing a signal intensity ratio of 2 fold
or more. Thus, it was clearly demonstrated that the ability to
detect a mutation improves by designing more than one probe having
several tens of bps, which is shorter than a genomic DNA fragment
serving as a sample.
Example 4
[0147] In this Example, to validate availability of a DNA
microarray having a probe designed based on known sequence data of
another organism, a DNA microarray having a probe designed based on
the total sequence data of Sorghum was prepared and a mutation of
sugar cane genomic DNA was detected.
[0148] (1) Material
[0149] Sugar cane varieties NiF8 and Ni9 were used.
[0150] (2) Acquisition of Sorghum genomic sequence data from genome
DB
[0151] From Sorghum total genomic sequence data of genome DB
(Gramene: http://www.gramene.org/), sequence data between PstI
recognition sequences were obtained.
[0152] (3) Preparation of Probe
[0153] Based on the sequence data of step (2), a probe having 50 to
75 bp was designed.
[0154] (4) Array preparation, labeling, hybridization-signal
detection
[0155] A DNA microarray was prepared in accordance with the steps
(7) to (9) of Example 1. A sample was prepared and a hybridization
reaction and the following signal detection were performed.
[0156] (5) Calculation of the Number of Mutation-Site Probes
[0157] When a signal intensity ratio of an array using Ni9 as a
sample to an array using NiF8 as a sample is twice or more or 1/2
or less, the probe was determined as a mutation-site probe.
[0158] (6) Results and Discussion
[0159] In this Example, 1,744,104 probes were designed based on
Sorghum genomic sequence data, as shown in Table 4.
TABLE-US-00004 TABLE 4 Number of probes Total number of test Signal
Detection number Chromosome samples (1,000 or more) of mutations
Chr. 1 215,534 14,988 3,959 Chr. 2 191,280 12,627 3,383 Chr. 3
214,387 13,138 3,629 Chr. 4 183,499 10,658 2,794 Chr. 5 161,513
6,810 1,952 Chr. 6 164,830 8,830 2,330 Chr. 7 161,463 6,846 1,959
Chr. 8 138,922 5,819 1,656 Chr. 9 153,484 7,426 1,930 Chr. 10
159,192 8,278 2,155 All 1,744,104 95,420 25,747
[0160] Of them, the number of sequence data having a probe
providing a signal intensity of 1,000 or more was 95,420. The ratio
of this to the number of sequence data used was 4.2% to 7.0% per
Sorghum chromosome. In total, it was 5.5%. From the results, it was
considered that a homologous region to these probe sequences is
present each in sugar cane NiF8 and Ni9. Furthermore, of these
probes, the number of probes in the case where a signal intensity
ratio was beyond 2, in NiF8 and Ni9, was 25,747. It was 1.2% to
1.8% per chromosome of the test probes. In total, it was 1.5%. In
the region of a probe providing a signal intensity ratio exceeding
2, it is considered that a mutation is present between NIF8 and
Ni9. From the results in the foregoing, it is clearly demonstrated
that designing a probe by use of genome information of another
organism can be used for analyzing gene mutation in a predetermined
organism.
[0161] All publications and patents and patent applications cited
in the specification are incorporated by reference in its entirety.
Sequence CWU 1
1
14118DNAArtificial SequenceSynthetic DNA 1cacgatggat ccagtgca
18216DNAArtificial SequenceSynthetic DNA 2ctggatccat cgtgca
16316DNAArtificial SequenceSynthetic DNA 3gatggatcca gtgcag
164260DNASaccharum officinarum 4aatacccctc tctaggcttt ggaattgtgc
tgtgatgata aaatgaatgt gatgcaaatg 60ctcatgcttt ggaattagag cctttcagtc
ctgagctagg taggctttac tagctgttat 120tgtttctttc ctattgctta
tttcgagacc agtatcccta agagtggcat tttttttctg 180cccctaagag
agtacattca tgtgtcttgt gatgtaacaa atcacgtgtt ccttcgctaa
240aataaatatg catggtcctc 2605320DNASaccharum officinarum
5acccgttatt atcatatgtt tactgtagca caatattgtc taattacgga ctgattaggc
60tcaaaaaaat catctcgcaa aatatacgca atttgtgtaa ttaattattt tttagtctac
120atttaatact tcatatgtgt gtcaaacatc cgatgtgata gggtagggga
gaaactaaaa 180gtcccaccat tatttcgtac cagtgaagct gacgcatctt
aattgcttct gaccaaatgt 240ttagtagcag cagtactatc atattcttcc
tgctgctcat aatatgattt tgtcttgcat 300attttcagga gactactgag
3206192DNASaccharum officinarum 6aatatctggg agcctttcag tcctgagcta
ggtaggcttt actagctgtt attgtttctt 60tcctattgct tatttcgaga ccagtatccc
taagagtggc atttttttgc tgcccctaag 120agagcacatt catgtgtctt
gtgatgtaac aaatcacgtg ttccttcgct aaaataaata 180tgcatggtcc tc
1927331DNASaccharum officinarum 7acctgttatt atcatatgtt tactgtagca
caacattgtc taattacgga ctaattaggc 60tcaaaaaaat cgtctagcaa aatacacgca
atctgtgcaa ttaattattt ttttagtcta 120catttaatac ttcatacgtg
tatcaaacat ccgatgtgat agggtagagg aggaactaaa 180caagtcctta
gttgccagca ttatttcgta ccagtgaagc tgatgcatct taattgcttc
240tgaccaaatg tttagtagca gcagtactat catattcttc ctgctactca
taatatgatt 300ttgtcttgca tattttcagg agactactga g
331850DNAArtificial SequenceSynthetic DNA 8gccgtcgctc acaaggacca
acgaacggaa aggcatgcat gcagagagtt 50957DNAArtificial
SequenceSynthetic DNA 9tatgagctat atgtaatgta agtgtactac tctcctgtca
ccttgcactt gacagca 571063DNAArtificial SequenceSynthetic DNA
10cctctctttg ctccgaaatt ggtcatgtac tcatgttata tgcaatatat acggagtagt
60act 631164DNAArtificial SequenceSynthetic DNA 11tcagaaacgc
aacattctgc actctgattt tactatatgc atcgcttctc attttactga 60cttg
641273DNAArtificial SequenceSynthetic DNA 12aagtaatgtt atcaatcggc
aaatcaaata tggccagaat caacataaga aactgagatt 60tggcacagaa atg
731375DNAArtificial SequenceSynthetic DNA 13ttcatctaca tttagtactc
catgcatata tcgcaagttt gatgtgacgg aaatcttttg 60tttgcacaat acttt
751458DNAArtificial SequenceSynthetic DNA 14agtatcccta agagtggcat
ttttttgctg cccctaagag agtacattca tgtgtctt 58
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References