U.S. patent application number 10/438338 was filed with the patent office on 2003-10-02 for snp detection method and snp detection apparatus.
This patent application is currently assigned to Hitachi, Ltd.. Invention is credited to Kamahori, Masao, Kambara, Hideki, Okano, Kazunori, Zhou, Guohua.
Application Number | 20030186314 10/438338 |
Document ID | / |
Family ID | 18782241 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186314 |
Kind Code |
A1 |
Kambara, Hideki ; et
al. |
October 2, 2003 |
SNP detection method and SNP detection apparatus
Abstract
To provide a method and an apparatus for detecting DNA mutation
without using electrophoresis, the presence or absence of specific
sequence is detected by a method comprising a step of serially
hybridizing a 5-base-long to 8-base-long short oligomer 6, which is
capable of being engaged in the complementary strand extension
reaction, and a long oligomer 61, which is capable of hybridizing
with a target DNA but incapable of being engaged in the
complementary strand extension reaction, with the target DNA 63; a
step of carrying out the complementary strand extension reaction 66
starting from the short primer using four kinds of nucleic acid
substrates and polymerase; and a step of detecting photo-emission,
in which pyrophosphate 68 produced as a reaction by-product of the
complementary strand extension reaction is converted into ATP and
the photo-emission reaction is carried out using an enzyme.
Inventors: |
Kambara, Hideki; (Hachioji,
JP) ; Zhou, Guohua; (Koganei, JP) ; Okano,
Kazunori; (Shiki, JP) ; Kamahori, Masao;
(Kokubunji, JP) |
Correspondence
Address: |
REED SMITH LLP
Suite 1400
3110 Fairview Park Drive
Falls Church
VA
22042
US
|
Assignee: |
Hitachi, Ltd.
|
Family ID: |
18782241 |
Appl. No.: |
10/438338 |
Filed: |
May 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10438338 |
May 13, 2003 |
|
|
|
09945703 |
Sep 5, 2001 |
|
|
|
Current U.S.
Class: |
435/6.18 ;
435/6.1; 435/91.2 |
Current CPC
Class: |
C12Q 1/6858 20130101;
C12Q 1/6858 20130101; C12Q 2565/301 20130101; C12Q 2565/537
20130101; C12Q 2535/125 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 2000 |
JP |
2000-300577 |
Claims
1. A DNA mutation detection method characterized in that a primer
which has a sequence complementary to a region of a specific
sequence containing anticipated mutations in a target DNA
hybridizes with a sample and whether a complementary strand
extension reaction with nucleic acid substrates and polymerase
starting from said primer proceeds or not is detected by detecting
chemiluminescence utilizing pyrophosphate produced as a reaction
by-product of the complementary strand extension to detect the
presence or absence of said specific sequence.
2. A DNA mutation detection method as claimed in claim 1 wherein
multiple kinds of said nucleic acid substrates or analogues thereof
are simultaneously added and said complementary strand extension
reaction is repeated.
3. A DNA mutation detection method as claimed in claim 1 wherein
four kinds of said nucleic acid substrates, which are capable of
being engaged in the complementary strand extension, or analogues
thereof are simultaneously added.
4. A DNA mutation detection method as claimed in claim 1 wherein
the complementary strand extension is proceeded or not depending on
whether the end base of said primer mismatches or matches with the
base of the site of mutation and further the primer is designed to
have a nucleotide which is not complementary to the corresponding
site of said target DNA in the vicinity of its 3'-end so as to more
securely control the complementary strand extension in response to
the match or mismatch of the 3' end of the primer to detect single
nucleotide polymorphisms (SNPs).
5. A DNA mutation detection method as claimed in claim 1 wherein
the extended complementary strand by said complementary strand
extension reaction is decomposed with an enzyme and said primer is
repeatedly hybridized with said target DNA to proceed said
complementary strand extension reaction and pyrophosphate produced
in response to the extension reaction is detected.
6. A DNA mutation detection method as claimed in claim 5 wherein
the enzyme to decompose said extended complementary strand is an
enzyme which decompose a DNA strand from the 5'-end.
7. A single nucleotide polymorphism detection method characterized
in that a DNA primer, which is complementary to a target DNA and is
hybridizable at the 3'-end with a site of single nucleotide
polymorphism of interest or the vicinity thereof, is used to
proceed a complementary strand extension reaction starting from
said primer, which is designed to successively incorporate at least
multiple nucleic acid substrates, chemiluminescence is generated
utilizing pyrophosphate produced as a result of the reaction, and
the photo-emission is detected thereby detecting single nucleotide
polymorphisms (SNPs).
8. A reagent kit used for a method of claim 1 comprising at least a
DNA primer, which is complementary to said target DNA and the
3'-end of which is designed to be hybridizable at a site of single
nucleotide polymorphism of interest or the vicinity thereof, and
nucleic acid substrates.
9. A reagent kit used for a method of claim 1 characterized in that
said kit comprises a DNA primer, which is complementary to said
target DNA and the 3'-end of which is designed to be hybridizable
at a site of single nucleotide polymorphism of interest or the
vicinity thereof; nucleic acid substrates; and an enzyme to
decompose pyrophosphate contained as an impurity, and that said kit
is used for detecting single polymorphisms (SNPs).
10. A method for detecting single nucleotide polymorphisms
characterized in that two kinds of primers having the end
nucleotide respectively complementary to wild type and mutant DNA
samples are held separately in different cells on the solid surface
or different reaction chambers by kind of said primers, and a
mixture of multiple kinds of dNTP is added to said cells or a
reaction solution of said reaction chambers to provide conditions
where a complementary strand extension reaction starting from said
primers can take place, pyrophosphate produced as a result of
actual complementary strand extension reaction starting from said
primers which hybridize with a target DNA is converted into ATP,
and chemiluminescence produced by reacting the ATP with luciferin
or the like is detected to discriminate the kind of primers which
are engaged in the complementary strand extension reaction, whereby
single nucleotide polymorphisms in said target DNA are
detected.
11. A method for detecting single nucleotide polymorphisms
characterized in that multiple kinds of long oligomers, which are
different in sequence from each other and are capable of
hybridizing with a target DNA but incapable of proceeding a
complementary strand extension reaction, are held separately by
kind of said oligomers in different cells on the solid surface or
in different reaction chambers, 5-base-long to 8-base-long short
oligomers capable of proceeding the complementary strand extension
reaction are added to said cells or said reaction chambers, said
short oligomers and said long oligomers are serially hybridized
with said target DNA, the complementary strand extension reaction
starting from the short primers proceeds by further adding a
mixture of multiple kinds of dNTP, pyrophosphate produced as a
result of the reactions is converted into ATP, and
chemiluminescence produced by reacting the ATP with a
chemiluminescence reagent is detected to discriminate the kind of
the primers which are engaged in the complementary strand extension
reaction, whereby single nucleotide polymorphisms (SNPs) contained
in said target DNA or the ratio of DNA substitutions in samples can
be detected.
12. A mutation detection apparatus for detecting DNA mutations
characterized in that said apparatus comprises a reaction chip
furnished with multiple cells on the solid surface, which hold at
least two different primers having end nucleotides each
complementary to wild type and mutant DNA samples, separately by
kind of said primers, or a reaction chamber supporting base on
which multiple reaction chambers holding said primers separately by
kind of said primers are placed; and a light detector to detect
chemiluminescence which is generated by reacting ATP with a
chemiluminescence reagent, said ATP being converted from
pyrophosphate produced as a result of actual complementary strand
extension reaction starting from said primers hybridized with said
target DNA under the conditions where the complementary strand
extension reaction starting from the primers can take place by
adding a mixture of multiple kinds of dNTP to said cells or a
reaction solution in said reaction chambers, whereby the kind of
said primers which are engaged in said complementary strand
extension reaction is discriminated to detect mutations contained
in said target DNA.
13. A mutation detection apparatus for detecting DNA mutations
characterized in that said apparatus comprises a reaction chip
furnished with multiple cells on the solid surface, which hold one
or more kinds of long oligomers, which are capable of hybridizing
with a target DNA but incapable of being engaged in a complementary
strand extension reaction and are different in sequence from each
other, separately by kind of said oligomers, or a reaction chamber
supporting base on which multiple reaction chambers holding said
long oligomers separately by kind of said oligomers are placed; and
a light detector to detect chemiluminescence which is generated by
reacting ATP with a chemiluminescence reagent, said ATP being
converted from pyrophosphate produced by a serial hybridization of
short oligomers and said long oligomers with the target DNA by
adding 5-base-long to 8-base-long short oligomers capable of being
engaged in the complementary strand extension reaction respectively
to said cells or said reaction chambers followed by the subsequent
complementary strand extension reaction starting from said short
primers by further adding a mixture of multiple kinds of dNTP,
whereby the kind of said short primers which are engaged in said
complementary strand extension reaction is discriminated to detect
mutations contained in said target DNA.
14. A method for detecting DNA mutations comprising (1) a step of
hybridizing a primer which contains a sequence complementary to a
region of a specific sequence of a target DNA; (2) a step of
carrying out a complementary strand extension reaction starting
from said primer using four kinds of nucleic acid substrates and
polymerase; and (3) a step of detecting photo-emission in which
pyrophosphate produced as a reaction by-product of said
complementary strand extension reaction is converted into ATP and a
photo-emission reaction is carried out using an enzyme, whereby the
presence or absence of said specific sequence is detected.
15. A method for detecting mutations comprising (1) a step of
hybridizing a primer which contains a sequence complementary to a
region of a specific sequence of a target DNA and a sequence not
complementary to the sequence of the target DNA at the second base
or the third base from the 3'-end; (2) a step of carrying out a
complementary strand extension reaction starting from said primer
using four kinds of nucleic acid substrates and polymerase; and (3)
a step of detecting photo-emission in which pyrophosphate produced
as a reaction by-product of said complementary strand extension
reaction is converted into ATP and a photo-emission reaction is
carried out using an enzyme, whereby the presence or absence of
said specific sequence is detected.
16. A method for detecting mutations comprising (1) a step of
serially hybridizing a 5-base-long to 8-base-long short oligomer,
which are capable of being engaged in a complementary strand
extension reaction, and a long oligomer, which is capable of
hybridizing with a target DNA but incapable of being engaged in a
complementary strand extension reaction, with the target DNA; (2) a
step of carrying out the complementary strand extension reaction
starting from said short primer using four kinds of nucleic acid
substrates and polymerase; and (3) a step of detecting
photo-emission in which pyrophosphate produced as a reaction
by-product of said complementary strand extension reaction is
converted into ATP and a photo-emission reaction is carried out
using an enzyme, whereby the presence or absence of said specific
sequence is detected.
17. A method characterized in that a sample is examined using at
least two probes or primers which contain a part of a sequence of a
DNA region of interest and correspond to a wild type and a mutant
respectively, and the presence or absence of mutations or the ratio
of mutations are examined by comparing results thus obtained.
18. A method as claimed in claim 17 wherein the wild type and the
mutant are discriminated at least by the difference in kind of
bases at the end of the probes or the primers.
19. A method as claimed in claim 17 wherein the wild type and the
mutant are discriminated by a 5 mar or a 6 mar which is
respectively complementary and contains a site of mutation.
20. A method for detecting SNPs as claimed in claim 17 wherein the
primers or the probes for discriminating mutants are hybridized
with the target DNA, the complementary strands are synthesized, and
the resulting pyrophosphate is detected.
21. A method for detecting SNPs as claimed in claim 17 wherein the
primers or the probes for discriminating mutants are hybridized
with the target DNA, the complementary strands are synthesized, and
the resulting synthesized DNA strands are analyzed.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to detection of specific
sequences contained in DNAs of test subjects, detection of
polymorphisms of genes, analysis of single nucleotide polymorphisms
(SNPs), and the like.
[0002] The progress of the human genome mapping project is
prompting a trend towards applying DNA sequence information to
medical diagnoses and various industries, for example, in producing
new drugs. Particularly, in a medical field, noticeable is a trend
towards revealing and applying gene functions. In particular,
analyses of gene expression profiles, in which genes active in a
body are examined, and analyses of single nucleotide polymorphisms
(SNPs) in genomes, which may be a cause of variations in gene
expression, are drawing attention.
[0003] In genomes, single nucleotide polymorphisms are said to be
present approximately one per 1000 bases, which means every
individual has a huge number of single nucleotide polymorphisms.
These single nucleotide polymorphisms are considered to be
associated with characteristics of individuals so that the analysis
of these single nucleotide polymorphisms is expected to provide a
therapy guide or the like for the treatment of diseases for the
individuals.
[0004] The number of single nucleotide polymorphisms present in
genomes of one human being is enormous, and the type of single
nucleotide polymorphisms in genomes of many people are polymorphic,
in which two kinds of bases appear in particular sites. It is
necessary to examine single nucleotide polymorphisms occurring at
sites of mutation and further to reveal their correlation with
diseases or the like, which requires the accumulation of enormous
amounts of DNA data. Thus, development of appropriate methods to
meet this, requirement is in need.
[0005] At present, various methods for analyzing base substitutions
are available. They are generally divided into two types, i.e.,
methods for detecting unknown base substitutions and methods for
examining, for example, whether certain known base substitutions
are found in many people or whether these known substitutions have
causal relationships with certain diseases.
[0006] Examples of the methods for detecting unknown base
substitutions include a method in which a base sequence is
determined using a DNA sequencer, and a method in which
hybridization of a DNA probe and a target DNA is detected using a
DNA probe array (DNA chip or gene chip).
[0007] On the other hand, in order to detect single nucleotide
polymorphisms at known positions, methods, such as an SSCP (single
strand configuration polymorphism) method, an invader assay (Nature
Biotechnology, 17, 292-296 (1999)), and DASH (Nature Biotechnology,
17, 87-88 (1999)), have been developed and implemented for
practical use. All of these methods use laser or the like as a
light source.
[0008] In synthesizing a DNA complementary strand using a primer,
complementary strand extension may or may not proceed depending on
whether the 3'-end of the primer is complementary to the target or
not. Accordingly, a method, in which a PCR amplification is
performed using a primer having the 3'-end at an accordant position
of mutation and products are analyzed using gel electrophoresis or
the like (ARMS: amplified refractory mutation system; Nucleic Acids
Research, 17, 2503-2515 (1989)), has also been used. In this case,
a key point is how exclusively the complementary strand synthesis
occurs in the presence of a target of interest.
[0009] In the SSCP method, single nucleotide polymorphisms can be
detected based on the observation that the configurations of
single-stranded DNAs with and without mutation during
electrophoresis are different, and these configurations influence
electrophoretic mobility.
[0010] In the invader assay, a triple-stranded chain is formed with
a DNA probe of a DNA probe array, a target DNA and a DNA probe, and
whether a part of the probe is cleaved with an enzyme is
detected.
[0011] In DASH, a target DNA and a probe DNA are hybridized under
the presence of an intercalator, and light generated from the
intercalator by irradiation is observed while changing a
temperature. The double stranded chain is dissociated and the
emission ceases if mutation is present in the target DNA.
[0012] On the other hand, a new technique is about to be
implemented, in which a short DNA base sequence is determined in a
short time and then mutations are examined using the determined DNA
base sequence.
[0013] For example, Nyren et al. have proposed a method for
determining a DNA base sequence (pyrosequencing), in which a target
DNA is hybridized with a primer, pyrophosphate produced by a
complementary strand extension reaction is converted into ATP, the
ATP is reacted with luciferin to emit light, and this
chemiluminescence is detected to discriminate the substrate (dNTP)
incorporated during the complementary strand extension reaction.
The pyrosequencing has drawn an attention as a simple and easy
method for determining a DNA sequence without the need for gel
electrophoresis. Recently, detection of SNPs using this
pyrosequencing has been reported (Anal Biochemistry, 280, 103-110
(2000). This method does not require the use of a new light source
because it utilizes chemiluminescence.
SUMMARY OF THE INVENTION
[0014] Mutations which appear in genome DNAs are associated with
characteristics of organisms, sensitivity to diseases and
medicines, and the like, and thus a huge number of subjects have to
be analyzed. Since individuals to be tested are numerous, there is
a demand for a method and an apparatus which suit for a simple and
large scale operation at a low running cost.
[0015] Further, SNP measurements are necessary for individual
genome samples, as well as for samples from a patient group and a
healthy subject group for comparison. Namely, in order to study a
causative relationship between particular SNPs and a certain
disease it is necessary to compare the incidence of the SNPs
(allele frequency) in the two groups. This comparison study can be
most efficiently and easily carried out by combining DNAs of each
group and quantitatively analyzing the SNPs contained in the
combined DNAs. However, a quantitative method accurate enough for
such analyses remains to be developed. Further problems arise upon
measurement of SNPs even with individual samples, as described
below.
[0016] In various methods which have been proposed previously, a
targeted DNA region is amplified by PCR and the resulting DNA
fragments are used as a target. Accordingly, the use of PCR is
inevitable.
[0017] In the DASH, it is necessary to use a detector equipped with
a laser light source and a filter to remove scattering light, which
requires a large-scale apparatus.
[0018] In a pyrosequencing for detecting chemiluminescence,
pyrophosphate is converted into ATP to generate chemiluminescence
and the resulting photo-emission is detected. However, the
pyrosequencing has disadvantages such that four kinds of dNTP
(dATP, dTTP, dGTP and dCTP) have to be added independently in a
designated order to a reaction part, that the photo-emission
intensity is weak, that a system for dNTP injection is necessary,
and that a considerably large-scale apparatus is required.
[0019] Further problems in this method are that because reagents
are injected in a designated order, the time required for the
analysis is 10-20 minutes although it is shorter than that with the
use of electrophoresis, and that sample DNAs have to be
PCR-amplified to increase the number of DNA copies of the target
region because the method is not sensitive enough without
amplification.
[0020] Furthermore, in pyrosequencing, if a multiple number of base
substitutions exist in a sample to be tested, the resulting
spectrum may be too complicated to perform accurate analyses.
[0021] In order to apply the pyrosequencing for the detection of
DNA mutations, it is important to further improve the sensitivity
so that the DNA mutations can be readily detected and DNA strands
containing multiple kinds of mutations can be analyzed.
[0022] An object of the present invention is to provide a tool
necessary to create a database applicable for genetic diagnoses,
new drug manufacturing using genes, and gene therapies, and further
to provide a method, an apparatus, test reagents and the like for
examining gene DNAs.
[0023] More specifically, an object of the present invention is to
provide a highly sensitive, easy method and an apparatus for
detecting DNA mutations, in which the use of excitatory light
source is not necessary and the reaction is simple and quick, and
further to provide a method and an apparatus for detecting DNA
mutations, in which a sample containing multiple mutations can be
analyzed at a high reliability.
[0024] Another object of the present invention is to provide a
technique for readily screening interrelations between a certain
disease and SNPs.
[0025] In the present invention, in order to examine the presence
or absence of a target DNA of interest, or the presence or absence
of mutations, a DNA probe (primer) capable of being engaged in
complementary strand extension is hybridized with the target DNA,
pyrophosphate generated as a result of the complementary strand
extension reaction is converted into ATP to emit light, and this
photo-emission is detected. When the target DNA sequence is not
present in the test sample, the complementary strand extension does
not take place so that no photo-emission is observed.
[0026] Further, in the present invention, probes are prepared to
have the 3'-end base at a position accordant to mutations and have
different 3'-end sequences so that the complementary strand
extension reaction occurs only if mutation exists or the
complementary strand extension reaction does not occur only when
mutation exists. Thus, this method can be used also for detecting
mutations. This is because the complementary strand extension
reaction starting from the primer highly depends on whether the
hybridization matching at the 3'-ends is perfect or imperfect (Kwok
S. et al., Nucleic Acids Res., 18, 999-1005 (1990); Huang M. M.,
Arnhein N., Goodman M. R., Nucleic Acids Res., 20, 4567-4573
(1992)).
[0027] In order to accurately switch on/off of the primer
complementary strand extension reaction in response to a DNA
template for targeted SNP measurements, an artificial mismatch is
introduced near the 3'-end of the primer. The presence or absence
of the complementary strand synthesis is examined by converting the
produced pyrophosphate into ATP, reacting the ATP with a
chemiluminescent reagent and optically detecting the resulting
chemiluminescence. In order to accomplish a highly accurate
detection by this method, pyrophosphate contained as an impurity
and pyrophosphate resulting from heat decomposition of nucleic acid
substrates, which generate a background light, are minimized as
much as possible, and at the same time, the chemiluminescence
resulting from the complementary strand extension is detected using
two primers having the end bases complementary to a wild type and a
mutant.
[0028] In a conventional pyrosequencing, photo-emission intensity
is weak because the photo-emission uses pyrophosphate resulting
from a complementary strand extension reaction with only one base.
Contrastingly in the present invention, the complementary strand
extension reaction proceeds with more than dozens of bases and
chemiluminescence is detected by converting the resulting
pyrophosphate into ATP so that the photo-emission intensity is more
than two orders stronger than that attained by the conventional
pyrosequencing.
[0029] Further, it is possible to enhance the photo-emission
intensity by repeating the decomposition of the double-stranded
chain resulting from the complementary strand extension reaction
using an enzyme and the process of the complementary strand
extension. Thus, the presence or absence of specific sequences or
mutations, and the ratio of mutation contained in combined genome
samples can be detected without PCR-amplification of the
samples.
[0030] In the present invention, substrates for the complementary
strand synthesis, i.e., dNTPs, are added in a controlled manner
upon the complementary strand extension starting from a primer,
pyrophosphate produced in response to the complementary strand
extension reaction is detected by chemiluminescence emission, and
thus the presence or absence of base mutations and the presence of
a wild type and a mutant in a target DNA are detected. The presence
or absence of substitution of base sequences or base mutation can
be examined by a simple and highly sensitive method using primers
designed such that their 3'-ends come to the position of the
anticipated base substitution in the target DNA and the
complementary strand extension may or may not occur depending on
the presence or absence of the substitution.
[0031] When the complementary strand extension occurs, multiple
dNTPs are simultaneously added to proceed the complementary strand
extension all the way through and produce a large amount of
pyrophosphate so that the intensity of chemiluminescence can be
enhanced to attain a highly sensitive detection of base
mutations.
[0032] Multiple primers are prepared taking the sequence of a
target DNA into consideration, and the progress of complementary
strand extensions starting from these various primers are detected
to discriminate the presence of multiple kinds of mutations.
Further, multiple kinds of base substitutions can be analyzed with
high reliability by sorting and holding multiple primers for the
reaction according to known standard DNA sequences. When a sample
contains various mutants, multiple primers are prepared referring
to the standard base sequence and immobilized on a solid surface by
kind, and complementary strand extension reactions are carried out
using these primers to monitor the presence of mutants.
[0033] An analysis of test samples containing various mutations has
been extremely difficult with conventional methods. However, the
mutations can be detected using a simple method and an apparatus
according to the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 illustrates Example 1.
[0035] FIG. 2 illustrates examples of the structures of primers
used in Example 1 and relative photo-emission intensities in
response to the formation of extended complementary strands by
respective primers.
[0036] FIG. 3 illustrates examples of detecting target DNAs having
mutations, in which relative photo-emission intensity by luciferase
is shown to indicate the presence or absence of pyrophosphate
produced as a reaction by-product upon the complementary strand
extension reaction in response to primers.
[0037] FIG. 4 illustrates the outline of chemical reactions in
Examples of the present invention.
[0038] FIG. 5 shows the comparison of the photo-emission intensity
in the complementary strand extension reaction by a conventional
pyrosequencing with the photo-emission intensity in the
complementary strand extension reaction by the method in Example
1.
[0039] FIG. 6 illustrates Example 2.
[0040] FIG. 7 illustrates Example 3.
[0041] FIG. 8 is a perspective view showing an example of the
structure of the DNA mutation detection apparatus of Example 4 of
the present invention.
[0042] FIG. 9 is a cross-sectional view showing an example of the
structure of the DNA mutation detection apparatus of Example 4 of
the present invention.
[0043] FIG. 10 illustrates another example of the structure of the
DNA mutation detection apparatus of Example 4 of the present
invention.
[0044] FIG. 11 illustrates Example 5.
[0045] FIG. 12 shows the structures of probes incapable of being
engaged in the complementary strand extension reaction and
6-base-long primers for detecting mutations shown in FIG. 11.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0046] Examples of the present invention will be hereinafter
explained in detail referring to the attached drawings.
[0047] First, a photo-emission mechanism, the premise of the
present invention, will be explained referring to a related
technique, pyrosequencing. A primer is hybridized with a target
DNA. DNA polymerase and dNTPs, substrates for a complementary
strand synthesis in a complementary strand extension reaction, are
added to proceed the complementary strand extension reaction.
Substrates complementary to the DNA strand are incorporated one by
one and the complementary strand is extended by DNA polymerase. At
the same time, pyrophosphate is produced as a reaction by-product.
One molecule of pyrophosphate is produced every time when one dNTP
is incorporated.
[0048] Pyrophosphate is converted into ATP by sulfurylase and the
resulting ATP oxidizes luciferin in the presence of luciferase to
emit light. Progress of the complementary strand extension starting
from the primer can be monitored by detecting this photo-emission.
Four kinds of dNTP are added one by one in a designated order to a
reaction part. The photo-emission with an added dNTP implies the
incorporation of this particular dNTP, whereby a DNA sequence is
determined.
[0049] At this case, it is necessary to decompose excess dNTPs so
that the succeeding reaction is not interfered. An addition of
apylase is reported as an essential technique (Science, 281,
363-364 (1998)). In this case, the photo-emission intensity
decreases by one order by the addition of apylase since apylase
also decomposes ATP and thus the photo-emission reaction and the
decomposition reaction occur competitively. The sequencing using
this reaction requires about 0.2 pmol of target DNA.
[0050] The method of the present invention largely differs from the
conventional pyrosequencing in that a primer for the complementary
strand extension is designed to have the 3'-end or the vicinity of
the 3'-end at the position complementary to a position of mutation
upon hybridization with the sample. A DNA strand portion which is
engaged in the complimentary strand synthesis does not contain a
complementary portion at the position of mutation. Furthermore, the
method of the present invention is characterized in that the
complementary strand synthesis is proceeded by successive
incorporation of multiple bases, a large number of the resulting
pyrophosphate react with a chemiluminescence reagent to generate
chemiluminescence for highly sensitive detection without the need
for recognizing the extending DNA sequence. Furthermore, the method
of the invention is characterized in that in order to obtain more
accurate SNP information or quantitative mutant information, the
complementary strand synthesis is carried out using two kinds of
primers having the end bases in accord with complementary bases of
mutant and wild type DNAs to ultimately measure
chemiluminescence.
EXAMPLE 1
[0051] FIGS. 1(a) through (d) are drawings to illustrate Example 1
showing that the hybridization proceeds completely or incompletely
depending on the kind of the base at the 3'-end of the primer, and
thus the complementary strand synthesis is controlled.
[0052] A single-stranded target DNA 1 is prepared from a DNA
containing mutation, and a single-stranded target DNA 2 is prepared
from a DNA without mutation. In this example, two different DNAs
having a base length of about 400 are used; longer DNAs can also be
used without any difficulty. The target DNAs 1 and 2 are hybridized
with a 10-base-long primer 5 in which the base at the 3'-end 6 is
designed to be in accord with the base at a position 3 of the
target DNAs 1 and 2, where the presence or absence of mutation is
to be examined. The complementary strand extension reaction is
carried out by adding DNA polymerase and substrates for the
complementary strand synthesis, i.e., dATP, dTTP, dGTP and dCTP.
The primer shown in FIG. 1 is 10 base long; however, primers having
a base length of 10 to 30 are generally used.
[0053] In the example shown in FIG. 1(a), the base (G) at the
3'-end 6 of the primer 5 and the base (C) at the position 3, where
the presence or absence of mutation of the target DNA 2 is to be
examined, are complementary, so that substrates complementary to
the DNA strand are successively incorporated by DNA polymerase to
form the extended complementary strand as indicated with the arrow
8. As a result, pyrophosphate is produced as a reaction
by-product.
[0054] In the example shown in FIG. 1(b), the base (G) at the
3'-end 6 of the primer 5 and the base (A) at the position 3 of the
target DNA 1, where the presence or absence of mutation is to be
examined, are not complementary. Accordingly, substrates
complementary to the DNA strands are hardly incorporated by DNA
polymerase so that the complementary strand extension hardly
occurs. As a result, the amount of pyrophosphate produced as a
reaction by-product is small.
[0055] As described above, pyrophosphate is detected using
photo-emission by luciferase. Strong signals are detected in the
example shown in FIG. 1(a), but not in the example shown in FIG.
1(b).
[0056] However, the strand extension may occur with some targets at
a considerably high probability even if bases at their 3'-ends are
not complementary.
[0057] Therefore, a primer 9 is used instead of the primer 5 in
order to detect mutation more accurately, or to switch the
complementary strand synthesis more accurately in response to the
kind of end bases. The sequence of the primer 9 is the same as that
of the primer 5 except that the base at the second or third base
position from the 3'-end 6 of the primer 5 is replaced by the base
(A) which is not complementary to the corresponding base of the
target DNAs 1 and 2.
[0058] The third base from the 3'-end of the primer 9 shown in
FIGS. 1(c) and 1(d) is the base (A) which is not complementary to
the corresponding base (C) of the target DNAs 1 and 2, which
generate an artificial mismatch at the third base from the 3'-end
6. As a consequence, the 3'-end 6 of the primer 9 is detached from
the target DNA when the 3'-end 6 of the primer 9 is mismatched.
Thus, the complementary strand extension reaction of the 3'-end of
the primer 9 is sensitively affected by the presence or absence of
the mismatch.
[0059] In the example shown in FIG. 1(c), the base (G) at the
3'-end 6 of the primer 9 is complementary to the base (C) at the
position 3 of the target DNA 1, where the presence or absence of
mutation is to be examined. In this case, even if the artificially
mismatched base (A) at the third base from the 3'-end 6 of the
primer 9, where the presence or absence of mutation of the target
DNA 1 is to be examined, is not complementary to the base at the
corresponding position of the target DNA 2, substrates
complementary to the DNA strand are successively incorporated by
DNA polymerase to form the extended complementary strand 8. As a
result, pyrophosphate is produced as a reaction by-product.
[0060] In contrast, in the example shown in FIG. 1(d), the base (G)
6 at the 3'-end of the primer 9 is not complementary to the base
(A) at the position 3 of the target DNA 1, where the presence or
absence of mutation is to be examined. In this case, the
complementary strand extension reaction hardly occurs so that no
extended complementary strand will be formed when the additional
mismatch exists at the 3'-end of the primer 9, since the
artificially mismatched base (A) at the third base from the 3'-end
6 of the primer 9, where the presence or absence of mutation of the
target DNA 1 is to be examined, is not complementary to the base at
the corresponding position of target DNA 2. As a result,
pyrophosphate is not produced as a reaction by-product. Although in
both FIGS. 1(b) and 1(d) no complementary strand is shown to be
extended from the 3'-end 6 where the presence or absence of
mutation of the target DNA 1 is to be examined, complementary
strand extension may occur with certain target base sequences in
the case of FIG. 1(b). For example, FIG. 2 shows the probability
(frequency) of complementary strand extension using various
primers. Relative photo-emission intensities for primers having
mismatch-rendering bases (changes) at their ends are shown while a
photo-emission intensity obtained using a primer SNP1-1, which has
a completely complementary sequence, is set to be 100. The figures
show that the complementary strand extension occurs at a
considerably high probability even in the presence of
mismatch-rendering end bases. On the other hand, in FIG. 1(d), an
artificial mismatch is introduced at the third base from the
3'-end. As a consequence, the complementary strand extension is
more securely suppressed since two mismatches altogether exist in
the vicinity of the end. In fact, this suppression is shown in FIG.
2 as the photo-emission intensity obtained by using primer SNP2-i
(i=1 through 4). The photo-emission intensity obtained using the
primer SNP2-1 having a match at the end is almost the same with
that obtained using the completely matched primer (SNP1-1). However
the photo-emission intensity is extremely weak if a mismatch is
introduced at the end and the resulting figures are almost the same
as that attributable to impurities contained in reagents. Namely,
the complementary strand synthesis hardly occurs when at least two
mismatches are present in the vicinity of the end. Further, a
similar reaction occurs by using a primer which introduces a
mismatch at the end of the primer while a site of the primer
corresponding to the position of mutation is set to be the third
base from the end. Namely, in FIG. 1(d), in contrast to the
structure in FIG. 1(b), the complementary strand extension is more
securely suppressed by making the primer 9 not complementary at the
third base from the 3'-end 6 where the presence or absence of
mutation of the target DNA 1 is to be examined and at the 3'-end
6.
[0061] As explained above, ATP and luciferin are reacted using
luciferase to emit light and the resulting photo-emission is
detected to examine mutations. Namely, mutation is detected in the
example shown in FIG. 1(c), but not in the example shown in FIG.
1(d).
[0062] FIG. 2 shows examples of the structure of the primer 5 and
primer 9 and the photo-emission intensity in response to the
formation of the extended complementary strand by each of these
primers.
[0063] For the primer 5, the following four kinds of primers (name
of the primers: SPN1-i; i=1, 2, 3, and 4) having different bases at
the 3'-end are used for evaluation. Each of the primer SPN1-i (i=1,
2, 3, and 4) is a 20-base-long sequence, i.e., Sequence
Identification Number (SEQ ID NO:) 1, 2, 3, or 4.
1 AGTTTTAAGA GGGTTGTTGT (SEQ ID NO: 1) AGTTTTAAGA GGGTTGTTGC (SEQ
ID NO: 2) AGTTTTAAGA GGGTTGTTGG (SEQ ID NO: 3) AGTTTTAAGA
GGGTTGTTGA (SEQ ID NO: 4)
[0064] For the primer 9 (SPN2-i (i=1, 2, 3, or 4)), the following
four kinds of primers (name of the primers: SPN2-i, i=1, 2, 3, and
4) which are the same with the primer 5 except that the third base
from the 3'-end of the primer 5, i.e., T, is replaced by A are used
for evaluation. Each of the primer SPN1-i (i=1, 2, 3, and 4) is a
20-base-long sequence, i.e., Sequence Identification Number (SEQ ID
NO:) 5, 6, 7, or 8.
2 AGTTTTAAGA GGGTTGTAGT (SEQ ID NO: 5) AGTTTTAAGA GGGTTGTAGC (SEQ
ID NO: 6) AGTTTTAAGA GGGTTGTAGG (SEQ ID NO: 7) AGTTTTAAGA
GGGTTGTAGA (SEQ ID NO: 8)
[0065] One of the results of the test in which sample DNAs having
various mutations were practically used will be explained as
follows.
[0066] FIG. 3 illustrates an example of detecting a target DNA
having a mutation, showing the relative photo-emission intensity by
luciferase, which shows the presence or absence of pyrophosphate
produced as a reaction by-product in response to the complementary
strand extension reaction according to the primers.
[0067] The target DNA has a mutation and have a 20-base-long
sequence, SEQ ID NO: 9. The base C at the end of the base sequence
shown in SEQ ID NO: 9 is replaced by A by mutation.
[0068] TCAAAATTCT CCCAACAACA (SEQ ID NO: 9)
[0069] FIG. 3(a) shows the relative photo-emission intensity
obtained by using the primer 5 (primer SPN1-i (i=1, 2, 3, or 4)) in
which no artificial mismatch is introduced into the third base from
the 3'-end. FIG. 3(b) shows the relative photo-emission intensity
obtained by using the primer 9 (primer SPN2-i (i=1, 2, 3, or 4)) in
which an artificial mismatch is introduced into the third base from
the 3'-end. The vertical axis shows the relative photo-emission
intensity setting the photo-emission intensity obtained by using
SPN1-1 to be 100. Figures in parentheses below the names of
primers, i.e., SPN1-i (i=1, 2, 3, or 4) and SPN2-i (i=1, 2, 3, or
4), are relative photo-emission intensities. The relative emission
intensities shown in FIG. 2 are these figures.
[0070] The horizontal axis shown in FIGS. 3(a) and 3(b) shows the
time elapsed for the change in the relative photo-emission
intensity. The scale shown below the axis indicates one minute. DNA
polymerase and substrates for the complementary strand synthesis,
i.e., dATP, dTTP, dGTP and dCTP, are added to a buffer solution
containing the target DNA and primers at the time corresponding to
the starting point of each curve showing the relative
photo-emission intensity, i.e., curves 37, 38, 39, 40 and curves
41, 42, 43, and 44, to start the complementary strand extension.
Figures show that the reaction proceeds with the time elapsed.
[0071] As shown with curves 37 through 40 in FIG. 3(a), as for the
primer 5 with no artificial mismatch introduced (primer SPN1-i
(i=1, 2, 3, or 4)), the curve 37 for the complementary strand
extension using primer SPN1-1 cannot be clearly distinguished from
the curves 38 and 39 for the complementary strand extension using
primers SPN1-i (i=2 and 3, respectively). Namely, the complementary
strand extension proceeded in spite of the fact that the bases of
the 3'-end of primers SPN1-2 and SPN1-3 mismatch with the base of
the target DNA in the corresponding position. Of course, the
photo-emission with primers SPN1-2 and SPN1-3 are weaker than that
with SPN1-1 so that they are not entirely be undistinguishable.
However, mutant and wild type cannot accurately distinguished and
their ratio in a sample cannot be accurately estimated.
[0072] On the other hand, as shown with curves 41 through 44 in
FIG. 3(b), as for the primer 9 (primer SPN1-i (i=1, 2, 3, or 4)) in
which an artificial mismatch is introduced into the third base from
the 3'-end, the selectivity of primers is improved. Namely, while
the photo-emission with primer SPN2-1 shown with the curve 41 is
similar to that shown with the curve 37, the photo-emission is
hardly detected with primers SPN2-i (i=2, 3, and 4) as shown with
curves 42, 43, and 44.
[0073] Thus, the mutation in the base sequence can be more
accurately detected by using the primer 9 (primer SPN2-i (i=1, 2,
3, or 4)) in which a mismatch is artificially introduced into the
third base from the 3'-end.
[0074] In FIGS. 3(a) and 3(b), the curves 37, 38 and 39 for the
primers 5 (SPN1-i (i=2, 3 and 4)) and the curve 41 for the primer 9
(SPN2-1) show that about 400 complementary strands are synthesized
by the complementary strand extension reaction and that the
photo-emission intensity reaches at its peak and then declines. The
photo-emission detected here results from chemiluminescence
emission with pyrophosphate released by the complementary strand
extension reaction.
[0075] In order to detect SNPs more accurately or to estimate the
ratio of those SNPs in a sample in which wild type and mutant genes
coexist, at least two kinds of primers which are most suitable to
each of the genes are to be used. In this case, primer SNP2-1 and
SNP2-3 are used since mutation occurs in a form of C/A. SNP2-1 is
hybridizable with the mutant sequence, but not with the wild type
sequence, to proceed the complementary strand synthesis. On the
other hand, SNP2-3 is hybridizable with the wild type sequence, but
not with the mutant sequence, to proceed the complementary strand
synthesis. The photo-emission intensity observed upon hybridization
with the mutant sequence shown in Table is the background light
derived from impurities and pyrophosphate produced by decomposition
of nucleotide substrates (dNTPs), and becomes zero by background
adjustment. It is important to know the ratio of the wild type and
mutant in a genome mixed samples or the like, which contain both
the wild type and mutant sequences. In particular, in order to
study relationship between diseases and SNPs, it is important to
compare the incidence of SNPs of interest in genomes of patients
and in healthy subjects. For that, it is necessary to analyze SNPs
in an extremely large number of samples. A huge number of data have
to be obtained since SNPs are numerous. In such a case, a
correlation with a certain disease can be efficiently examined by
measuring SNPs in pooled genomes of patients and pooled genomes of
healthy subjects. Generally, the significance level for the
incidence of SNPs is considered to be more than 1% and the
significance level for the correlation with disease is considered
to be more than 5%. Therefore, in a mixed genome sample, the ratio
of the incidence of SNPs has to be measured at the accuracy level
of about 1%, which has been difficult using conventional methods.
According to a method of the present invention, the ratio of the
incidence can be determined at the accuracy level of about 1% by
measuring the ratio of respective complementary strand extensions
using two primers. FIG. 3(c) shows the result of the experiment
discussed above. Namely, FIG. 3(c) shows results of measurement for
a mixed genome sample containing both the wild type and mutant,
according to the present invention.
[0076] The horizontal axis of FIGS. 3(c) is the presence ratio of
mutation while maintaining the sum of wild type and mutant to be
constant. The signal intensity derived from wild type (open circle)
and the signal intensity derived from mutant (open triangle) are
shown. The reaction for the measurement was carried out using
primers each specific to wild type and mutant sequences. As the
results show, when the ratio of the mutant increases, the
photo-emission intensity for the wild type decreases and that for
the mutant increases. Taking the S/N ratio into consideration,
detection is possible when the ratio of mutation is as low as about
1%. It is evident that a sufficiently accurate detection is
possible according to the invention since the significant level of
detection ratio necessary to determine the correlation between
normality and disease is 5%, as described above.
[0077] In order to improve the accuracy of measurement, it is
effective to detect SNPs using the total of four kinds of primers
for both double-stranded DNAs.
[0078] FIG. 4 explains the outline of the chemical reactions in
Example 1 of the present invention. In FIG. 4(a), a target DNA 45
and a primer 46 are introduced into a buffer solution in a reaction
vessel 200. In FIG. 4(b), DNA polymerase and substrates for
complementary strand synthesis, i.e., dNTPs (dATP, dTTP, dGTP and
dCTP) are added to the buffer solution in the reaction vessel and
the complementary strand extension proceeds as shown with an array
48. At this time, APS (adenosine 5'-phosphosulfate), ATP
sulfurylase and luciferin are also added. FIG. 4(c) shows chemical
reactions in the case where the complementary strand extension
proceeds, in which as a result of the reaction with DNA polymerase
on the target DNA 45, the primer 46 and the substrates, unreacted
target DNA 45, a double-stranded DNA synthesized with the extended
complementary strand 48 and the target DNA 45, and pyrophosphate
(PPi) 49 are obtained. The number of molecules of the resulting
pyrophosphate (PPi) 49 is equivalent to that of dNTP 47
incorporated in proportion to the base length upon the synthesis of
the extended complementary strand 48. In FIG. 4(d), each molecule
of resulting pyrophosphate (PPi) 49 is converted into ATP 50 by ATP
sulfurylase in the presence of APS. At the same time, sulfuric acid
ion is produced. In FIG. 4(e), the converted ATP 50 oxidizes
luciferin in the presence of luciferase to emit light (hi) 51. At
this time, carbon dioxide is produced, pyrophosphate is converted
into ATP and the ATP oxidizes luciferin to produce pyrophosphate
(PPi) 49'. The PPi 49' is again converted into ATP 50 by ATP
sulfurylase and the ATP reacts with luciferin. If the complementary
strand extension proceeds up to a 400 base length, 400 molecules of
pyrophosphate 49 are to be produced by the complementary strand
extension reaction for one target DNA. The reaction converts ATP
into AMP (adenosine monophosphate) and pyrophosphate. The
pyrophosphate is again converted into ATP and thus the emission
reaction is repeated to continue the emission.
[0079] As evident from the data on the photo-emission intensity
obtained in FIG. 3, the reaction for ATP production after the
complementary strain extension reaction transitionally continues as
long as sufficient APS, luciferin and O2 are present, whereby the
emission reaction further continues and the emission 51 is
maintained. The photo-emission intensity 51 is proportional to the
amount of produced pyrophosphate 49, i.e., the length of DNA chain
of the extended complementary stand 48.
[0080] In this Example 1, the mixture of all four kinds of
substrates for the complementary strand synthesis, i.e., dNTPs 47
(N=A, T, G and C), can be put along with DNA polymerase into the
reaction solution containing the single-stranded target DNA 45 and
the primer 46. Therefore, this method can attain a highly sensitive
detection by obtaining chemiluminescence having intensity as high
as double digits as compared to the pyrosequencing in which DNA
sequencing is carried out by a step-by-step reaction. In the
pyrosequencing, substrates dNTP (N=A, T, G and C) for the
complementary strand synthesis must be added independently by kind
in a designated order into the reaction part.
[0081] FIG. 5(a) shows the comparison of the photo-emission
intensity obtained by pyrosequencing (a graph marked with dGTP) and
the method of Example 1 (a graph marked with dNTP) in which the
primer 9 (with an artificial mismatch) shown in FIG. 2 is bound to
a single-stranded target DNA. In pyrosequencing, only one kind of
substrate dNTP or ddNTP (dideoxy nucleotide triphosphate) (N=either
one of A, T, G and C) is used to proceed the complementary strand
extension for only one base. In the method of Example 1, all four
kinds of substrate dNTPs (N=A, T, G and C) are added simultaneously
to proceed the complementary strand extension over a broad region.
A sample used is 3 fmol of M13. FIG. 5(b) shows background data
obtained under the same conditions as those for FIG. 5(a) except
that no sample is added.
[0082] In this example, since apyrase is added in the sample to
decompose ATP and pyrophosphate, emission and decomposition
compete. As a result, the emission intensity is low and cannot be
maintained. Background emission is generated because pyrophosphate
is often contained in sample DNAs, enzymes, dNTP and ddNTP, as an
impurity. Such pyrophosphates can be decomposed by adding apyrase
and the background emission disappears. If these contaminating
pyrophosphates are removed in advance, the level of the signal
intensity several times higher than the peak value can be
maintained and there is no need for adding apyrase.
[0083] In FIG. 5(a) and FIG. 5(b), the vertical axes indicate
photo-emission intensity and the horizontal axes indicate the time
elapsed for the photo-emission intensity. FIG. 5(a) shows that the
photo-emission intensity obtained by the complementary strand
extension using four kinds of substrates dNTP to release an
abundant pyrophosphate for chemiluminescence generation is about 50
times higher than that obtained by extension of only one base using
only one kind of dGTP. This comparison also shows that the
sensitivity can be highly improved by the method of this Example
1.
[0084] More specifically, if the background data in FIG. 5(b) is
taking into consideration, the sample DNA (M13) cannot virtually be
detected when only dGTP is added, since the signal intensity in
FIG. 5(a) and background data in FIG. 5(b) are equivalent. In
contrast, when four kinds of dNTP are added, the signal intensity
in FIG. 5(a) is 12 times higher than that of the background data in
FIG. 5(b). In fact, one DNA base mutation can be detected at the
order of 10-18 mol since the measurement noise was {fraction
(1/78)} signal intensity. This signal level is considered to be
sufficient for the detection without PCR amplification.
[0085] Pyrophosphoric acid contained in samples or phosphoric acid
generated from the decomposition of dNTP or ddNTP are responsible
for the background emission which may thus interfere the
measurement of such a trace amount of DNA. These phosphoric acids
were removed by treating reagents to be used in the reaction in
advance with apyrase to reduce the background light.
EXAMPLE 2
[0086] Example 2 is an example in which two adjacent DNA primers
are used like a single primer. Namely, two adjacent primers, long
and short, are hybridized with a target DNA instead of introducing
a mismatch in a primer itself like the primer 9 shown in FIG. 2.
This method is based on the observation that a short primer is
difficult to hybridize if a mismatch is present.
[0087] FIG. 6 illustrates Example 2. In Example 2, two probes
placed side by side act like a single primer; the first probe is
long enough to stably hybridize with a target DNA and the second
probe is too short to hybridize and synthesize a complementary
strand by itself but becomes hybridizable with the target DNA when
placed closely adjoining to the 3'-end of the first probe. FIG. 6
exemplifies the detection of mutation, in which the complementary
strand extension reaction is carried out starting from the 3'-end
of the second probe of a nominal primer, in which the 3'-end of the
first primer and the 5'-end of the second probe are closely
adjoined, and the photo-emission is detected using luciferase.
[0088] As shown in FIG. 6(a), the first probe 61 is 15 to 30 bases
long, which is long enough to stably hybridize with a
single-stranded target DNA 63. The 3'-end of the first probe is
made to be dideoxynucleotide 64 to prevent complementary strand
extension, since the first probe hybridizes with any target DNA
regardless of the presence or absence of SNPs.
[0089] In another method, a short oligomer and a long oligomer are
first hybridized in a series. Then, they are bound by a ligation
reaction. For this reaction, a short oligomer complementary to a
wild type sequence and a short oligomer complementary to a mutant
sequence are prepared. The 3'-end of the oligomer complementary to
the wild type is made to be dideoxynucleotide so as not to proceed
the complementary strand extension. The oligomers ligate and the
complementary strand extension proceeds only when the sample is
mutant. However, the extension does not occur if the sample is wild
type. In this way, the wild type and the mutant can be
distinguished.
[0090] As shown in FIG. 6(b), the second probe 62 having 5 or 6
bases, which is placed closely adjoining to the 3'-end of the first
probe 61, hybridizes with the target DNA 63. The 5'-end of the
second probe 62 is made to be phosphoric acid group 67 which
readily adheres to the 3'-end of the first probe 61. The 3'-end of
the second probe 62 can be a start of the complementary strand
synthesis but the probe is made too short to proceed the
complementary strand synthesis by itself although it is
hybridizable.
[0091] The second probe 62 can stably function as a primer only
when it is adjoined close to the first probe 61 and hybridized with
the target DNA. In the method of Example 2, only when the first
probe 61 and the second probe 62 hybridize with the target DNA 63,
an extended complementary strand 66 having n bases is formed and n
molecules of pyrophosphate 68 are generated. Namely, a high
selectivity can be attained by the hybridization of the two closely
adjoined probes with the target DNA. Also, in the same manner as in
Example 1, the base 6l at the 3'-end of the primer consisting of
the first probe 61 and the second probe 62 is designed to be
brought into the position 35 where the presence or absence of
mutation of the target DNA 63 is to be examined. In FIG. 6(b), the
complementary strand extension takes place when the primer
consisting of the first probe 61 and the second probe 62 is
hybridized with the target DNA 63, since base 6 (G) at the 3'-end
of the primer and base 35 (C) on the target position of the target
DNA are complementary as shown in FIG. 1(a). Namely, this is an
example in which the complementary strand extension takes place for
the wild type without substitution.
[0092] In contrast, FIG. 6(c) illustrates an example in which no
complementary strand extension reaction takes place when a primer
hybridizes with a mutant DNA having a base substitution. In the
example shown in FIG. 6(c), base 6 (G) at the 3'-end of the primer
and base 35 (A) on the corresponding position of the target DNA are
not complementary. Therefore, as explained in FIG. 1(b), no
extended complementary strand is formed by the hybridization of the
primer. In this Example, unlike the case explained in FIG. 1(b), no
extended complementary strand is formed despite that the primer is
not complementary, as primer SNP1-2 or SNP1-3 of FIG. 3(a). This is
an effect of using the two probes 61 and 62 as a single primer.
[0093] As shown in FIG. 6(d), in this Example 2, hybridization is
unstable because the mutation 69 (base G in Figure (d)) is located
on any one of the positions on the target DNA, where the second
probe 62 is to be hybridized, and the third base from the 3'-end of
the second probe 62 is T. Namely, since the primer consisting of
the first probe 61 and the second probe 62 is not stably formed, no
extended complementary strand is formed even if base 6 (G) at the
3'-end of the primer and base 35 (C) on the corresponding position
on the target DNA are complementary. Again, this is an effect of
composing a primer with two probes 61 and 62. In the case where the
mutation 69 (G in the example in FIG. 6(d)) exists, it is needless
to say that hybridization is stable and an extended complementary
strand is formed only when the third base from the 3'-end of the
second probe 62 is C in the case where the sequence of site 65 of
the target DNA 63 is perfectly complementary to the sequence of the
second probe 62, in the example of FIG. 6(d).
[0094] Accordingly, the presence or absence of the mutation can be
detected by examining the presence or absence of formation of
pyrophosphate 68 by examining the presence or absence of
photo-emission in the same manner as described in Example 1. Of
course, alternatively, the primer 62 can be designed so as to
proceed the complementary strand extension reaction only when
mutation exists.
[0095] Practically, it is preferable to carry out independent
reactions using short primers each complementary to wild type and
mutant sequences and analyze SNPs or estimate their ratio using the
results of the reactions.
[0096] Alternatively, the position of mutation is designed to
correspond to the 3'-end of the first probe so that the second
probe cannot hybridize with the target DNA adjoining close to the
first probe if mutation exists.
EXAMPLE 3
[0097] In order to examine SNPs, DNA is extracted from a blood
sample and stored as a library for use in various tests. Also, the
extracted DNA is cleaved into an average length of several kb and
the resulting DNA fragments are PCR-amplified and stored as test
samples. For various SNP tests, a portion of the stored DNA sample
is taken and a necessary short DNA region (100-200b) is again
PCR-amplified for use. However, it is too labor-intensive,
time-consuming and costly to carry out amplification every time for
measurement. Therefore, a method which requires no PCR
amplification has been desired. In the present method, a high
sensitivity can be attained because a large number of pyrophosphate
are produced simultaneously upon the complementary strand extension
and used for the chemiluminescence reaction. A complementary strand
of about 1 kb is synthesized, and the measurement can be carried
out with a sample less than 0.1 fmol and occasionally without PCR.
Example 3 is an example in which sensitivity is further improved so
that no PCR is required.
[0098] In Example 3, extended complementary strands produced in
Example 1 and Example 2 are cleaved with an enzyme and the
complementary strand extension is again carried out so that further
more amounts of pyrophosphate are produced to improve the detection
sensitivity.
[0099] FIG. 7 illustrates Example 3. The Figure explains the
principle in which the extended complementary strand is cleaved
with an enzyme and the complementary strand extension is repeated
to produce an enormously large number of pyrophosphate molecules to
improve the detection sensitivity.
[0100] First, as shown on top of the FIG. 7, a target DNA 85, which
is immobilized on a solid carrier 81, such as a bead, at the
5'-end, and a primer 86 are placed with DNA polymerase in a buffer
solution. As a result, as explained in FIG. 4(c), pyrophosphate 87
is produced upon the formation of an extended complementary strand
82 of the primer 86. Unlike Example 1, in this Example 3, enzymes
for photo-emission are not placed in the buffer solution in the
stage of extension of the complementary strand 82 of the primer
86.
[0101] Then the complementary strand extension reaction ends when
the extension of the complementary strand 82 proceeds to a certain
extent, and the complementary strand 83 consisting of the extended
complementary strand 82 of the primer 86 and the target DNA 85 form
a double-stranded DNA. In this state, exonuclease is put into the
buffer solution to cleave the complementary strand. As a result,
the complementary strand 83 formed with the extended complementary
strand 82 of the primer 86 is decomposed into nucleotides 84 one by
one by exonuclease reaction, and ultimately the target DNA 85 is
recovered.
[0102] Exonucleases to cleave complementary strands will be
explained as follows. A 5'3' exonuclease cuts off nucleotides one
by one in the direction from the 5'-end to the 3'-end of a DNA
strand while a 3'5' exonuclease cuts off nucleotides from the
3'-end of a DNA strand. In the case where the match or mismatch of
the 3'-end of primer is used to detect the presence or absence of
the complementary strand synthesis, the 3'5' exonuclease is not
appropriate because the mismatch site of the primer is cut off and
the wild-type and mutant cannot be distinguished. The 5'3'
exonuclease decomposes the DNA stand from the 5'-end of a
double-stranded DNA. The 5'-end of the target DNA 85 is immobilized
to the solid carrier 81 such as a bead or is modified so that the
5'3' exonuclease does not decompose the target DNA. The 5'3'
exonuclease is suitable for distinguishing mutation using the
mismatch of the 3'-end.
[0103] The primer 86 excessibly present in the buffer solution
again hybridizes with this target DNA 85 and thus the extended
complementary strand 82 of the primer 86 is again formed. Thus,
pyrophosphate 87 is newly produced.
[0104] Supposing that n molecules of pyrophosphate 87 are produced
by the formation of the extended complementary strand 82 of the
primer 86 and that the formation of the extended complementary
strand 82 and decomposition of the complementary strand 83 by the
5'3' exonuclease reaction are repeated m times, (m.times.n)
molecules of pyrophosphate 88 are to be produced as a result of the
total of m times of formation of the extended complementary strand
82.
[0105] After the production of (m.times.n) molecules of
pyrophosphate 88, enzymes for photo-emission are added to the
buffer solution. Pyrophosphate 88 is then converted to ATP and the
ATP oxidizes luciferin to newly produce pyrophosphate PPi. The
newly produced PPi turns into ATP by the action of ATP sulfurylase
and again reacts with luciferin. Accordingly, if m=10, namely the
repeat is 10 times, the photo-emission by this method is about
10000 times stronger than that by pyrosequencing or the like, since
about 1000 molecules of pyrophosphate are produced when one DNA
complementary strand is formed.
[0106] The formation of the extended complementary strand 82 and
decomposition of the complementary strand 83 can be simultaneously
proceeded. In this case, DNA polymerase having a 5' exonuclease
activity is preferably used.
[0107] In the case where the formation of the extended
complementary strand 82 and the decomposition of the complementary
strand 83 are repeated in the same manner as described above using
a 3'5' exonuclease instead of the abovementioned 5'3' exonuclease,
the primer 86 is modified to have a sulfur bonding at the 3'-end
instead of a phosphoric acid bonding. In this way, a nucleotide in
the primer, which is complementary to the base at the position to
be examined for mutation, can be protected from decomposition and
cut-off by the exonuclease. A DNA polymerase having a strong 3'
exonuclease activity is conveniently used.
[0108] In either cases, the primer 86 hybridizes with the target
DNA 85. The complementary strand extension proceeds or does not
proceed according to match or mismatch of the 3'-end of the primer
86, which determines the presence or absence of mutation. Mutation
can be accurately examined, for example, by modifying the sequence
to have an artificial mismatch near the 3'-end of the primer 86, if
necessary.
EXAMPLE 4
[0109] Example 4 is an example in which a multiple number of
primers are used. A simple examination to detect the incidence of
formation of the extend complementary strand using a single primer
is not satisfactory to determine the type of mutation or to exactly
know the kind or the frequency of mutation present in a sample
DNA.
[0110] Therefore, it is desirable to examine mutations which occur
both strands of a double-stranded DNA of interest. It is effective
to examine whether an extended complementary strand is formed using
a total of four kinds of primers which are complementary to the
wild type and mutant of both strands.
[0111] Complementary strand extension reactions starting from these
four kinds of primers are carried out independently but under the
same conditions, and the intensity of the resulting
chemiluminescence is compared to obtain information such as the
ratio of existing mutation. In order to equalize the reaction
conditions, the four kinds or multiple numbers of primers are
immobilized on different positions of the solid surface by kind or
on different beads by kind and then the complementary strand
extension is carried out in the same reaction solution in a
spatially separated state. Of course, the reaction can be carried
out by placing the primers individually in partitioned small
reaction cells. Further, a reaction part can be used, in which the
reaction solution can move between the cells while the primers are
immobilized on beads or the like and cannot move between the cells.
Further, not only the four kinds of primers but also a group of
primers corresponding to various SNPs can be examined
simultaneously so that various SNPs can be detected simultaneously.
Further, genomes of many people can be examined for one or several
SNPs. Pyrophosphate produced by the complementary strand extension
reaction and chemiluminescence generated through ATP produced from
the pyrophosphate are observed discriminately according to the kind
of primers.
[0112] FIGS. 8 and 9 illustrate an example of the structure of the
SNP detection apparatus of Example 4. FIG. 8 is a perspective view
and FIG. 9 is a cross-sectional view taken on the dotted broken
line 9-9 of FIG. 8 viewing in the direction shown with an arrow (Y
direction).
[0113] In this example, partitioned reaction cells 102-i, 103-i,
104-i, and 105-i (i=1, 2, . . . , N) are formed on a reaction chip
101 having the transparent bottom, and beads 106-i, 107-i, 108-i,
and 109-i (i=1, 2, . . . , N) on which primers are immobilized, are
held in the cells according to the kind of the primers. On the
other hand, reaction fluid cells 100-i (i=1, 2, . . . , N) are
formed parallel to the reaction cells 102-i, 103-i, 104-i, and
105-i (i=1, 2, . . . , N). The reaction fluid cells 100 and the
reaction cells 102 in corresponding positions are connected at the
bottom. Reaction fluids 110-i (i=1, 2, . . . , N) containing a
single-stranded target DNA are injected into the reaction fluid
cells 100-i (i=1, 2, . . . , N). Since the reaction fluid cells 100
and the reaction cells 102 are connected at the bottom, reaction
fluids 110-i (i=1, 2, . . . , N) injected into the reaction fluid
cells 100-i (i=1, 2, . . . , N) are supplied into the reaction
cells 102-i, 103-i, 104-i, and 105-i (i=1, 2, . . . , N).
[0114] Prior to the complementary strand extension reaction,
primer-i (i=1, 2, . . . , N) and target DNA-i (i=1, 2, . . . , N)
are thoroughly hybridized. After that, a mixed solution of the four
kinds of dNTP is added to each of the reaction cells 100-i (i=1, 2,
. . . , N) to start the complementary strand extension
reaction.
[0115] Chemiluminescence generated as a result of the complementary
strand extension reaction is detected using a lens 111 placed at
the transparent bottom of the reaction chip 101 or a line sensor or
two-dimensional sensor photo-detector 112 through the transparent
bottom of the reaction chip 101. In the case where the line sensor
112 is used, the photo-emission is measured by scanning a detector
support 113 along the bottom of the reaction chip 101. In the case
where the two-dimensional sensor is used, scanning of the detector
support 113 is not necessary.
[0116] Of course, the abovementioned light-detecting system can be
placed on the top of the reaction chip 101. Further, the light can
be detected by scanning the bottom or the top surface of the
reaction chip 101 using a photon multiplier tube and measuring the
photo-emission for every reaction cell in a designated order.
[0117] In the SNP detection apparatus shown in FIG. 8 and FIG. 9,
multiple kinds of target DNAs can be analyzed simultaneously.
[0118] The abovementioned example refers to primers having
different kinds of bases at the 3'-end. This system can also be
used effectively to detect base substitutions which occur different
sites of the target DNA, in which multiple kinds of different
primers are prepared and base substitutions on different sites of
the target DNA can be collectively detected. In this case, the
reaction chip 101 is constructed to have a multiple number, at
least the number of the kind of primers, of reaction cells in the X
direction.
[0119] Further, in order to simplify the structure of the
apparatus, the reaction part can be placed on a turntable.
[0120] FIG. 10 illustrates another embodiment of the structure of
the SNP detection apparatus of Example 4. In the structure shown in
FIG. 10, four reaction chambers 122-i, 123-i, 124-i, and 125-i
having transparent bottoms are held on a round turntable 121 in the
radial direction. Beads each having different primers immobilized
on the surface are held in the reaction chambers. The bottom areas
of the reaction chambers 122-i, 123-i, 124-i, and 125-i are the
same.
[0121] A multiple number of the reaction chambers 122-i, 123-i,
124-i, and 125-i (i=1, 2, . . . , N) are placed on the round
turntable 121 in the peripheral direction. A reaction solution
containing a target DNA is added to the reaction chambers 122-i,
123-i, 124-i, and 125-i (i=1, 2, . . . , N) using an injection
nozzle 126 having four flow-out openings, and the target DNA is
hybridized with the primers immobilized on the beads. Then, a dNTP
mixed solution is added and the complementary strand extension
proceeds in each reaction chamber.
[0122] Chemiluminescence generated as a result of the complementary
strand extension is examined one by one for the multiple reaction
chambers placed on the turntable 121 in the peripheral direction
using photon multiplier tubes 127, 128, 129, and 130 placed under
the turntable 121, while the turntable 121 turns.
EXAMPLE 5
[0123] In Example 5, the complementary strand extension reaction is
carried out by hybridizing the two oligomers illustrated in Example
2 in a series with the target. Mutations are examined by changing
the kind of the second oligomer (the second probe illustrated in
Example 2) instead of using the four kinds of primers illustrated
in Example 4, which are immobilized on a solid.
[0124] In a method of Example 5, the first oligomer (the first
probe illustrated in Example 2) is immobilized on the surface of
solid carriers, such as beads, so that the kinds of primers to
immobilize on the solid carriers can be advantageously reduced. The
solid carriers on which the first oligomer (probe) is immobilized
are placed in the reaction cells or the reaction chambers
illustrated in Example 4.
[0125] FIG. 11 illustrates an example of the structure of primers,
in which a single-stranded DNA derived from the p53 gene, DNA 200,
containing the sequence of the exon 8, is used as a target DNA. The
DNA 200 has a standard base sequence of 158 base length as shown in
SEQ ID NO: 10. Of course, DNA 200 is a DNA strand which contains a
158-base long standard base sequence shown in SEQ ID NO: 10 and the
following discussion for the detection of mutations 150, 151, 152,
153, and 154 is applicable also in the case where a base sequence
derived from the p53 gene is linked at the 5'-end and/or the 3'-end
in addition to the sequence shown in FIG. 11.
3 TGGTAATCTA CTGGGACGGA AACAGCTTTG SEQ ID NO: 10 AGGTGCGTGT
TTGTGCCTGT CCTGGGAGAG ACCGGCGCAC AGAGGAAGAG AATCTCCGCA AGAAAGGGGA
GCCTCACCAC GAGCTGCCCC CAGGGAGCAC TAAGCGAGGT AAGCAAGCAG GACAAGAA
[0126] Long oligomers 142, 144, 146, and 148 corresponding to
mutations are prepared as shown in FIG. 11. Both chains of the
double-stranded DNA can be detected to improve the accuracy
although only one side of the DNA chains is illustrated in the
Figure for simplification. The ends of these long oligomers are
treated to be incapable of proceeding the complementary strand
extension in the same manner with the first probe 61 shown in FIGS.
6(a), 6(b), 6(c) and 6(d).
[0127] Six-base-long oligomers 141, 143, 145, and 147 are prepared
to be hybridized with the target DNA adjoining closely to the long
oligomers 142, 144, 146, and 148. The 6-base-long oligomers (141,
143, 145, and 147) correspond to the second probe 62 shown in FIGS.
6(b), 6(c), and 6(d). As illustrated in Example 2, the 6-base-long
oligomers (141, 143, 145, and 147) alone hybridize with the target
DNA and synthesize complementary strands in a low probability, but
they can hybridize and synthesize complementary strands when they
closely adjoin to the long oligomers 142, 144, 146, and 148.
[0128] However, in the case where the 3'-end of the long oligomers
142, 144, 146, and 148 mismatches to the target DNA, or the 3'-end
of the 6-base-long oligomers (141, 143, 145, and 147) or either one
of the bases in the sequences of the 6-base-long oligomers
mismatches to the target DNA, the 6-base-long oligomers do not
hybridize with the target DNA. Of course, 5-base-long oligomers can
also be used.
[0129] In this case, the primers are designed such that the 3'-end
of the 6-base-long oligomers comes to the point to be tested for
mutation of the target DNA, and the examination is carried out. Not
only the presence or absence of mutations but also their ratio can
be recognized by synthesizing complementary strands using
6-base-long primers each complementary to the wild type and mutant
sequences.
[0130] Therefore, the primers are designed such that the 3'-end of
the 6-base-long oligomers comes to the point to be tested for
mutation of the target DNA, and the examination is carried out. A
readiness of occurring the complementary strand extension reaction
or the like depending on the kind of bases at the 3'-end of
6-base-long oligomers can also be examined by changing the kind of
6-base-long oligomers.
[0131] Actually in the exon 8 of the p53, mutations at positions
150, 151, 152, 153, and 154 indicated with arrows in FIG. 11 can be
detected. The mutations at positions 150, 151, 152, 153, and 154
indicated with arrows in FIG. 11 are T A, G A, C T, GC AT, and C T
when the substitution of standard base X by base Y is expressed X Y
(in which X and Y are either A, T, G, or C).
[0132] FIG. 12 shows structures of probes which are incapable of
proceeding the complementary strand extension and 6-base-long
primers for the detection of the mutations shown in FIG. 11.
[0133] The probe 142 incapable of proceeding the complementary
strand extension binds complementarily to the region from position
43 to 58 from the 5'-end of the sequence of SEQ ID NO: 10 and has
the 16-base-long sequence of SEQ ID NO: 11. Of course, the probe
142 can be a 20- to 30-base-long oligomer. N is a base, either G or
T, corresponding to the wild type or mutant sequence.
[0134] CTCCCANGAC AGGCAC (SEQ ID NO: 11)
[0135] The 6-base-long primer 141 binds complementarily to the
region from position 37 to 42 from the 5'-end of the sequence of
SEQ ID NO: 10 and has the 6-base-long sequence of SEQ ID NO: 11'. N
is either A or T.
[0136] NAACAC (SEQ ID NO: 11')
[0137] It is understood that the mutation 150 exists if signal is
obtained when N=T and not when N=A.
[0138] The probe 144 incapable of proceeding the complementary
strand extension binds complementarily to the region from position
58 to 72 from the 5'-end of the sequence of SEQ ID NO: 10 and has
the 16-base-long sequence of SEQ ID NO: 12. The probe can have any
base length as long as the length is sufficient for stable
hybridization, and thus longer oligomer can be used if necessary. N
at eleventh base from 5' terminal is G or A, and NN at sixth to
seventh base from 5' terminal is GC or AT.
[0139] TCTGTNNGCC NGTCTC (SEQ ID NO: 12)
[0140] The 6-base-long primer 143 binds complementarily to the
region from position 52 to 57 from the 5'-end of the sequence of
SEQ ID NO: 10 and has the 6-base-long sequence of SEQ ID NO: 12'. N
is either T or G.
[0141] TCCCAN (SEQ ID NO: 12')
[0142] The probe 146 incapable of proceeding the complementary
strand extension binds complementarily to the region from position
69 to 84 from the 5'-end of the sequence of SEQ ID NO: 10 and has
the 16-base-long sequence of SEQ ID NO: 13.
[0143] GTTTCTCTTC CTCTGT (SEQ ID NO: 13)
[0144] The 6-base-long primer 145 binds complementarily to the
region from position 63 to 68 from the 5'-end of the sequence of
SEQ ID NO: 10 and has the 6-base-long sequence of SEQ ID NO: 13'. N
at 3' terminal is G or A, and NN at the first and second bases from
5' terminal is GC or AT.
[0145] NNGCCN (SEQ ID NO: 13')
[0146] In order to discriminate mutations of different positions,
four primers including ones for the wild type are prepared (eight
primers if both chains of the double-stranded DNA are to be
examined). Primers corresponding to the wild type sequence can be
used as a mixture when only the presence or absence of mutation is
to be examined. Of course, one or more mutations can be detected
one by one by shifting the positions of the primers which bind
complementarily to the target DNA.
[0147] The probe 148 incapable of proceeding the complementary
strand extension binds complementarily to the region from position
141 to 156 from the 5'-end of the sequence of SEQ ID NO: 10 and has
the 16-base-long sequence of SEQ ID NO: 14.
[0148] CTTGTCCTGC TTCGTT (SEQ ID NO: 14)
[0149] The 6-base-long primer 147 binds complementarily to the
region from position 135 to 140 from the 5'-end of the sequence of
SEQ ID NO: 10 and has the 6-base-long sequence of SEQ ID NO: 14'. N
is either G or A.
[0150] ACCTCN (SEQ ID NO: 14')
[0151] If N=A, the presence of the mutation 154 can be
detected.
[0152] Of course, the SNP detection apparatus illustrated in
Example 4 can be used for the detection of mutations illustrated in
Examples above.
[0153] As illustrated above, according to the present invention,
the number of pyrophosphate molecules produced at a time can be
increased by simultaneously adding the four kinds of dNTP to the
reaction part to proceed the complementary strand extension, and
the resulting photo-emission intensity can be increased by several
orders higher than that obtained by the conventional pyrosequencing
method, by repeating the pyrophosphate production process.
[0154] Accordingly, the presence or absence of the target DNA and
the presence or absence of mutations can be examined without the
need for an amplifying process such as PCR. Further, when the wild
type and mutant are present in the target DNA at a certain ratio,
various base substitutions can be examined by using respectively
corresponding primers each immobilized on the surface of different
solid carriers. In particular, substitutions on multiple sites
contained in a long DNA can be discriminated. The method of the
present invention can be applicable to a high throughput apparatus
to simultaneously analyze a multiplicity of DNA samples, for
example, by immobilizing the target DNAs on beads or on partitioned
solid surface.
[0155] Characteristics of mutation (SNP) detection methods of the
present invention are listed as follows.
[0156] In a mutation detection method of the present invention, a
primer, which has a sequence complementary to a region of a
specific DNA sequence or a region of a specific RNA sequence to be
detected, hybridizes with a DNA sample or an RNA sample and whether
a complementary strand extension reaction with nucleic acid
substrates (dATP, dTTP, dGTP and dCTP, or analogues thereof) using
DNA polymerase in the case of DNA sample or reverse transcriptase
in the case of RNA samples proceed or not is detected by converting
pyrophosphate produced as a reaction by-product of the
complementary strand extension into ATP, carrying out a
photo-emission reaction using luciferase or the like and detecting
the resulting photo-emission, whereby the presence or absence of
the specific sequence is detected.
[0157] In the complementary strand extension reaction using a
primer which directly hybridizes with the RNA sample, four kinds of
nucleic acid substrates, and reverse transcriptase, cDNA is formed
and at this moment RNA mutations can be detected by detecting
dissociating pyrophosphate.
[0158] In the abovementioned method, in addition to the primer
which hybridizes with the specific sequence, multiple kinds of
nucleic acid substrates are simultaneously added, the complementary
strand extension reaction is repeated, the resulting pyrophosphate
is converted into ATP, and thereby a photo-emission reaction is
carried out to detect the photo-emission. Further, in addition to
the primer which hybridizes with the specific sequence, four kinds
of nucleic acid substrates are simultaneously added, the
complementary strand extension proceeds, the resulting ATP is
converted into pyrophosphate, and a photo-emission reaction is
carried out for photo-emission detection.
[0159] Further in the abovementioned method, a nucleotide which is
not complementary to the target DNA or the target RNA is
artificially inserted in the vicinity of the 3'-end of the primer,
the resulting pyrophosphate is converted into ATP, and a
photo-emission reaction is carried out with a photo-emission
reagent such as luciferin using luciferase or the like to detect
the photo-emission, whereby SNPs are detected by examining whether
the complementary strand extension is proceeded or not.
[0160] Further, in the abovementioned method, the complementary
strand synthesis and chemiluminescence detection are each
independently carried out using at least two kinds of primers,
which are capable of hybridizing with wild type and mutant
sequences and proceeding the complementary strand synthesis, and
the presence or absence and the ratio of mutation can be accurately
determined using the resultant information. Further, the
abovementioned two kinds of primers are prepared for each of a
double-stranded DNA (totally four kinds), and the complementary
strand synthesis and photo-emission detection are carried out to
accurately determine the ratio of mutation. This is effective when
a correlation between diseases and SNPs is to be statistically
studied for a sample which is a mixture of various genome DNAs.
[0161] Further, in the abovementioned method, the extended
complementary strand by the complementary strand extension reaction
is decomposed from its ends by an enzyme and the primers repeatedly
hybridize with a target DNA or a target RNA to repeatedly proceed
the complementary strand extension reaction, and a multiplicity of
PPi produced by a series of repeated complementary strand extension
reaction are converted into ATP, which is reacted with luciferin to
generate photo-emission to be detected. The enzyme to decompose the
extended complementary strand is one which decomposes a DNA strand
or RNA strand from the 5'-end.
[0162] Further, in the abovementioned method, the complementary
strand extension is carried out using a primer which is
complementary to a target DNA strand or RNA strand and the 3'-end
of which is bound to targeted two-base substitution site or its
vicinity, pyrophosphate resulting from the complementary strand
extension is converted into ATP or the like, and a reaction with a
photo-emitting substrate such as luciferin is carried out to
generate chemiluminescence to detect SNPs.
[0163] A reagent kit of the present invention for detecting SNPs or
examining the SNP ratio at least includes a primer, which is
complementary to a target DNA strand or RNA strand and the 3'-end
of which is bound to a targeted single base substitution site or
its vicinity, and nucleic acid substrates (dATP, dTTP, dGTP, and
dCTP, or analogues thereof).
[0164] Further, a reagent kit of the present invention for
detecting SNPs or examining the SNP ratio at least includes a
primer, which is complementary to a target DNA strand or RNA strand
and the 3'-end of which is bound to a targeted single base
substitution site or its vicinity, nucleic acid substrates (dATP,
dTTP, dGTP, and dCTP, or analogues thereof), and an enzyme which
decomposes pyrophosphate contained as an impurity.
[0165] In the method for detecting SNPs of the present invention,
primers having different kinds of nucleotides at their ends are
held separately by kind of the primers in different cells on the
solid surface or in different reaction chambers, and a mixture of
multiple kinds of dNTP is added to the cells or a reaction solution
of the reaction chambers to provide conditions where the
complementary strand extension reaction starting from the primers
can take place, pyrophosphate produced as a result of actual
complementary strand extension reaction starting from the primers
which hybridize with a target DNA or a target RNA is converted into
ATP, and chemiluminescence produced by reacting the ATP with
luciferin or the like is detected to discriminate the kind of
primers which are engaged in the complementary strand extension
reaction, whereby mutations in the target DNA or target RNA are
detected.
[0166] Further, in the method for detecting SNPs of the present
invention, multiple kinds of long oligomers, which are different in
sequence from each other and are capable of hybridizing with a
target DNA or a target RNA but incapable of proceeding the
complementary strand extension reaction, are held separately by
kind of the long oligomers in different cells on the solid surface
or in different reaction chambers, 5-base-long to 8-base-long short
oligomers capable of proceeding the complementary strand extension
reaction are added to the cells or the reaction chambers, the short
oligomers and said long oligomers are serially hybridized with the
target. DNA or the target RNA, the complementary strand extension
reaction starting from the short primers proceeds by further adding
a mixture of multiple kinds of dNTP, pyrophosphate produced as a
result of the reactions is converted into ATP, and
chemiluminescence produced by reacting the ATP with luciferin or
the like is detected to discriminate the kind of the primers which
are engaged in the complementary strand extension reaction, whereby
mutations in the target DNA or the target RNA are detected. In this
case, the chemiluminescence reagent is not limited to
luciferin.
[0167] A mutation detection apparatus of the present invention
comprises a reaction chip furnished with multiple cells on the
solid surface, which holds primers differing in kinds of end
nucleotides from each other, separately by kind of primers, or a
reaction chamber supporting base on which multiple reaction
chambers holding the primers separately by kind are placed; and a
light detector to detect chemiluminescence which is produced by
reacting ATP with luciferin or the like; said ATP being converted
from pyrophosphate produced as a result of actual complementary
strand extension reaction starting from the primers hybridized with
a target DNA or a target RNA under the conditions where the
complementary strand extension reaction starting from the primers
can take place by adding a mixture of multiple kinds of dNTP to the
cells or a reaction solution in the reaction chambers, whereby the
kind of primers which are engaged in the complementary strand
extension reaction is discriminated to detect mutations contained
in the target DNA or the target RNA.
[0168] A mutation detection apparatus of the present invention for
detecting mutations in a target DNA or a target RNA comprises a
reaction chip furnished with multiple cells on the solid surface,
which hold multiple kinds of long oligomers, which are capable of
hybridizing with the target DNA or the target RNA but incapable of
being engaged in the complementary strand extension and are
different in sequence from each other, separately by kind of the
oligomers, or a reaction chamber supporting base on which multiple
reaction chambers holding the long oligomers separately by kind of
the oligomers are placed; and a light detector to detect
chemiluminescence which is produced by reacting ATP with luciferin
or the like; said ATP being converted from pyrophosphate produced
by a serial hybridization of short oligomers and the long oligomers
with the target DNA or the target RNA by adding 5-base-long to
8-base-long short oligomers capable of being engaged in the
complementary strand extension reaction respectively to the cells
or the reaction chambers, followed by the subsequent complementary
strand extension reaction starting from the short primers by
further adding a mixture of multiple kinds of dNTP, whereby the
kind of the short primers which are engaged in the complementary
strand extension reaction is discriminated to detect mutations
contained in the target DNA or the target RNA
[0169] Further, a method for detecting mutations according to the
present invention comprises a step of hybridizing a primer which
contains a sequence complementary to a region of a specific
sequence of a target DNA or a target RNA; a step of carrying out
the complementary strand extension reaction starting from the
primer using four kinds of nucleic acid substrates and polymerase
or reverse transferase; and a step of detecting photo-emission in
which pyrophosphate produced as a reaction by-product of the
complementary strand extension reaction is converted into ATP and a
photo-emission reaction is carried out using an enzyme, whereby the
presence or absence of the specific sequence is detected.
[0170] Further, a method for detecting mutations according to the
present invention comprises a step of hybridizing a primer which
contains a sequence complementary to a region of a specific
sequence of a target DNA or a target RNA and a sequence not
complementary to the sequence of the target DNA or the target RNA
at the second base or the third base from the 3'-end; a step of
carrying out the complementary strand extension reaction starting
from the primer using four kinds of nucleic acid substrates and
polymerase or reverse transferase; and a step of detecting
photo-emission in which pyrophosphate produced as a reaction
by-product of the complementary strand extension reaction is
converted into ATP and a photo-emission reaction is carried out
using an enzyme, whereby the presence or absence of the specific
sequence is detected.
[0171] Further, a method for detecting mutations according to the
present invention comprises a step of serially hybridizing a
5-base-long to 8-base-long short oligomer, which is capable of
being engaged in the complementary strand extension reaction, and a
long oligomer, which is capable of hybridizing with a target DNA or
a target RNA but incapable of being engaged in the complementary
strand extension reaction, with the target DNA or the target RNA; a
step of carrying out the complementary strand extension reaction
starting from the short primer using four kinds of nucleic acid
substrates and polymerase or reverse transferase; and a step of
detecting photo-emission, in which pyrophosphate produced as a
reaction by-product of the complementary strand extension reaction
is converted into ATP and the photo-emission reaction is carried
out using an enzyme, whereby the presence or absence of the
specific sequence is detected.
[0172] A detection method of the present invention uses two probes
(primers) each corresponding to target wild type and mutant
sequences to improve accuracy, and its use is not limited to the
detection of pyrophosphate produced as a result of the
complementary strand synthesis. Products, i.e., DNA strands, can be
labeled with different labeling fluorescent substances and analyzed
for comparison by electrophoresis or the like. Further, the method
can be applied to other SNP detection methods. Namely, a pair of
probes, one acting on a wild type sequence and the other acting on
the mutant sequence to generate signals, are effectively used to
accurately measure the ratio of mutation.
[0173] As illustrated above, according to the present invention,
mutation can be readily detected with great accuracy by (1) using
primers which are specifically designed considering the presence or
absence of mutations contained in DNAs, (2) judging whether the
primers form extended complementary strands by the complementary
extension reaction; and (3) increasing the number of pyrophosphate
molecules released by the complementary strand extension reaction
by two orders greater than that obtained using the conventional
pyrosequencing, in the detection of the extended complementary
strand. Further, the mutation ratio in multiple samples containing
wild type and mutant sequences can be accurately examined by the
use of multiple probes or primers.
[0174] Further, according to the present invention, DNA samples
containing multiple kinds of DNA mutations can be analyzed. Namely,
various information required for DNA mutation measurements (e.g.,
whether only an anticipated base sequence exists, whether a
mutation exists, what kind of mutations appears and where mutations
appear, or how many sites mutations appear) can be readily obtained
by examining the mutations using multiple kinds of primers taking
the anticipated base sequences into consideration.
[0175] The present invention can provide a method and an apparatus
for detecting DNA mutations without the need for gel
electrophoresis, in which no emission light resource is necessary
because of its high sensitivity, and engaging reactions are simple
and quick, and further a method and an apparatus for detecting DNA
mutations, in which a sample containing multiple mutations can be
analyzed with great accuracy. Accordingly, various information
required for DNA mutation measurements can be readily obtained.
[0176] Sequence Listing
[0177] <110> HITACHI, LTD.
[0178] <120> Mutation Detection Method of DNA and Apparatus
for Mutation Detection
[0179] <130> H00011571A
[0180] <160> 18
[0181] <210> 1
[0182] <211> 20
[0183] <212> DNA
[0184] <213> Artificial Sequence
[0185] <220>
[0186] <223> DNA primer used for forming an extended
complementary strand
[0187] <400> 1
[0188] agttttaaga gggttgttgt 20
[0189] <210> 2
[0190] <211> 20
[0191] <212> DNA
[0192] <213> Artificial Sequence
[0193] <220>
[0194] <223> DNA primer used for forming an extended
complementary strand
[0195] <400> 2
[0196] agttttaaga gggttgttgc 20
[0197] <210> 3
[0198] <211> 20
[0199] <212> DNA
[0200] <213> Artificial Sequence
[0201] <220>
[0202] <223> DNA primer used for forming an extended
complementary strand
[0203] <400> 3
[0204] agttttaaga gggttgttgg 20
[0205] <210> 4
[0206] <211> 20
[0207] <212> DNA
[0208] <213> Artificial Sequence
[0209] <220>
[0210] <223> DNA primer used for forming an extended
complementary strand
[0211] <400> 4
[0212] agttttaaga gggttgttga 20
[0213] <210> 5
[0214] <211> 20
[0215] <212> DNA
[0216] <213> Artificial Sequence
[0217] <220>
[0218] <223> DNA primer having base sequence replaced T at 18
of SEQ ID NO: 1 by A for introducing a mismatch between DNA primer
and template DNA
[0219] <400> 5
[0220] agttttaaga gggttgtagt 20
[0221] <210> 6
[0222] <211> 20
[0223] <212> DNA
[0224] <213> Artificial Sequence
[0225] <220>
[0226] <223> DNA primer having base sequence replaced T at 18
of SEQ ID NO: 2 by A for introducing a mismatch between DNA primer
and template DNA
[0227] <400> 6
[0228] agttttaaga gggttgtagc 20
[0229] <210> 7
[0230] <211> 20
[0231] <212> DNA
[0232] <213> Artificial Sequence
[0233] <220>
[0234] <223> DNA primer having base sequence replaced T at 18
of SEQ ID NO: 3 by A for introducing a mismatch between DNA primer
and template DNA
[0235] <400> 7
[0236] agttttaaga gggttgtagg 20
[0237] <210> 8
[0238] <211> 20
[0239] <212> DNA
[0240] <213> Artificial Sequence
[0241] <220>
[0242] <223> DNA primer having base sequence replaced T at 18
of SEQ ID No: 4 by A for introducing a mismatch between DNA primer
and template DNA
[0243] <400> 8
[0244] agttttaaga gggttgtaga 20
[0245] <210> 9
[0246] <211> 20
[0247] <212> DNA
[0248] <213> Artificial Sequence
[0249] <220>
[0250] <223> Template DNA
[0251] <400> 9
[0252] tcaaaattct cccaacaaca 20
[0253] <210> 10
[0254] <211> 158
[0255] <212> DNA
[0256] <213> Artificial Sequence
[0257] <220>
[0258] <223> Template DNA originating from p53 and including
base sequence of exon 8
[0259] <400> 10
4 tggtaatcta ctgggacgga aacagctttg aggtgcgtgt ttgtgcctgt cctgggagag
60 accggcgcac agaggaagag aatctccgca agaaagggga gcctcaccac
gagctgaaaa 120 cagggagcac taagcgaggt aagcaagcag gacaagaa 158
[0260] <210> 11
[0261] <211> 16
[0262] <212> DNA
[0263] <213> Artificial Sequence
[0264] <220>
[0265] <221> unsure
[0266] <222> (7)
[0267] <223> DNA probe complementary with base sequence
between 43 and 58 of SEQ ID NO: 10 (n is a or c or g or t) and DNA
probe being not able to be extended
[0268] <400> 11
[0269] ctcccangac aggcac 16
[0270] <210> 12
[0271] <211> 16
[0272] <212> DNA
[0273] <213> Artificial Sequence
[0274] <220>
[0275] <221> unsure
[0276] <222> (6)-(7), (11)
[0277] <223> DNA probe complementary with base sequence
between 58 and 72 of SEQ ID NO: 10 (the base sequences of nn at 6
to 7.sup.th position from 5' terminal are cg or ta and the base
sequence of n at 11.sup.th position from 5' terminal is g or a.)
and DNA probe being not able to be extended
[0278] <400> 12
[0279] tctgtnngcc ngtctc 16
[0280] <210> 13
[0281] <211> 16
[0282] <212> DNA
[0283] <213> Artificial Sequence
[0284] <220>
[0285] <223> DNA probe complementary with base sequence
between 69 and 84 of SEQ ID NO: 10 and DNA probe being notable to
be extended
[0286] <400> 13
[0287] gtttctcttc ctctgt 16
[0288] <210> 14
[0289] <211> 16
[0290] <212> DNA
[0291] <213> Artificial Sequence
[0292] <220>
[0293] <223> DNA probe complementary with base sequence
between 141 and 156 of SEQ ID NO: 10 and DNA probe being not able
to be extended
[0294] <400> 14
[0295] cttgtcctgc ttcgtt 16
[0296] Sequence Listing Free Text
[0297] (1) Other related information on the sequence of SEQ ID NO:
1
[0298] A DNA primer used for forming an extended complementary
strand.
[0299] (2) Other related information on the sequence of SEQ ID NO:
2
[0300] A DNA primer used for forming an extended complementary
strand.
[0301] (3) Other related information on the sequence of SEQ ID NO:
3
[0302] A DNA primer used for forming an extended complementary
strand.
[0303] (4) Other related information on the sequence of SEQ ID NO:
4
[0304] A DNA primer used for forming an extended complementary
strand.
[0305] (5) Other related information on the sequence of SEQ ID NO:
5
[0306] A DNA primer having a base sequence in which base T at the
18th from the 5'-end of the sequence of SEQ ID NO: 1 is replaced by
base A for introducing a mismatch.
[0307] (6) Other related information on the sequence of SEQ ID NO:
6
[0308] A DNA primer having a base sequence in which base T at the
18th from the 5'-end of the sequence of SEQ ID NO: 2 is replaced by
base A for introducing a mismatch.
[0309] (7) Other related information on the sequence of SEQ ID NO:
7
[0310] A DNA primer having a base sequence in which base T at the
18th from the 5'-end of the sequence of SEQ ID NO: 3 is replaced by
base A for introducing a mismatch
[0311] (8) Other related information on the sequence of SEQ ID NO:
8
[0312] A DNA primer having a base sequence in which base T at the
18th from the 5'-end of the sequence of SEQ ID NO: 4 is replaced by
base A for introducing a mismatch.
[0313] (9) Other related information on the sequence of SEQ ID NO:
9
[0314] A target DNA.
[0315] (10) Other related information on the sequence of SEQ ID NO:
10
[0316] A target DNA which is a single-stranded DNA derived from the
p53 gene containing the sequence of the exon 8.
[0317] (11) Other related information on the sequence of SEQ ID NO:
11
[0318] A 6-base-long DNA probe which is complementarily bound to
the sequence from the 43rd to 58th from the 5'-end of the sequence
of SEQ ID NO: 10 and is incapable of being engaged in the
complementary strand extension reaction (n is either a, c, g, or
t).
[0319] (12) Other related information on the sequence of SEQ ID NO:
12
[0320] A 16-base-long DNA probe which is complementarily bound to
the sequence from the 58th to 72nd from the 5'-end of the sequence
of SEQ ID NO: 10 and is incapable of being engaged in the
complementary strand extension reaction (n1 is g or a, and n2n3 is
cg or ta).
[0321] (13) Other related information on the sequence of SEQ ID NO:
13
[0322] A 16-base-long DNA probe which is complementarily bound to
the sequence from the 69th to 84th from the 5'-end of the sequence
of SEQ ID NO: 10 and is incapable of being engaged in the
complementary strand extension reaction.
[0323] (14) Other related information on the sequence of SEQ ID NO:
14
[0324] A 16-base-long DNA probe which is complementarily bound to
the sequence from the 141st to 156th from the 5'-end of the
sequence of SEQ ID NO: 10 and is incapable of being engaged in the
complementary strand extension reaction.
Sequence CWU 1
1
14 1 20 DNA Artificial Sequence DNA primer used for forming an
extended complementary strand 1 agttttaaga gggttgttgt 20 2 20 DNA
Artificial Sequence DNA primer used for forming an extended
complementary strand 2 agttttaaga gggttgttgc 20 3 20 DNA Artificial
Sequence DNA primer used for forming an extended complementary
strand 3 agttttaaga gggttgttgg 20 4 20 DNA Artificial Sequence DNA
primer used for forming an extended complementary strand 4
agttttaaga gggttgttga 20 5 20 DNA Artificial Sequence DNA primer
having base sequence replaced T at 18 of SEQ ID NO 1 by A for
introducing a mismatch between DNA pri 5 agttttaaga gggttgtagt 20 6
20 DNA Artificial Sequence DNA primer having base sequence replaced
T at 18 of SEQ ID NO 2 by A for introducing a mismatch between DNA
primer and template DNA 6 agttttaaga gggttgtagc 20 7 20 DNA
Artificial Sequence DNA primer having base sequence replaced T at
18 of SEQ ID NO 3 by A for introducing a mismatch between DNA
primer and template DNA 7 agttttaaga gggttgtagg 20 8 20 DNA
Artificial Sequence DNA primer having base sequence replaced T at
18 of SEQ ID No 4 by A for introducing a mismatch between DNA
primer and template DNA 8 agttttaaga gggttgtaga 20 9 20 DNA
Artificial Sequence Template DNA 9 tcaaaattct cccaacaaca 20 10 158
DNA Artificial Sequence Template DNA originating from p53 and
including base sequence of exon 8 10 tggtaatcta ctgggacgga
aacagctttg aggtgcgtgt ttgtgcctgt cctgggagag 60 accggcgcac
agaggaagag aatctccgca agaaagggga gcctcaccac gagctgaaaa 120
cagggagcac taagcgaggt aagcaagcag gacaagaa 158 11 16 DNA Artificial
Sequence unsure (7) DNA probe complementary with base sequence
between 43 and 58 of SEQ ID NO 10 (n is a or c or g or t) and DNA
pro 11 ctcccangac aggcac 16 12 16 DNA Artificial Sequence unsure
(6)-(7), (11) DNA probe complementary with base sequence between 58
and 72 of SEQ ID NO 10 (the base sequences of nn at 6 to 7 and the
base sequence of n at 1lth position from 5' terminal is g or a.)
and DNA 12 tctgtnngcc ngtctc 16 13 16 DNA Artificial Sequence DNA
probe complementary with base sequence between 69 and 84 of SEQ ID
NO 10 and DNA probe being not able to be 13 gtttctcttc ctctgt 16 14
16 DNA Artificial Sequence DNA probe complementary with base
sequence between 141 and 156 of SEQ ID NO 10 and DNA probe being
not able to be extended 14 cttgtcctgc ttcgtt 16
* * * * *