U.S. patent application number 10/514558 was filed with the patent office on 2006-08-03 for novel method of assaying nucleic acid using labeled nucleotide.
This patent application is currently assigned to Kankyo Engineering Co, Ltd.. Invention is credited to Shinya Kurata, Kazunori Nakamura, Kyoto Takatsu.
Application Number | 20060172293 10/514558 |
Document ID | / |
Family ID | 29706555 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172293 |
Kind Code |
A1 |
Kurata; Shinya ; et
al. |
August 3, 2006 |
Novel method of assaying nucleic acid using labeled nucleotide
Abstract
A novel method is provided to assay at least one nucleic acid.
According to this method, a nucleic acid polymerization reaction is
conducted in a nucleic acid polymerization reaction system, which
contains (A) the at least one nucleic acid as a template, (B) at
least one nucleotide monomer labeled with at least one label
selected from the group consisting of (a) fluorescent dyes, (b)
quenchers and (c) immune related substances with a fluorescent dye
or quencher contained therein, and (C) at least one nucleic
acid-synthesizing enzyme. The template nucleic acid or a nucleic
acid, which has been synthesized using the template nucleic acid as
a template, is then assayed from a change or an amount of a change
in an optical character of the nucleic acid polymerization system.
This method makes it possible to specifically and accurately assay
at least one nucleic acid, which is contained in a single system
and can be an unknown nucleic acid and/or a known nucleic acid,
with excellent sensitivity, in short time and with ease.
Inventors: |
Kurata; Shinya; (Tokyo,
JP) ; Takatsu; Kyoto; (Tokyo, JP) ; Nakamura;
Kazunori; (Ibaraki, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Kankyo Engineering Co, Ltd.
1-9-8, Higashi-kanda, Chiyoda-ku
Tokyo
JP
101-0031
Nat Institute of Advance Indust Science & Tech.
3-1, Kasumigaseki 1-chome, Chiyoda-ku
Tokyo
JP
100-8921
|
Family ID: |
29706555 |
Appl. No.: |
10/514558 |
Filed: |
May 30, 2003 |
PCT Filed: |
May 30, 2003 |
PCT NO: |
PCT/JP03/06896 |
371 Date: |
November 30, 2004 |
Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 2563/107 20130101;
C12Q 1/6844 20130101; C12Q 1/6844 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2002 |
JP |
2002-160659 |
Claims
1. A method of assaying at least one nucleic acid, which comprises:
conducting a nucleic acid polymerization reaction in a nucleic acid
polymerization reaction system comprising (A) said at least one
nucleic acid as a template, (B) at least one nucleotide monomer
labeled with at least one label selected from the group consisting
of (a) fluorescent dyes, (b) quenchers and (c) immune related
substances with a fluorescent dye or quencher contained therein,
and (C) at least one nucleic acid-synthesizing enzyme; and assaying
said template nucleic acid or a nucleic acid, which has been
synthesized using said template nucleic acid as a template, from a
change or an amount of a change in an optical character of said
nucleic acid polymerization system.
2. A method according to claim 1, wherein said label is a
combination of at least one donor fluorescent dye and at least one
acceptor fluorescent dye.
3. A method according to claim 1, wherein said label is a
combination of at least one fluorescent dye and at least one
quencher.
4. A method according to claim 1, wherein said nucleic acid
polymerization system further comprises (D) at least one nucleotide
monomer not labeled with any label.
5. A method according to claim 1, wherein said nucleic acid
polymerization system further comprises (E) a nucleic acid primer
capable of specifically hybridizing to said template nucleic acid
and comprising at least one nucleotide monomer.
6. A method according to claim 5, wherein said nucleic acid primer
is labeled with (E') a label as defined in claim 1.
7. A method according to claim 1, wherein said nucleic acid
polymerization system further comprises a non-labeled
nucleotide.
8. A method according to claim 7, wherein said fluorescence-labeled
and/or non-labeled nucleotide comprises guanine (g) and/or said
template nucleic acid comprises at least one guanine (g).
9. A method according to claim 1, wherein said nucleic acid
polymerization system further comprises a non-labeled nucleotide
and a fluorescence-labeled nucleic acid primer without any
ingredient (B) as defined in claim 1.
10. A method according to claim 9, wherein said non-labeled
nucleotide comprises a guanine (g) base.
11. A method according to any one of claims 1, 7 and 9, wherein
said non-labeled nucleotide and/or labeled nucleotide is a
triphosphate.
12. A method of assaying a nucleic acid, which comprises:
conducting a nucleic acid polymerization reaction in a nucleic acid
polymerization system comprising said nucleic acid as a template,
at least one dideoxynucleotide monomer labeled with at least one
fluorescent dye and/or at least one quencher, and a nucleic
acid-synthesizing enzyme; and assaying said template nucleic acid
or a nucleic acid polymer, which has been synthesized using said
template nucleic acid as a template, from a change or an amount of
a change in fluorescence intensity.
13. A method according to claim 12, wherein said nucleic acid
polymerization system is a system further comprising a labeled
nucleotide or a non-labeled nucleotide or both of them.
14. A method according to claim 12, wherein said nucleic acid
polymerization system further comprises a non-labeled nucleic acid
primer.
15. A method according to any one of claims 1, 7, 9 and 12, wherein
said nucleic acid polymerization system further comprises (F) a
fluorescent dye capable of emitting fluorescence upon binding to a
nucleic acid.
16. A method of assaying a nucleic acid, which comprises:
conducting a nucleic acid polymerization reaction in a nucleic acid
polymerization system comprising said nucleic acid as a template, a
non-labeled dideoxynucleotide monomer, a labeled nucleotide, a
non-labeled nucleic acid primer, at least one fluorescent dye cable
of emitting fluorescence upon binding to a nucleic acid, and a
nucleic acid-synthesizing enzyme; and assaying said template
nucleic acid or a nucleic acid polymer, which has been synthesized
using said template nucleic acid as a template, from a change or an
amount of a change in fluorescence intensity.
17. A method according to claim 12 or 16, wherein said non-labeled
dideoxynucleotide, labeled dideoxynucleotide, non-labeled
nucleotide and/or labeled nucleotide is a triphosphate.
18. A method according to any one of claims 1, 7, 9, 12 and 16,
wherein said nucleic acid-synthesizing enzyme is at least one
enzyme selected from DNA polymerases, RNA polymerases, reverse
transcriptases, and modifications thereof.
Description
TECHNICAL FIELD
[0001] This invention relates to a method of assaying plural
nucleic acids, and specifically to a method of assaying at least
one of unknown nucleic acids and/or a known nucleic acids (target
nucleic acid) by using a nucleotide labeled with a substance such
as a fluorescent dye. In the case of plural nucleic acids, they can
be assayed at the same time.
BACKGROUND ART
[0002] Numerous methods are known for the assay of a target nucleic
acid by using a nucleic acid probe. Many examples can be mentioned
including those represented by (1) methods making use of a probe
which utilizes the FRET (fluorescence resonance energy transfer)
phenomenon (see, for example, Morrison et al., Anal. Biochem., 183,
231-244, 1989, and Xiangnin Chen et al., Proc. Natl. Acad. Sci.
USA, 94, 10756-10761, 1977); (2) and methods making use of a probe
which utilizes the characteristic of a fluorescence dye that the
intensity of fluorescence emission is quenched as a result of its
interaction with a particular nucleic acid base (see, for example,
KURATA et al., Nucleic Acids Research, 29(6), e34, 2001). These
methods measures a change or the amount of a change in an optical
character (fluorescence intensity) of a fluorescence dye or the
like, with which a nucleic acid probe is labeled, by hybridizing
the labeled nucleic acid probe with a target nucleic acid and/or
amplifying the target nucleic acid in a homogeneous system. Such a
nucleic acid probe will hereinafter be called "a nucleic acid probe
for a homogeneous solution system" throughout the specification. As
an alternative, it may also be called simply "a nucleic acid probe"
in some instances.
[0003] However, a nucleic acid probe for a homogeneous solution
system, said probe being required in any one of the above-described
methods, requires an oligonucleotide to be labeled with a
fluorescence substance and/or a quencher substance. On top of this
requirement, there is no standardized method for the designing of
the probe. These circumstances have led to a waste of time and
money. There is also an outstanding demand for further improvements
in the assay sensitivity, although the assay sensitivity has been
increasingly improved. Moreover, plural nucleic acids, which exist
in a single system in the natural world and include unknown nucleic
acids and/or known nucleic acids, cannot be assayed at the same
time, simply and easily, specifically, accurately, in a short time,
and with excellent sensitivity.
[0004] With the foregoing circumstances in view, the present
invention has as an object to provide a novel method which makes it
possible to assay at least one of unknown nucleic acids and/or
known nucleic acids, which exist in a single system, simply and
easily, specifically, accurately, in a short time, and with
excellent sensitivity.
DISCLOSURE OF THE INVENTION
[0005] As a result of an extensive investigation, the present
inventors have found that, when a fluorescence-labeled nucleotide
or quencher-labeled nucleotide is incorporated in a nucleic acid
polymer in the course of synthesis of a nucleic acid, the
fluorescence character of the fluorescence dye changes
significantly compared with that before the incorporation. The
present invention has been completed on the basis of the above
finding.
[0006] Described specifically, the present invention provides:
[0007] 1) A method of assaying at least one nucleic acid, which
comprises: conducting a nucleic acid polymerization reaction in a
nucleic acid polymerization reaction system comprising (A) the at
least one nucleic acid as a template, (B) at least one nucleotide
monomer labeled with at least one label selected from the group
consisting of (a) fluorescent dyes, (b) quenchers and (c) immune
related substances with a fluorescent dye or quencher contained
therein, and (C) at least one nucleic acid-synthesizing enzyme; and
assaying the template nucleic acid or a nucleic acid, which has
been synthesized using the template nucleic acid as a template,
from a change or an amount of a change in an optical character of
the nucleic acid polymerization system.
[0008] In the above-described method of the present invention, it
is preferred:
[0009] 2) that the label is a combination of at least one donor
fluorescent dye and at least one acceptor fluorescent dye;
[0010] 3) that the label is a combination of at least one
fluorescent dye and at least one quencher;
[0011] 4) that the nucleic acid polymerization system further
comprises (D) at least one nucleotide monomer not labeled with any
label;
[0012] 5) that the nucleic acid polymerization system further
comprises (E) a nucleic acid primer capable of specifically
hybridizing to said template nucleic acid and comprising at least
one nucleotide monomer;
[0013] 6) that in the above method 5), said nucleic acid primer is
labeled with (E') a label as described above in 1);
[0014] 7) that in the above method 1), said nucleic acid
polymerization system further comprises a non-labeled
nucleotide;
[0015] 8) that in the above method 7), said fluorescence-labeled
and/or non-labeled nucleotide comprises guanine (g) and/or said
template nucleic acid comprises at least one guanine (g);
[0016] 9) that in the above method 1), said nucleic acid
polymerization system further comprises a non-labeled nucleotide
and a fluorescence-labeled nucleic acid primer without any
ingredient (B) as described above in 1);
[0017] 10) that in the above method 9), said non-labeled nucleotide
comprises a guanine (g) base; and
[0018] 11) that in the above method 1), 7) and/or 9), said
non-labeled nucleotide and/or labeled nucleotide is a
triphosphate.
[0019] The present invention also provides 12) a method of assaying
a nucleic acid, which comprises: conducting a nucleic acid
polymerization reaction in a nucleic acid polymerization system
comprising said nucleic acid as a template, at least one
dideoxynucleotide monomer labeled with at least one fluorescent dye
and/or at least one quencher, and a nucleic acid-synthesizing
enzyme; and assaying said template nucleic acid or a nucleic acid
polymer, which has been synthesized using said template nucleic
acid as a template, from a change or an amount of a change in
fluorescence intensity.
[0020] It is preferred in:
[0021] 13) that in the above method 12), said nucleic acid
polymerization system is a system further comprising a labeled
nucleotide or a non-labeled nucleotide or both of them;
[0022] 14) that in the above method 12, said nucleic acid
polymerization system further comprises anon-labeled nucleic acid
primer;
[0023] 15) that in the above method 1, 7, 9 and/or 12, said nucleic
acid polymerization system further comprises (F) a fluorescent dye
capable of emitting fluorescence upon binding to a nucleic
acid.
[0024] The present invention further provides 16) a method of
assaying a nucleic acid, which comprises: conducting a nucleic acid
polymerization reaction in a nucleic acid polymerization system
comprising said nucleic acid as a template, a non-labeled
dideoxynucleotide monomer, a labeled nucleotide, a non-labeled
nucleic acid primer, at least one fluorescent dye cable of emitting
fluorescence upon binding to a nucleic acid, and a nucleic
acid-synthesizing enzyme; and assaying said template nucleic acid
or a nucleic acid polymer, which has been synthesized using said
template nucleic acid as a template, from a change or an amount of
a change in fluorescence intensity.
[0025] It is also preferred:
[0026] 17) that in the above method 12) and/or 16), said
non-labeled dideoxynucleotide, labeled dideoxynucleotide,
non-labeled nucleotide and/or labeled nucleotide is a triphosphate;
and
[0027] 18) that in the above method 1), 7), 9), 12) and/or 16),
said nucleic acid-synthesizing enzyme is at least one enzyme
selected from DNA polymerases, RNA polymerases, reverse
transcriptases, and modifications thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 illustrates an outline of a method A according to the
present invention: a method of assaying a nucleic acid by making
use of an interaction between florescent dyes.
[0029] FIG. 2 illustrates an outline of a method B according to the
present invention: a method of assaying a nucleic acid by making
use of an interaction between a fluorescent dye specific to a
nucleic acid (hereinafter referred to as "nucleic-acid-specific
fluorescent dye") and a florescent dye.
[0030] FIG. 3 illustrates an outline of a method C according to the
present invention: a method of assaying a nucleic acid by making
use of an interaction between the base G and a florescent dye.
[0031] FIG. 4 illustrates an outline of a method D according to the
present invention: a method of assaying a nucleic acid by making
use of an interaction between a quencher and a florescent dye.
[0032] FIG. 5 illustrates an outline of a method E according to the
present invention: (1) a method of assaying a nucleic acid by
making use of a labeled specific primer.
[0033] FIG. 6 illustrates an outline of the method E according to
the present invention: (2) a method of assaying a nucleic acid by
making use of a labeled specific primer.
[0034] FIG. 7 illustrates an outline of a method F according to the
present invention: a method of assaying a nucleic acid by making
use of an interaction between a fluorescence dye labeled with a
specific primer and the base G.
[0035] FIG. 8 illustrates an outline of the method G according to
the present invention: a method of assaying a nucleic acid by
making use of a nucleotide labeled with an antigen or antibody.
[0036] FIG. 9 illustrates an outline of the method H according to
the present invention: a method of assaying a nucleic acid by
making use of specific primers immobilized on a surface of a
solid.
[0037] FIG. 10 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between fluorescent dye
specific to a double-stranded nucleic acid (hereinafter referred to
as "double-stranded-nucleic-acid-specific fluorescent dye") and a
fluorescence-labeled nucleotide: changes in the fluorescence
intensities at F1 of Models 1 to 10. TABLE-US-00001
-.circle-solid.- Model 1 -.box-solid.- Model 2 -.diamond-solid.-
Model 3 -- Model 4 -.largecircle.- Model 5 -.quadrature.- Model 6
-.diamond.- Model 7 -.DELTA.- Model 8 -- Model 9 -- Model 10
[0038] FIG. 11 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between fluorescent dye
specific to a double-stranded nucleic acid (hereinafter referred to
as "double-stranded-nucleic-acid-specific fluorescent dye") and a
fluorescence-labeled nucleotide: changes in the fluorescence
intensities at F3 of Models 1 to 10. TABLE-US-00002
-.circle-solid.- Model 1 -.box-solid.- Model 2 -.diamond-solid.-
Model 3 -- Model 4 -.largecircle.- Model 5 -.quadrature.- Model 6
-.diamond.- Model 7 -.DELTA.- Model 8 -- Model 9 -- Model 10
[0039] FIG. 12 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between fluorescent dye
specific to a double-stranded nucleic acid (hereinafter referred to
as "double-stranded-nucleic-acid-specific fluorescent dye") and a
fluorescence-labeled nucleotide: changes in the fluorescence
intensities at F1 of Models 11 to 20. TABLE-US-00003
-.circle-solid.- Model 11 -.box-solid.- Model 12 -.diamond-solid.-
Model 13 -- Model 14 -.largecircle.- Model 15 -.quadrature.- Model
16 -.diamond.- Model 17 -.DELTA.- Model 18 -- Model 19 -- Model
20
[0040] FIG. 13 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between fluorescent dye
specific to a double-stranded nucleic acid (hereinafter referred to
as "double-stranded-nucleic-acid-specific fluorescent dye") and a
fluorescence-labeled nucleotide: changes in the fluorescence
intensities at F2 of Models 11 to 20. TABLE-US-00004
-.circle-solid.- Model 11 -.box-solid.- Model 12 -.diamond-solid.-
Model 13 -- Model 14 -.largecircle.- Model 15 -.quadrature.- Model
16 -.diamond.- Model 17 -.DELTA.- Model 18 -- Model 19 -- Model
20
[0041] FIG. 14 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between fluorescent dye
specific to a double-stranded nucleic acid (hereinafter referred to
as "double-stranded-nucleic-acid-specific fluorescent dye") and a
fluorescence-labeled nucleotide: changes in the fluorescence
intensities at F1 of Models 21 to 30. TABLE-US-00005
-.circle-solid.- Model 21 -.box-solid.- Model 22 -.diamond-solid.-
Model 23 -- Model 24 -.largecircle.- Model 25 -.quadrature.- Model
26 -.diamond.- Model 27 -.DELTA.- Model 28 -- Model 29 -- Model
30
[0042] FIG. 15 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between fluorescent dye
specific to a double-stranded nucleic acid (hereinafter referred to
as "double-stranded-nucleic-acid-specific fluorescent dye") and a
fluorescence-labeled nucleotide: changes in the fluorescence
intensities at F2 of Models 21 to 30. TABLE-US-00006
-.circle-solid.- Model 21 -.box-solid.- Model 22 -.diamond-solid.-
Model 23 -- Model 24 -.largecircle.- Model 25 -.quadrature.- Model
26 -.diamond.- Model 27 -.DELTA.- Model 28 -- Model 29 -- Model
30
[0043] FIG. 16 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between fluorescent dye
specific to a double-stranded nucleic acid (hereinafter referred to
as "double-stranded-nucleic-acid-specific fluorescent dye") and a
fluorescence-labeled nucleotide: changes in the fluorescence
intensities at F1 of Models 31 to 40. TABLE-US-00007
-.circle-solid.- Model 31 -.box-solid.- Model 32 -.diamond-solid.-
Model 33 -- Model 34 -.largecircle.- Model 35 -.quadrature.- Model
36 -.diamond.- Model 37 -.DELTA.- Model 38 -- Model 39 -- Model
40
[0044] FIG. 17 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between two
fluorescence-labeled nucleotides: changes in the fluorescence at F1
of Models 1 to 8. TABLE-US-00008 -.circle-solid.- Model 1
-.box-solid.- Model 2 -.diamond-solid.- Model 3 -- Model 4
-.largecircle.- Model 5 -.quadrature.- Model 6 -.diamond.- Model 7
-.DELTA.- Model 8
[0045] FIG. 18 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between two
fluorescence-labeled nucleotides: changes in the fluorescence at F3
of Models 1 to 8. TABLE-US-00009 -.circle-solid.- Model 1
-.box-solid.- Model 2 -.diamond-solid.- Model 3 -- Model 4
-.largecircle.- Model 5 -.quadrature.- Model 6 -.diamond.- Model 7
-.DELTA.- Model 8
[0046] FIG. 19 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between two
fluorescence-labeled nucleotides: changes in the fluorescence at F1
of Models 9 to 16. TABLE-US-00010 -.circle-solid.- Model 9
-.box-solid.- Model 10 -.diamond-solid.- Model 11 -- Model 12
-.largecircle.- Model 13 -.quadrature.- Model 14 -.diamond.- Model
15 -.DELTA.- Model 16
[0047] FIG. 20 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between two
fluorescence-labeled nucleotides: changes in the fluorescence at F2
of Models 9 to 16. TABLE-US-00011 -.circle-solid.- Model 9
-.box-solid.- Model 10 -.diamond-solid.- Model 11 -- Model 12
-.largecircle.- Model 13 -.quadrature.- Model 14 -.diamond.- Model
15 -.DELTA.- Model 16
[0048] FIG. 21 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between two
fluorescence-labeled nucleotides: changes in the fluorescence at F1
of Models 17 to 24. TABLE-US-00012 -.circle-solid.- Model 17
-.box-solid.- Model 18 -.diamond-solid.- Model 19 -- Model 20
-.largecircle.- Model 21 -.quadrature.- Model 22 -.diamond.- Model
23 -.DELTA.- Model 24
[0049] FIG. 22 illustrates an assay of a nucleic acid by making use
of an interaction (FRET phenomenon) between two
fluorescence-labeled nucleotides: changes in the fluorescence at F2
of Models 17 to 24. TABLE-US-00013 -.circle-solid.- Model 17
-.box-solid.- Model 18 -.diamond-solid.- Model 19 -- Model 20
-.largecircle.- Model 21 -.quadrature.- Model 22 -.diamond.- Model
23 -.DELTA.- Model 24
[0050] FIG. 23 illustrates real-time monitoring of PCR
amplification product by making use of two fluorescence-labeled
nucleotides (changes in the fluorescence at F1). TABLE-US-00014
-.DELTA.- 0 copy -.box-solid.- 1E+06 copy -.circle-solid.- 1E+07
copy -.diamond-solid.- 1E+08 copy -- 1E+09 copy
[0051] FIG. 24 illustrates real-time monitoring of PCR
amplification product by making use of two fluorescence-labeled
nucleotides (changes in the fluorescence at F3). TABLE-US-00015
-.DELTA.- 0 copy -.box-solid.- 1E+06 copy -.circle-solid.- 1E+07
copy -.diamond-solid.- 1E+08 copy -- 1E+09 copy
[0052] FIG. 25 illustrates real-time monitoring of PCR
amplification product by making use of two fluorescence-labeled
nucleotides (changes in the fluorescence at F1 after data
processing). TABLE-US-00016 -.box-solid.- 1E+06 copy
-.circle-solid.- 1E+07 copy -.diamond-solid.- 1E+08 copy -- 1E+09
copy
[0053] FIG. 26 illustrates real-time monitoring of PCR
amplification product by making use of two fluorescence-labeled
nucleotides (changes in the fluorescence at F3 after data
processing). TABLE-US-00017 -.box-solid.- 1E+06 copy
-.circle-solid.- 1E+07 copy -.diamond-solid.- 1E+08 copy -- 1E+09
copy
[0054] FIG. 27 illustrates a calibration line by a real-time
quantitative PCR method making use of two fluorescence-labeled
nucleotides (data employed for the preparation of the calibration
line: fluorescence values at F1 after data processing).
[0055] FIG. 28 illustrates a calibration line by a real-time
quantitative PCR method making use of two fluorescence-labeled
nucleotides (data employed for the preparation of the calibration
line: fluorescence values at F3 after data processing).
[0056] FIG. 29 illustrates real-time monitoring of PCT
amplification product by using a
double-stranded-nucleic-acid-specific fluorescent dye and a
fluorescence-labeled nucleotide (changes in the fluorescence at F3
after data processing). TABLE-US-00018 -.largecircle.- 1E+05 copy
-.box-solid.- 1E+06 copy -.circle-solid.- 1E+07 copy
-.diamond-solid.- 1E+08 copy -- 1E+09 copy
[0057] FIG. 30 illustrates a calibration line by a real-time
quantitative PCR method making use of a
double-stranded-nucleic-acid-specific fluorescent dye and a
fluorescence-labeled nucleotide (changes in the fluorescence at F3
after data processing).
[0058] FIG. 31 is a diagram illustrating changes in the
fluorescence intensity of FITC when primers 6 of three genotypes
(-.diamond-solid.- C-allele homozygote, -.quadrature.- T-allele
homozygote, -.tangle-solidup.-heterozygote) were used.
[0059] FIG. 32 is a diagram illustrating changes in the
fluorescence intensity of CY5 when primers 6 of three genotypes
(-.diamond-solid.- C-allele homozygote, -.quadrature.- T-allele
homozygote, -.tangle-solidup.- heterozygote) were used.
[0060] FIG. 33 is a diagram illustrating changes in the
fluorescence intensity of FITC when primers 7 were used.
[0061] -.diamond-solid.- C-allele homozygote, -.quadrature.-
heterozygote, -.tangle-solidup.- T-allele homozygote
[0062] FIG. 34 is a diagram illustrating changes in the
fluorescence intensity at CY5 of FITC when primers 7 were used.
-.diamond-solid.- C-allele homozygote, -.quadrature.- heterozygote,
-.tangle-solidup.- T-allele homozygote
LEGEND
[0063] N: Nucleotide monomer
BEST MODES FOR CARRYING OUT THE INVENTION
[0064] The present invention will hereinafter be described in
detail. Before describing the present invention in detail, however,
definitions will be provided for certain terms used throughout the
application including the claims. It is to be noted that the terms
employed in the present invention have the same meanings as those
used commonly in biology, molecular biology, genetics or genetic
engineering, or microbiology or microbial engineering unless
otherwise specifically indicated.
[0065] The term "nucleotide monomer" means a nucleotide which can
be incorporated into a nucleic acid polymer by at least one nucleic
acid-synthesizing enzyme. It can preferably be a mononucleotide of
a nucleic acid constituent of an oligonucleotide. In the present
invention, however, the term "nucleotide monomer" encompasses
oligonucleotides of from 2 to 30 nucleotide units in length in
addition to mononucleotides. Preferred examples can include
nucleoside monophosphates (NMPs), nucleoside diphosphates (NDP),
and nucleoside triphosphate (NTPS), with nucleoside triphosphates
being more preferred. As a base, one contained in a nucleic acid
constituent, specifically adenine, guanine, uracil, cytosine,
thymine, a derivative thereof, a trace component contained in RNA,
or the like can be mentioned. A sugar can be ribose or deoxyribose.
Insofar as the above-described oligonucleotide is hybridizable with
a template nucleic acid, it can be incorporated into a nucleic acid
polymer in a nucleic acid polymerization system by using a nucleic
acid-synthesizing enzyme having no exonuclease activity (for
example, DNA polymerase) and ligase.
[0066] As a reason for the usability of a nucleoside monophosphate
or diphosphate, a nucleic acid polymerization system may contain a
kinase or phosphorylase, which converts the phosphate into the
corresponding triphosphate, or a production system therefor. For
example, an unpurified crude template nucleic acid or crude nucleic
acid-synthesizing enzyme contain such an enzyme and/or its
production system in many instances. When ATP is excessively
contained in a nucleic acid polymerization system, the formation of
triphosphates other than ATP is facilitated. The term "nucleic acid
polymerization system" in the present invention is, therefore,
defined such that it can encompass these enzymes and/or their
production systems. This also applies equally to labeled
nucleotides and nucleotides labeled with immune related substances,
and hence, their triphosphates are more preferred. This also
applies likewise to dideoxynucleotide monomers and labeled or
unlabeled dideoxynucleotides, and accordingly, their triphosphates
are more preferred.
[0067] The term "labeled nucleotide" means a nucleotide monomer
labeled with at least one of fluorescent dyes, quencher substances
and the like, which will be described subsequently herein. A
nucleotide labeled with a fluorescent dye is called a
"fluorescence-labeled nucleotide", while a nucleotide labeled with
a quencher is called a "quencher-labeled nucleotide". Further, a
fluorescent-labeled nucleotide labeled with a donor fluorescent dye
is called a "donor-labeled nucleotide", while a fluorescent-labeled
nucleotide labeled with an acceptor fluorescent dye is called an
"acceptor-labeled nucleotide". About these labeled nucleotides, a
detailed description will be made subsequently herein.
[0068] The term "unlabeled nucleotide" means a nucleotide monomer
not labeled with such a labeling substance as described above.
[0069] The term "nucleic acid primer" means a primer which
specifically hybridizes to a template nucleic acid. Nucleic acid
primers labeled with a fluorescent dye and a quencher are called a
"fluorescence-labeled nucleic acid primer" and a "quencher-labeled
nucleic acid primer", respectively. Collectively, they are also
called "labeled nucleic acid primers". Adenine, guanine, uracil,
cytosine and thymine re designated "A" or "a", "G" or "g", "U" or
"u", "C" or "c", or "T" or "t", respectively. A fluorescent dye,
which emits fluorescence when bound to a nucleic acid, is defined
as a nucleic-acid-specific fluorescent dye.
[0070] The term "template nucleic acid" means one that can serve as
a template for a nucleic acid polymer. In the present invention, it
indicates a nucleic acid which is unknown (which may also be called
an "unknown nucleic acid"), a known nucleic acid (which may also be
called a "target nucleic acid"), or a mixture thereof. It is a DNA
and/or RNA. The term "template nucleic acid" used in the present
invention, therefore, is not only limited to any specific nucleic
acid(s) (target nucleic acid(s)) to be assayed, but also includes
non-specific nucleic acid(s). Needless say, it encompasses genes
and the like. These nucleic acids may exist together. In addition,
no limitation is imposed on the concentration or size of the
template nucleic acid. Accordingly, the term "template nucleic
acid" also means one or more specific and/or non-specific nucleic
acids existing in a single system. Specifically, the term "template
nucleic acid" means a nucleic acid which can be detected or assayed
by polymerization and/or amplification in accordance with the
method of the present invention.
[0071] A nucleic acid-synthesizing enzyme can be any synthase
insofar as it has ability to synthesize a nucleic acid polymer by
polymerizing the above-described unlabeled nucleotide and/or
labeled nucleotide while using the above-described nucleic acid
template as a template. Representative examples can include DNA
polymerases, RNA polymerases, reverse transcriptases, ligases,
various kinases, nucleotide triphosphate production systems, and
enzymes containing their modified proteins obtained by genetic
engineering. These DNA polymerases, RNA polymerases and reverse
transcriptases, ligases, various kinases, and enzymes containing
nucleotide triphosphate production systems are suitably usable in
the present invention. In the present invention, these nucleic
acid-synthesizing enzyme can be used either singly or in
combination.
[0072] Of course, these enzymes may or may not contain various
factors which allow the enzymes to fully exhibit their activities.
In the case of DNA polymerases, they may or may not be provided
with the exonuclease activity, and they may be either in a purified
form or in the form of an unpurified, crude enzyme. No particular
limitation is imposed on the origin (microorganism, animal or
plant) of the enzyme. However, those having heat resistance are
preferred. Preferred specific examples can include Vent(exo-)DNA
polymerase (derived from Thermococcus litoralis), Tgo(exo-)DNA
polymerase, "THERMOSEQUENASE DNA POLYMERASE" (product of Amersham
Biosciences Corp.), AmpliTagGold polymerase, and T7 Sequenase DNA
polymerase, all of which have been rendered deficient in
3'.fwdarw.5' exonuclease activity.
[0073] A hybridization complex between a nucleic acid polymer or
nucleic acid primer labeled with a florescent dye or the like and a
corresponding nucleic acid such as a template or nucleic acid
polymer is called a "hybrid (or hybrid) complex", "nucleic acid
polymer-template complex", "nucleic acid primer-template complex",
or "nucleic acid primer-nucleic acid polymer complex".
[0074] The expression "to assay a nucleic acid" or "to measure the
concentration of a nucleic acid" as used herein means to perform a
quantitative detection of the nucleic acid, to perform a
qualitative detection of the nucleic acid, to simply measure or
simply monitor the intensity of fluorescence from a nucleic acid
polymerization system, to perform a simple detection of
fluorescence, to analyze or study the nucleic acid, to measure,
study and/or analyze a polymorphism (including SNP) and/or
mutation, or to perform a like detection, measurement, study or
analysis, to say nothing of quantitatively measuring the
concentration of the target nucleic acid. The above expression
should also be interpreted to encompass an operation or the like
that the data obtained as described above is studied by the known
method of Kurata et al. (EP 1 046 717 A9) to determine the
concentration (the number of copies or the like) of a nucleic acid
existing in a single system. Further, the above expression should
also be interpreted to encompass an operation or the like that the
sequence of a base is determined by a known method ("KISO SEIKAGAKU
JIKKENHO (Fundamental Biochemical Experiments)", Vol. 4
(Experiments on Nucleic Acids and Genes), Compiled by the Japanese
Biochemical Society, Published by Tokyo Kagaku Dojin Kabushiki
Kaisha) or the like.
[0075] The term "polymerization reaction of a nucleic acid"
encompasses not only mere polymerization (synthesis or elongation)
reactions but also amplification reactions of the nucleic acid, for
example, PCR techniques, real-time quantitative PCR techniques,
ICAN techniques, LAMP techniques, NASBA techniques, TAMA
techniques, LCR techniques, and hybridization reaction,
elongations, modifications and the like making use of such
techniques. As specific examples of the polymerization reaction,
the following examples can be mentioned:
[0076] (1) a reaction in which a template nucleic acid is a DNA, a
nucleic acid-synthesizing enzyme is a DNA polymerase or modified
RNA polymerase, a nucleotide monomer or a fluorescence-labeled
nucleotide or quencher-labeled nucleotide is a deoxyribonucleotide,
and a nucleic acid polymer is a DNA;
[0077] (2) a reaction in which a template nucleic acid is a DNA, a
nucleic acid-synthesizing enzyme is an RNA polymerase or modified
DNA polymerase, a nucleotide monomer or a fluorescence-labeled
nucleotide or quencher-labeled nucleotide is a ribonucleotide, and
a nucleic acid polymer is an RNA;
[0078] (3) a reaction in which a template nucleic acid is an RNA, a
nucleic acid-synthesizing enzyme is a reverse transcriptase, a
nucleotide monomer or a fluorescence-labeled nucleotide or
quencher-labeled nucleotide is a deoxyribonucleotide, and a nucleic
acid polymer is a DNA;
[0079] (4) a reaction in which a template nucleic acid is an RNA, a
nucleic acid-synthesizing enzyme is a reverse transcriptase or RNA
polymerase, a nucleotide monomer or a fluorescence-labeled
nucleotide or quencher-labeled nucleotide is a ribonucleotide or
deoxyribonucleotide, and a nucleic acid polymer is an RNA, that is,
a reaction which proceeds through a DNA synthesis reaction;
[0080] (5) a reaction system making combined use of a ligase in the
above-described reaction system; and
[0081] (6) a reaction system making combined use of one of various
kinases and nucleotide triphosphate production systems in the
above-described reaction system.
[0082] Among the above-described reaction systems, preferred are
the reaction systems (1) to (4), more preferred are the reaction
systems (1) to (3), and particularly preferred are the reaction
systems (1) and (2).
[0083] The term "optical character" means one of various absorption
spectra and fluorescence emission spectrum of a fluorescent dye,
quencher or the like, with which a nucleotide is labeled, or its
optical characteristic or the like such as absorption intensity,
polarization, fluorescence emission, fluorescence intensity,
fluorescence lifetime, fluorescence polarization or fluorescence
anisotropy (these optical characteristics will be collectively
called "fluorescence intensity"). It may also mean a characteristic
determined by totally analyzing one or more measurement values of
at least one fluorescent dye or the like, with which a labeled
nucleotide or the like is labeled, as measured at at least one
measurement wavelength. For example, a fluorescence intensity curve
or the like of a modification reaction of a nucleic acid can be
used as an optical character.
[0084] In the present invention, the expression "from a change or
an amount of a change in fluorescence intensity" shall embrace not
only a change in fluorescence intensity on the basis of a nucleic
acid polymer synthesized in the present invention, but also a
change or an amount of a change in fluorescence intensity when a
nucleic acid probe for a homogeneous solution system, said nucleic
acid probe having been labeled with a fluorescent dye and/or
quencher, is hybridized with the nucleic acid polymer.
[0085] A nucleic acid polymerization system may contain a labeled
or unlabeled dideoxynucleotide together with a labeled or unlabeled
nucleotide. When the dideoxynucleotide is used in the reaction, the
polymerization of a nucleic acid in this system terminates as soon
as the use of the dideoxynucleotide takes place. When a single
species of target nucleic acid serves as a template, many nucleic
acid polymers of different chain lengths are obtained using the
target nucleic acid as their template. By analyzing and studying
these nucleic acid polymers by electrophoresis, liquid
chromatography or the like, important information can be obtained
on the target nucleic acid. In such an analysis and study, a change
in the intensity of fluorescence from the labeling substance is
also used.
[0086] A detailed description will now be made about
fluorescence-labeled nucleotides, quencher-labeled nucleotides, and
nucleic-acid-specific fluorescent dyes. The term "fluorescent dye
(which may also be called `fluorescent substance`)" as used in the
present invention generally means a fluorescent dye which is
generally used to label a nucleic acid probe to assay or detect the
nucleic acid. Illustrative are fluoresce in and its derivatives
[for example, fluorescein isothiocyanate (FITC) and its
derivatives], Alexa 488, Alexa 532, cy3, cy5, 6-joe, EDANS,
rhodamine 6G (R6G) and its derivatives [for example,
tetramethylrhodamine (TMR), tetramethylrhodamine isothiocynate
(TMRITC), and x-rhodamine], Texas red, "BODIPY FL" ("BODIPY" is a
trademark, "FL" is a tradename; product of Molecular Probes
Corporation, U.S.A.; this will hereinafter apply equally), "BODIPY
FL/C3", "BODIPY FL/C6", "BODIPY 5-FAM", "BODIPY TMR", and their
derivatives (for example, "BODIPY TR", "BODIPY R6G", and "BODIPY
564"). Among the above-exemplified fluorescent dyes, FITC, EDANS,
Texas Red, 6-joe, TMR, Alexa 488, Alexa 532, "BODIPY FL/C3",
"BODIPY R6G", "BODIPY FL", Alexa 532, "BODIPY FL/C6", "BODIPY TMR",
5-FAM, "BODIPY 493/503", "BODIPY 564", "BODIPY 581", Cy3, Cy5,
Texas red, x-Rhodamine, and the like can be mentioned as preferred
ones.
[0087] The term "quencher" means a substance, which acts on the
above-described fluorescent dye and reduces or quenches the
emission of fluorescence from the fluorescent dye. Illustrative are
Dabcyl, "QSY7" (product of Molecular Probes Corporation), "QSY33"
(product of Molecular Probes Corporation), Ferrocene and its
derivatives, methyl viologen, and N,N'-dimethyl-2,9-diazopyrenium,
with Dabcyl being preferred.
[0088] The term "fluorescence-labeled nucleotide" as used in the
present invention means a nucleotide monomer labeled with at least
one fluorescent dye. The labeling can be on the 5'-position of the
sugar moiety and/or on the position of its phosphate group, on the
position of the base, or on the 3'-position of the sugar moiety
and/or on the position of its phosphate group. The term
"fluorescent dye" means such a dye as exemplified above, which can
act as a donor dye or an acceptor dye. Likewise, the term
"quencher-labeled nucleotide" means a nucleotide monomer labeled
with such a quencher as exemplified above. It is to be noted that a
fluorescence-labeled nucleotide and a quencher-labeled nucleotide
may also be collectively called "a labeled nucleotide". This
applies equally to fluorescence-labeled dideoxynucleotides and
quencher-labeled dideoxynucleotides. There is, however, no OH group
at the 3'-position of the sugar moiety in such a dideoxynucleotide.
When the dideoxynucleotide is used in the polymerization of a
nucleic acid, the polymerization reaction, therefore, terminates as
soon as the use of the dideoxynucleotide takes place.
[0089] When the 3'-OH group of the sugar moiety is labeled in the
labeled nucleotide and the nucleotide is used in the polymerization
reaction of the nucleic acid, the polymerization of the nucleic
acid terminates as soon as the use of the nucleotide takes place.
When a single species of target nucleic acid is used as a template,
many nucleic acid polymers of different chain lengths are obtained
using the target nucleic acid as their template. By analyzing and
studying these nucleic acid polymers by electrophoresis, liquid
chromatography or the like, important information can be obtained
on the target nucleic acid. In such an analysis and study, a change
in the intensity of fluorescence from the labeling substance is
also used.
[0090] To label a nucleotide monomer with a fluorescent dye or a
quencher, any desired one of conventionally known labeling methods
can be used. The position to be labeled can be one of the OH groups
in the 5'-phosphate group or an OH group or amino group in the
base. When labeling the amino group, it is convenient to use a kit
reagent, for example, "UNI-LINK AMINOMODIFIER" (product of CLONTECH
LABORATORIES, INC., U.S.A.) or "FLUOROREPORTER KIT F-6082",
"FLUOROREPORTER KIT F-6083", "FLUOROREPORTER KIT F-6084" or
"FLUOROREPORTER KIT F-10220" (all, products of Molecular Probes
Corporation, U.S.A.). By a method known per se in the art,
molecules of the labeling substance can be bonded to the nucleotide
monomer.
[0091] When labeling the OH group, "5' AMINO-MODIFIER C6 KIT" (Glen
Research Corporation, U.S.A.) or the like is used. When bonding a
molecule of the labeling substance, for example, to an OH group of
a base, --(CH.sub.2).sub.n--SH is firstly introduced as an
illustrative spacer to the OH group by a method known per se in the
art. In this case, n stands for 3 to 8, preferably for 6. A labeled
nucleotide monomer can be synthesized by binding the labeling
substance, which has reactivity to the SH group, or a derivative
thereof to the spacer. This procedure can be followed likewise upon
labeling the amino group. The 3'-OH groups of ribose and
deoxyribose, the 2'-OH group of ribose and the OH group in the
5'-phosphate group can be labeled in a similar manner as described
above. A variety of nucleotide monomers synthesized as described
above and labeled with the above-described labeling substances can
be purified by chromatography such as reverse phase chromatography
to provide labeled nucleotides which are useful the present
invention. Needless to say, they can also be obtained by relying
upon custom synthesis services.
[0092] The term "nucleic acid primer for use in the present
invention" means a primer which can serve as a precursor for a
nuclear acid polymer. It can be either in a deoxyribose form or in
a ribose form. No particular limitation is imposed on its chain
length insofar as it can be used for the synthesis of a known
nucleic acid. The chain length can be, for example, from 2 to 50
bases, more preferably from 3 to 40 bases, still more preferably
from 5 to 30 bases. It is possible to use either a nucleic primer
having such a base sequence as permitting specific hybridization to
a template nucleic acid or a nucleic primer having a common base
sequence or consensus sequence. A nucleic acid polymer making use
of a specific nucleic acid template as a template can be obtained
in the former case, while a non-specific nucleic acid polymer can
be obtained in the latter case.
[0093] The primer in the present invention is usable no matter
whether it is labeled with the above-described fluorescent dye or
quencher. The labeling is required to have been effected by at
least one of such labeling substances. A preferred nucleic acid
primer is one labeled at the 5' end and/or the base in the chain
but not labeled on the 3'-OH group at the 3' end of the sugar
moiety. In this case, the resulting nucleic acid polymer is in a
form labeled with the labeling substance employed to label the
primer. Needless to say, it is also possible to use such a nucleic
acid primer that the 3'-OH group at the 3' end of the sugar moiety
has been labeled. In this case, the primer is used simply as a
nucleic acid probe.
[0094] The oligonucleotide which makes up the nucleic acid primer
for use in the present invention can be produced by a process
commonly employed for producing general nucleotides. It can be
produced, for example, by a chemical synthesis process, a
biological process making use of a plasmid vector, pharge vector or
the like, or the like. The use of a nucleic acid synthesizer
currently available on the commercial market is suited.
[0095] Labeling of an oligonucleotide with a fluorescent dye or
quencher can be conducted in a similar manner as in the case of the
above-described labeled nucleotide monomer. In this case, the
target of labeling is the base at the 5' end of the
oligonucleotide, the base at its 3' end, the phosphate group at its
5' end, or the ribose or deoxyribose at its 3' end. Concerning the
synthesis of the oligonucleotide and that of the labeled nucleic
acid primer, it is simplest to rely upon custom synthesis
services.
[0096] The term "nucleic-acid-specific fluorescent dye" as used in
the present invention means a substance which emits fluorescence
when bound to a nucleic acid. No particular limitation is imposed
on the species of the nucleic acid to which the substance is bound,
insofar as it is a nucleic acid such as a labeled or unlabeled
nucleic acid primer-template complex, a single-stranded DNA, a
single-stranded RNA, a double-stranded nucleic acid formed of a DNA
and an RNA, or a double-stranded RNA. Examples of the
nucleic-acid-specific fluorescent dye include intercalators such as
ethidium bromide, Sybr green 1, Sybr green 2, YOYO, TOTO, and
YO-PRO-1. It is, however, to be noted that any substance can be
applied to the method of the present invention insofar as it emits
fluorescence when bound to a nucleic acid.
[0097] The present invention is directed to a method for assaying a
nucleic acid, which comprises the following procedure:
[0098] 1) To initiate a nucleic acid polymerization reaction
(alone) or a nucleic acid polymerization reaction and nucleic acid
amplification reaction (both) in any one of the following nucleic
acid polymerization systems (1) to (8), preferably (6), (7) and
(8), more preferably (6) and (7): [0099] (1) a nucleic acid
polymerization system containing a template nucleic acid, at least
one labeled nucleotide, and a nucleic acid-synthesizing enzyme,
[0100] (2) a nucleic acid polymerization system similar to the
nucleic acid polymerization system (1) except for the additional
inclusion of an unlabeled nucleotide, [0101] (3) a nucleic acid
polymerization system containing a template nucleic acid, at least
one labeled dideoxynucleotide, and a nucleic acid-synthesizing
enzyme, [0102] (4) a nucleic acid polymerization system similar to
the nucleic acid polymerization system (3) except for the
additional inclusion of at least one nucleotide selected from the
group consisting of labeled nucleotides and unlabeled nucleotides,
[0103] (5) a nucleic acid polymerization system containing a
template nucleic acid, an unlabeled dideoxynucleotide, a labeled
nucleotide, and a nucleic acid-synthesizing enzyme, [0104] (6) a
nucleic acid polymerization system similar to any one of the
nucleic acid polymerization system (1) to (5) except for the
additional inclusion of a labeled nucleic acid primer or an
unlabeled nucleic acid primer, [0105] (7) a nucleic acid
polymerization system containing a template nucleic acid, an
unlabeled nucleotide, a labeled nucleic acid primer, and a nucleic
acid-synthesizing enzyme, and [0106] (8) a nucleic acid
polymerization system similar to any one of the nucleic acid
polymerization system (1) to (7) except for the additional
inclusion of a nucleic-acid-specific fluorescent dye.
[0107] 2) To measure a change in the intensity of fluorescence from
a nucleic acid polymerization system or the amount of the change.
This change takes place because the labeled nucleotide and/or
nucleic-acid-specific fluorescent dye is incorporated in the
nucleic acid polymer as a reaction product in the course of the
above-described reaction. When a nucleic acid probe caused to
exist, the probe and the nucleic acid polymer are hybridized with
each other so that the intensity of fluorescence from the nucleic
acid polymerization system undergoes a unique change.
[0108] 3) To analyze the reaction product by electrophoresis or
HPLC as needed.
[0109] The above-described nucleic acid polymerization systems,
especially in the nucleic acid polymerization system (6) which
contains a nucleic acid primer, the assay method is suited when the
nucleic acid as the output of the nucleic acid polymerization
system is a DNA. The nucleic acid polymerization systems (1) and
(2), on the other hand, are suited when the outputs of the nucleic
acid polymerization systems are RNAs. Further, the nucleic acid
polymerization systems each of which contains a labeled or
unlabeled dideoxynucleotide are suited for use in assaying,
analyzing or studying polymorphisms (including SNP) or mutations to
be described subsequently herein.
[0110] The expression "in the presence or absence of at least one
nucleic acid primer" is used in the present invention, because many
of template nucleic acids (for example, crude template nucleic
acids), samples each of which contains at least one template
nucleic acid, and crude nucleic acid-synthesizing enzyme s each
contains an oligonucleotide which may hybridize to the template
nucleic acid to become a precursor for a nucleic acid polymer. It
is also to be noted that many of such crude template nucleic acids
and crude nucleic acid-synthesizing enzyme s contain an enzyme
which synthesizes the precursor.
[0111] A nucleic acid polymerization reaction for the production of
an RNA-type nucleic acid polymer may proceed even if the
above-described nucleic acid primer is not allowed to exist.
[0112] When a nucleic acid synthesis system is contained in a
template nucleic acid (for example, crude template nucleic acid) or
in a sample containing at least one template nucleic acid (for
example, a cell extract from one of various microorganisms), a
nucleic acid polymerization reaction takes place even if no nucleic
acid-synthesizing enzyme is added. In such a case, it is only
necessary to initiate the reaction by causing at least one labeled
nucleotide or nucleic-acid-specific fluorescent dye selected from
the group consisting of fluorescence-labeled nucleotides,
quencher-labeled nucleotides and nucleic-acid-specific fluorescent
dyes.
[0113] The above-described reaction can be conducted under known
reaction conditions. For example, the reaction temperature may be
10.degree. C. or higher but lower than a nucleic acid denaturing
temperature, and specifically relies upon the nucleic
acid-synthesizing enzyme. When a DNA polymerase is used, for
example, the reaction temperature can be 10.degree. C. or higher
but lower than the nucleic acid denaturing temperature, preferably
from 30 to 90.degree. C., more preferably from 30 to 80.degree. C.
When an RNA polymerase is used, the reaction temperature can be
30.degree. C. to 60.degree. C. When an reverse transcriptase is
used, the reaction temperature can be 30.degree. C. to 70.degree.
C. The reaction time is until the intensity of fluorescence from
the nucleic acid polymerization system reaches an equilibrium when
the intensity of fluorescence is monitored as a function of time.
For example, the reaction time can be from 10 seconds to 10 hours,
preferably from 10 seconds to 2 hours, more preferably from 10
seconds to 1 hour.
[0114] The above-described change in fluorescence intensity is
presumably induced by at least one of phenomena in the
below-described group. These phenomena interact with each other.
[0115] (1) Interaction between a nucleic-acid-specific fluorescent
dye and a fluorescent dye [FRET (fluorescence resonance energy
transfer) phenomenon], [0116] (2) Interaction between fluorescent
dyes (FRET phenomenon), [0117] (3) Interaction (fluorescence
quenching phenomenon) between a quencher and a fluorescent dye
[same as the interaction (2)], and [0118] (4) Interaction between
the guanine base and a fluorescent dye (fluorescence quenching
phenomenon).
[0119] The term "interaction" as used in the present invention
means a reaction in which excitation energy is transferred from one
of the reactants to the other. "Fluorescence quenching phenomenon"
may also be called simply "fluorescence quenching".
[0120] As a preferred, practical method for measuring a change in
fluorescence intensity or the amount of the change, it is preferred
and practical to measure in a real-time manner the intensity of
fluorescence from a nucleic acid polymerization system and to
determine the measurement value. In this method, it is desired to
use a commercial measurement instrument which emits at least one
incident light or excitation light and has at least one
light-receiving surface such as a photomultiplier, in other words,
which has multichannels. For example, "SMART CYCLER" (TAKARA BIO
INC.), "ABI PRISM.TM. 7700 SEQUENCE DETECTION SYSTEM" (PE Applied
Biosystems), "LIGHTCYCLER.TM. SYSTEM" (Roche Diagnostics, Mannheim,
Germany), or the like can be used. To obtain an actual measurement
value, at least one of the following measurements is conducted.
[0121] (1) Measurement of a nucleic acid polymerization system both
before and after a nucleic acid polymerization reaction.
[0122] (2) Measurement of a nucleic acid polymerization system by
using, as a control, a system in which no nucleic acid synthesis is
allowed to proceed (for example, a system in which neither a
template nucleic acid nor a nucleic acid-synthesizing enzyme is
added).
[0123] (3) The intensity of fluorescence from a nucleic acid
polymerization system in which the synthesis of a nucleic acid has
reached equilibrium is measured firstly. The nucleic acid
polymerization system is next subjected to nucleic acid
denaturation treatment (for example, treatment at from 90 to
98.degree. C.), followed by the measurement of the intensity of
fluorescence from the nucleic acid polymerization system. By
processing (analyzing) the measurement values, which have been
obtained by the above-described method, in accordance with the
below-described data analysis method, it is possible to ascertain
the species of templates (unknown nucleic acids, a target nucleic
acid) existing in a single system in the nature and also to
determine their concentrations such as the numbers of their copies
before the polymerization or amplification of these nucleic acids.
As a result, still better data are obtained.
[0124] The characteristic features of the present invention will
hereinafter be described with reference to the drawings.
1) Invention Method A (See FIG. 1)
[0125] (1) It is characterized in that a nucleic acid template or a
nucleic acid polymer synthesized by using the nucleic acid template
as a template is assayed by incorporating nucleotides--which are
labeled by at least one fluorescent dyes, respectively--in the
nucleic acid polymer and measuring a change in fluorescence
character of a nucleic acid polymerization system due to an
interaction between fluorescent dye (A) and fluorescent dye (B) in
the incorporated fluorescence-labeled nucleotide or monitoring the
change as a function of time (hereinafter simply called
"monitoring"). This method is an illustrative method applicable
when the nucleic acid polymerization system is the above-described
nucleic acid polymerization system (1) or (2), and corresponds to
the present invention as defined in claims 1 to 3.
[0126] (2) In the above-described method (1), a nucleic acid
polymerization reaction is conducted in a nucleic acid
polymerization system with a nucleic acid primer contained therein.
In this case, the nucleic acid primer is used as a precursor
(primer) for a nucleic acid polymer. This method is an illustrative
method applicable when the nucleic acid polymerization system is
the above-described nucleic acid polymerization system (6), and
corresponds to the present invention as defined in claim 5.
[0127] When a fluorescence-labeled nucleotide is incorporated in a
nucleic acid polymer as in the methods (1) and (2), the distance
between the fluorescent dyes (A, B) in the incorporated
fluorescence-labeled nucleotide significantly decreases so that an
interaction takes place between the fluorescent dyes although this
interaction did not occur in the state that the
fluorescence-labeled nucleotide was dispersed in the solution (see
FIG. 1). The nucleic acid template or the nucleic acid polymer
synthesized by using the nucleic acid template as a template can be
assayed by measuring or monitoring a change in fluorescence
intensity due to the interaction between the fluorescent dyes or
the amount of the change.
[0128] Of the fluorescent dyes interacting with each other in the
above-described method, one of the fluorescent dyes is a dye which
donates excitation energy for the FRET phenomenon, and is called a
"donor dye" (A). The other fluorescent dye is a dye which emits
fluorescence upon acceptance of the energy, and is called an
"acceptor dye" (B).
[0129] The acceptor dye can generally be any dye insofar as it can
act as an acceptor dye in the FRET phenomenon when paired with a
donor dye or it can accept a transfer of energy from a donor dye
(in other words, can give quenching effect to the donor dye). The
donor dye, on the other hand, can be any dye insofar as it can
transfer excitation energy to the acceptor dye. They can be
suitably chosen from the above-described dyes.
[0130] Preferred examples of donor dyes can include FITC, "BODIPY
FL", the above-described "BODIPY FL" series dyes, "BODIPY 493/503",
"5-FAM", "BODIPY 5-FAM", tetramethylrhodamine, and 6-TAMRA, with
FITC, "BODIPY FL", "BODIPY 493/503", "BODIPY 5-FAM",
tetramethylrhodamine, and 6-TAMRA being more preferred.
[0131] A preferred acceptor dye varies depending upon the donor dye
to be paired. When "BODIPY FL", the above-described "BODIPY FL"
series dyes, "BODIPY 493/503", "5-FAM", "BODIPY 5-FAM",
tetramethylrhodamine, 6-TAMRA and the like are used as donor dyes,
for example, rhodamine X, "BODIPY 581/591" and the like can be used
as acceptor dyes. This method measures an increase or decrease in
the intensity of fluorescence at a particular wavelength from
fluorescent dye(s) in a labeled nucleotide, that is, from a nucleic
acid polymerization system. A decrease in fluorescence intensity is
measured upon determining the intensity of fluorescence from a
donor dye, and an increase in fluorescence intensity is measured
upon determining the intensity of fluorescence from an acceptor
dye.
2) Invention Method B (See FIG. 2)
[0132] This method is an illustrative method applicable when a
nucleic acid polymerization system, to which the present invention
can be applied, contains a nucleic-acid-specific fluorescent dye.
Specifically, this method is an illustrative method applicable when
the nucleic acid polymerization system is the above-described
nucleic acid polymerization system (8). The nucleic-acid-specific
fluorescent dye (C) binds to a nucleic acid polymer, a nucleic acid
polymer-template complex, or a nucleic acid primer-template
complex. An interaction takes place between fluorescent dye (D) in
a fluorescence-labeled nucleotide, which has been incorporated in
the nucleic acid polymer, and the nucleic-acid-specific fluorescent
dye (C). The template nucleic acid or the nucleic acid polymer
synthesized by using the template nucleic acid as a template can be
assayed by measuring or monitoring a change in fluorescence
intensity due to the interaction. This method corresponds to the
present invention as defined in claim 15, and measures a decrease
in the intensity of fluorescence from the nucleic-acid-specific
fluorescent dye (C) or an increase in the intensity of fluorescence
from the fluorescent dye (D) in the labeled nucleotide.
Specifically, an increase or decrease in the intensity of
fluorescence at a particular wavelength from the nucleic acid
polymerization system is measured.
[0133] In the course of conducting a reaction to incorporate a
fluorescence-labeled nucleotide in a nucleic acid polymer in the
presence of a nucleic-acid-specific fluorescent dye (C), the
nucleic-acid-specific fluorescent dye (C) binds to the synthesized
nucleic acid polymer, and therefore, the distance between the
fluorescent dye (C) and a fluorescent dye (D) in the
fluorescence-labeled nucleotide incorporated in the course of the
reaction significantly decreases as described above (see FIG. 2).
Accordingly, a similar interaction takes place between the
fluorescent dyes.
[0134] As the fluorescent dye (D) in this method, the
above-described fluorescent dyes are all usable, and preferred
examples can include FITC, EDANS, 6-joe, TMR, Alexa 488, Alexa 532,
"BODIPY FL/C3", "BODIPY R6G", "BODIPY FL", Alexa 532, "BODIPY
FL/C6", "BODIPY TMR", 5-FAM, "BODIPY 493/503", "BODIPY 564",
"BODIPY 581", Cy3, Cy5, Texas red, and x-Rhodamine. The
above-described examples of the nucleic-acid-specific fluorescent
dye (C) are also all usable, with Sybr green 1 and YO-PRO-1 being
preferred. Preferred examples of the combination of fluorescent
dyes can include the combinations between Sybr green and Texas red,
6-joe, TMR, Alexa 532, BODIPY R6G, Alexa 532, BODIPY TMR, BODIPY
564, BODIPY 581, Cy3, Cy5 and x-Rhodamine; and the combinations
between YO-PRO-1 and Texas red, 6-joe, TMR, Alexa 532, "BODIPY
R6G", Alexa 532, "BODIPY TMR", "BODIPY 564", "BODIPY 581", Cy3,
Cy5, Texas red, x-Rhodamine.
3) Invention Method C
[0135] This is an assay method of a nucleic acid, which is
characterized by conducting a nucleic acid polymerization reaction
in a nucleic acid polymerization system--which contains a
fluorescence-labeled nucleotide, one or more unlabeled nucleotides,
a nucleic acid template and a nucleic acid-synthesizing enzyme with
or without an unlabeled nucleic acid primer--and assaying the
nucleic acid template or a nucleic acid polymer, which has been
synthesized using the nucleic acid template as a template, from a
decrease in the intensity of fluorescence from the nucleic acid
polymerization system or from the amount of the decrease. It is
preferred that at least one of the unlabeled nucleotides contains
guanine (g) an/or the template nucleic acid contains at least one
guanine (g). Described specifically, the base of the template
nucleic acid, said base corresponding to the base of the
fluorescent-labeled nucleotide incorporated in the nucleic acid
polymer, forms a gc(GC) pair, or the template nucleic acid contains
G at a position 1 to 3 bases apart from the base of the
fluorescence-labeled nucleotide (said corresponding base being
counted as "one" base) or the nucleic acid polymer contains an
unlabeled nucleotide the base of which is G. This assay method of
the nucleic acid relies upon a change in fluorescence intensity
based on an interaction between the fluorescent dye (E) and G. This
method is an illustrative method applicable when the nucleic acid
polymerization system is the above-described nucleic acid
polymerization system (2), and corresponds to the present invention
as defined in claim 7 or 8.
[0136] The decrease in the intensity of fluorescence from the
polymerization reaction system takes place in any one of the
following situations: [0137] (1) The base of a fluorescence-labeled
nucleotide is cytosine (c) or guanine (g), [0138] (2) The base of
at least one fluorescence-labeled nucleotide is guanine (g), and
[0139] (3) The template nucleic acid contains at least one guanine
(g).
[0140] In the course of conducting a nucleic acid polymerization
reaction by using a fluorescence-labeled nucleotide and/or an
unlabeled nucleotide, the distance between the guanine (g) in the
template or the guanine (g) in the guanine (g)-containing unlabeled
nucleotide incorporated in the synthesized nucleic acid polymer and
the fluorescent dye (E) in the incorporated, fluorescence-labeled
nucleotide significantly decreases as described above (see FIG. 3).
Accordingly, excitation energy is transferred from the fluorescent
dye (E) to the guanine (b) base although such a transfer did not
take place in the state that the fluorescence-labeled nucleotide
and/or an unlabeled nucleotide and the template were dispersed in
the solution.
[0141] As the fluorescent dye (E) labeling the fluorescence-labeled
nucleotide in this method, the above-described fluorescent dyes are
all usable, and preferred examples can include FITC, EDANS, Texas
red, 6-joe, TMR, Alexa 488, Alexa 532, "BODIPY FL/C3", "BODIPY
R6G", "BODIPY FL", Alexa 532, "BODIPY FL/C6", "BODIPY TMR", 5-FAM,
"BODIPY 493/503", "BODIPY 564", "BODIPY 581", Cy3, Cy5, Texas red,
and x-Rhodamine.
[0142] The preferred nucleotide monomer to be fluorescence-labeled
may be a nucleotide monomer containing cytosine as a base
(cytidylic acid, cytidine 5'-phosphate, cytidine 5'-diphosphate,
cytidine 5'-triphosphate, or a polymer thereof, or a polymer
containing cytidylic acid), and the position of labeling can be a
base (amino group), phosphate group (OH group), or a ribose moiety
(2'- or 3'-OH group). The preferred position is either a base or a
phosphate group.
4) Invention Method D (See FIG. 4)
[0143] This method is an illustrative method similar to the method
(1) or (2) of the invention method A except that a
fluorescence-labeled nucleotide and quencher-labeled nucleotide are
both used. Described specifically, when the fluorescence-labeled
nucleotide and quencher-labeled nucleotide are incorporated in a
nucleic acid polymer, a florescent dye (A) in the incorporated
fluorescence-labeled nucleotide and a quencher (Q) in the
quencher-labeled nucleotide come close to each other and interact
with each other (see FIG. 4), resulting in a decrease in the
intensity of fluorescence from the nucleic acid polymerization
system. This method is characterized in that a template nucleic
acid or a nucleic acid polymer synthesized by using the template
nucleic acid as a template is assayed by measuring this decrease in
fluorescence intensity or monitoring the decrease. This method is
an illustrative method applicable when the nucleic acid
polymerization system is the above-described nucleic acid
polymerization system (1) or (2), and corresponds to the present
invention as defined in claim 3 or 4. Practically, a decrease in
the intensity of fluorescence from the nucleic acid polymerization
system is measured.
[0144] Examples of the quencher (also called "fluorescence
quenching substance") usable in this method include Dabcyl, "QSY7"
(product of Molecular Probes Corporation), "QSY33" (product of
Molecular Probes Corporation), derivatives thereof, methyl
viologen, and N,N'-dimethyl-2,9-diazopyrenium.
[0145] When the nucleic acid polymerization reaction is conducted
in the presence of the fluorescence-labeled nucleotide and
quencher-labeled nucleotide in the nucleic acid polymerization
system, the fluorescence-labeled nucleotide and quencher-labeled
nucleotide are incorporated in the nucleic acid polymer as
described above. As a result of the incorporation in the nucleic
acid polymer, the distance between the fluorescence-labeled
nucleotide and quencher-labeled nucleotide significantly decreases
so that the distance between the fluorescent dye (A) and the
quencher (Q) also decreases significantly (see FIG. 4).
Accordingly, an interaction (a transfer phenomenon of
light-emitting energy) takes place between the quencher (Q) and the
fluorescent dye (A) although such an interaction did not take place
in the state that the fluorescence-labeled nucleotide and
quencher-labeled nucleotide were dispersed in the solution.
5) Invention Method E (See FIG. 5 and FIG. 6)
[0146] This method is an illustrative method similar to the method
(2) of the invention method A except that a labeled nucleic acid
primer is used as a nucleic acid primer. In the course of
conducting polymerization of a template nucleic acid, a labeled
nucleotide is incorporated in the nucleic acid polymer. By an
interaction between a fluorescent dye (A) or quencher (Q) in the
labeled nucleic acid primer and a fluorescent dye (A) or quencher
(Q) in the labeled nucleotide, the intensity of fluorescence
changes. According to this method, this change is measured or
monitored to assay the template nucleic acid or the nucleic acid
polymer synthesized by using the template nucleic acid as a
template. This method is an illustrative method applicable when the
nucleic acid polymerization system is the above-described nucleic
acid polymerization system (6), and corresponds to the present
invention as defined in claim 6. Practically, an increase or
decrease in the intensity of fluorescence from the nucleic acid
polymerization system is measured. In this case, the increase or
decrease in florescence intensity varies depending upon the
combination of fluorescent dye (A) or quencher (Q) in the labeled
nucleotide and the fluorescent dye (A) or quencher (Q) as a label
in the labeled nucleic acid primer. A donor dye and acceptor dye
have a similar interaction as in the invention method A. A quencher
and fluorescent dye have a similar interaction as in the invention
method D.
[0147] When a nucleic acid polymerization reaction is conducted in
a similar manner as in the invention method A except for the use of
the labeled nucleic acid primer and the fluorescence-labeled
nucleotide, the labeled nucleic acid primer and
fluorescence-labeled nucleotide are incorporated in the nucleic
acid polymer as described above. As a result of the incorporation
in the nucleic acid polymer, the distance between the labeled
nucleic acid primer and the fluorescent dye (A) or quencher (Q) in
the fluorescence-labeled nucleotide significantly decreases (see
FIG. 5 and FIG. 6). Accordingly, an interaction takes place between
the fluorescent dye (A) or quencher (Q) in the labeled nucleic acid
primer and the fluorescent dye (A) or quencher (Q) in the labeled
nucleotide although such an interaction did not take place in the
state that the labeled nucleic acid primer and labeled nucleotide
were dispersed in the solution.
[0148] As substance-substance interactions which may take place in
the present invention, the following three cases can be
contemplated: (1) an interaction between the fluorescent dye (A) in
the labeled nucleic acid primer and the fluorescent dye (B) in the
labeled nucleotide; (2) an interaction between the fluorescent dye
(A) in the labeled nucleic acid primer and the quencher (Q) in the
labeled nucleotide; and (3) an interaction between the quencher (Q)
in the labeled nucleic acid primer and the fluorescent dye (A) in
the labeled nucleotide [FIG. 5 illustrates the case (1), while FIG.
6 depicts the case (3)]. Different from a nucleic acid probe for a
homogeneous solution system according to the known technique, said
nucleic acid probe being to be described subsequently herein, it is
not necessary to design the above-described labeled nucleic acid
primer in such a way that the intensity of fluorescence changes
when hybridized to a template. This method, therefore, has an
advantage in that the establishment of an experiment system is
simple, easy and sure. Fluorescent dyes preferred for use in this
method are similar to those exemplified above in connection with
the invention method A. On the other hand, quenchers preferred for
use in this method are similar to those exemplified above in
connection with the invention method D.
6) Invention Method F (See FIG. 7)
[0149] This is an illustrative method similar to the method (2) of
the invention method A except that a labeled nucleic acid primer is
used as a nucleic acid primer and an unlabeled nucleotide is used
in place of a labeled nucleotide (in other words, no labeled
nucleotide is used). By an interaction between a fluorescent dye
(A) contained as a label in the labeled nucleic acid primer and the
G base in the unlabeled nucleotide with the G base incorporated
therein, the intensity of fluorescence from the nucleic acid
polymerization system decreases. This method is characterized in
that a template nucleic acid or a nucleic acid polymer synthesized
by using the template nucleic acid as a template is assayed by
measuring this decrease in fluorescence intensity or monitoring the
decrease. This method is an illustrative method applicable when the
nucleic acid polymerization system is the above-described nucleic
acid polymerization system (7), and corresponds to the present
invention as defined in claim 9 or 10.
[0150] Described specifically, an interaction takes place when an
unlabeled nucleotide contains G and moreover, when at a position 1
to 3 bases apart from the base labeled with the fluorescent dye (A)
of the fluorescence-labeled nucleic acid primer in a synthesized
nucleic acid polymer, said labeled base being counted as the
1.sup.st base, a newly polymerized nucleic acid polymer contains at
least one G (which is meant to be a base other than any one of the
bases in the chain of the primer) (see FIG. 7). Different from a
nucleic acid probe for a homogeneous solution system according to
the known technique, it is not necessary to design the
above-described labeled nucleic acid primer in such a way that the
intensity of fluorescence changes when hybridized to a template.
Similar to the invention method E, this method hence has an
advantage in that the establishment of an experiment system is
simple, easy and sure. Fluorescent dyes preferred for use in this
method are similar to those exemplified above in connection with
the invention method C.
7) Invention Method G (See FIG. 8)
[0151] This is an illustrative method similar to any one of the
invention methods A to F except that instead of using the
fluorescence-labeled nucleotide or quencher-labeled nucleotide, a
nucleotide monomer labeled with at least one immune-related
substance selected from the group consisting of antigens,
antibodies and anti-antibodies (immune-related, labeled nucleotide)
is used. Describing based on an example, a fluorescent dye or
quencher is bound on an antibody with which the nucleotide monomer
is labeled. An antigen or anti-antibody corresponding to the
above-described antibody is bound. As a result, the immune-related,
labeled nucleotide acts in a similar manner as a
fluorescence-labeled nucleotide or quencher-labeled nucleotide. To
an antigen, an antibody with a fluorescent dye or quencher bound
thereon binds. To an anti-antibody, an antibody with a fluorescent
dye or quencher bound thereon binds. As a result, these
immune-related, labeled nucleotides also act in a similar manner as
a fluorescence-labeled nucleotide or quencher-labeled
nucleotide.
[0152] To label a nucleotide with an immune-related substance,
specifically with an immune-related substance selected from the
group consisting of antigens, antibodies and anti-antibodies, the
above-described, conventionally known method can be used to achieve
the labeling. It is, however, more preferred to obtain it by
relying upon custom synthesis services (NIHON GENE RESEARCH
LABORATORIES, INC.; http://www.ngrl.co.jp) as described above.
[0153] Since this method is as described above, the immune-related,
labeled nucleotide is paired with an immune-related substance
corresponding to the immune-related substance in the
immune-related, labeled nucleotide, the latter immune-related
substance being labeled with the fluorescent dye or quencher. Even
when a nucleotide is not labeled, the immune-related substance with
which the immune-related, labeled nucleotide is labeled binds to
the above-described immune-related substance to which the
fluorescent dye or quencher is bound, so that a complex is formed
as such a pair. This complex can be considered to have a structure
with which the nucleotide is labeled. In the present invention,
this complex is hence taken as being equivalent to a fluorescent
dye or quencher for the sake of simplicity. The term "fluorescent
dye" as used in the present invention is, therefore, defined to
also embrace the complex, which contains the fluorescent dye, in
addition to the fluorescent dye. Likewise, the term "quencher" as
used in the present invention is defined to also embrace the
complex, which contains the quencher, in addition to the quencher.
A complex, that is, immune-related, labeled nucleotide, which
contains a fluorescent dye, is included within the concept of a
fluorescence-labeled nucleotide, and is also called "a
fluorescence-labeled nucleotide". By applying a similar concept to
a quencher, a nucleotide labeled with an immune-related substance,
which contains the quencher, is called "a quencher-labeled
nucleotide". Handling nucleic acid primers, which are labeled with
immune-related substances, respectively, in a similar manner as the
above-described nucleotide, a nucleic acid primer labeled with a
fluorescent-dye-containing, immune-related substance is called "a
fluorescence-labeled nucleic acid primer", and a nucleic acid
primer labeled with a quencher-containing, immune-related substance
is called "a quencher-labeled nucleic acid primer".
[0154] Therefore, the terms "immune-related, labeled nucleotide"
and "immune-related, labeled nucleic acid primer" are defined such
that the nucleotide and primer contain immune-related substances
corresponding to the immune-related substances of the nucleotide
and primer and carrying a fluorescent dye or quencher bound
thereto. Specifically, the intensity of fluorescence from the
nucleic acid polymerization system is measured by causing the
nucleotide and/or primer and the immune-related substance, to which
the fluorescent dye or quencher is bound, to exist together in the
nucleic acid polymerization system.
[0155] In a similar manner as in the above-described invention
methods A to F, the nucleic acid polymer or its template, or the
template or the nucleic acid polymer synthesized by using the
template as a template can be assayed. As appreciated from the
foregoing, this method, including the methods to be described
subsequently herein, corresponds to the present invention as
defined in any one of the claims, and measures an increase or
decrease in the intensity of fluorescence from the nucleic acid
polymerization system.
[0156] As described above, a nucleotide is labeled with an antigen,
antibody or anti-antibody, and in the course of a nucleic acid
polymerization reaction, the nucleotide labeled with the antigen,
antibody or anti-antibody is incorporated in a nucleic acid polymer
such that any one of the substance-substance interactions described
above in connection with the invention methods A to F,
respectively, can take place. FIG. 8 illustrates the use of such an
interaction between fluorescent dyes as described above in
connection with the invention method A. Different from the
conventional nucleic acid probe for homogeneous solution systems,
said nucleic acid probe being to be described subsequently herein,
it is not necessary to design a probe in such a manner that the
intensity of fluorescence changes when hybridized to a template.
This method, therefore, has an advantage in that the establishment
of an experiment system is simple, easy and sure.
8) Invention Method H (No Diagrammatic Illustration)
[0157] This method corresponds to two aspects of the present
invention.
[0158] i) This method is an illustrative method applicable when the
nucleic acid polymerization system is the above-described nucleic
acid polymerization system (3), and corresponds to the present
invention as defined in any one of claims 12 to 15. This method is
an assay method of a nucleic acid, which is characterized in that a
nucleic acid polymerization reaction is conducted in a nucleic acid
polymerization system, which contains a nucleic acid template, at
least one dideoxynucleotide monomer labeled with at least one
fluorescent dye and/or at least one quencher (the former being
called "a fluorescence-labeled dideoxynucleotide" while the latter
being called "a quencher-labeled dideoxynucleotide", and both of
them being collectively called "labeled dideoxynucleotides"), and a
nucleic acid-synthesizing enzyme, and the template nucleic acid or
a nucleic acid polymer synthesized by using the template nucleic
acid as a template is assayed from a change in fluorescence
intensity or the amount of the change. The nucleic acid
polymerization system contains at least one nucleotide selected
from the group consisting of labeled nucleotides and unlabeled
nucleotides. The nucleic acid polymerization system also contains
an unlabeled nucleic acid primer.
[0159] ii) This method is an illustrative method applicable when
the nucleic acid polymerization system is the above-described
nucleic acid polymerization system (7), and corresponds to the
present invention as defined in claim 16. This method is an assay
method of a nucleic acid, which is characterized in that a nucleic
acid polymerization reaction is conducted in a nucleic acid
polymerization system, which contains a nucleic acid template, at
least one dideoxynucleotide monomer (will be called "an unlabeled
dideoxynucleotide monomer"), a labeled nucleotide, an unlabeled
nucleic acid primer, a nucleic-acid-specific fluorescent dye, and a
nucleic acid-synthesizing enzyme, and the template nucleic acid or
the nucleic acid polymer synthesized by using the template nucleic
acid as a template is assayed from a change or the amount of the
change. In the above method i) or ii) of the invention, the
concepts of the fluorescent dye and quencher are similar to the
those shown in the invention process G.
[0160] In combination with the single-base elongation reaction
method, this method can suitably be used for the measurement, study
or analysis of a polymorphism (including SNP) and/or mutation.
Specifically, when a nucleic acid polymerization reaction or
nucleic acid amplification reaction in the present invention is
conducted using a nucleic acid primer, which has been designed such
that the base at the 3' end of the nucleic acid primer is located
adjacent to a base at the position of a target polymorphism
(including SNP) and/or mutation in a template nucleic acid, and an
unlabeled or labeled dideoxynucleotide having a base either
complementary (capable of forming a hydrogen bond with) or
non-complementary to the base at the position of the target
polymorphism (including SNP) and/or mutation in the template
nucleic acid, a difference in the intensity of fluorescence from
the nucleic acid polymerization system arises depending upon
whether or not the base at the position of the target polymorphism
(including SNP) and/or mutation exists. Based on this difference,
the objective can be achieved. This method is specifically
described in Example 5 and Example 6.
9) Invention Method I (See FIG. 9)
[0161] This method is similar to any one of the above-described
invention methods A to G except that it is characterized by
conducting the polymerization of a template nucleic acid from one
or more nucleic acid primers, which are immobilized on a surface of
a solid, with one of various nucleic acid-synthesizing enzyme
s.
[0162] As described above, one or more nucleic acids are
immobilized on a surface of a solid, and a polymerization reaction
of a template nucleic acid is conducted with one of various nucleic
acid-synthesizing enzyme s. Upon conducting the polymerization
reaction, at least one of a nucleic acid primer,
fluorescence-labeled nucleotide, a quencher-labeled nucleotide, a
nucleic-acid-specific fluorescent dye and the like are provided
such that the substance-substance interaction in any one of the
above-described invention methods A to H takes place. By monitoring
a change in fluorescence intensity, said change taking place upon
occurrence of the interaction, or the amount of the change, the
nucleic acid polymer alone or its template nucleic acid or the
nucleic acid polymer synthesized by using the template nucleic acid
as a template can be assayed (see FIG. 9; in the diagram, the
interaction between the fluorescent dyes described above in
connection with the invention method A is used).
[0163] When observed based on the single template nucleic acid or a
single complex of the template nucleic acid and the
newly-polymerized and synthesized nucleic acid polymer, the
newly-polymerized and synthesize nucleic acid polymer contains
plural fluorescent dyes as described above. This method is further
improved in the sensitivity of an assay over the method that uses a
nucleic acid probe for a homogeneous solution system and relies
upon a change in the intensity of fluorescence from a single
species of fluorescent dye. The sensitivity of an assay is
considerably improved in this method, because this method is an
assay method which is based on a change in fluorescence intensity
due to a transfer of energy from the nucleic-acid-specific
fluorescent dye, which has been incorporated in the complex between
the template nucleic acid and the unlabeled or labeled nucleic acid
primer or the newly-polymerized and synthesized nucleic acid
polymer, to the fluorescent dye in the labeled nucleotide.
Moreover, this method has another advantage in that it can assay
one or more nucleic acids, including a target gene or the like,
simply, easily and promptly.
[0164] In a method for the analysis of data obtained by the
above-described method of the present invention, more preferred
data can be obtained when the intensity of fluorescence from a
fluorescent dye or nucleic-acid-specific fluorescent dye, which can
play a role as a donor, in the FRET phenomenon of a nucleic acid
polymerization system is divided by the intensity of fluorescence
from another fluorescent dye capable of playing a role as an
acceptor, or vice versa. This data processing method is also
encompassed by the present invention.
[0165] The below-described, known nucleic acid probes (1) to (7)
for homogeneous solution systems (oligonucleotides each of which
hybridizes specifically to a template nucleic acid and is labeled
with a fluorescent dye or quencher) and known nucleic acid assay
methods can be preferably applied to the method of the present
invention, especially to labeled nucleic acid primers. When used as
such primers, they can be applied as described above. When used
simply as the probes described in connection with the nucleic acid
amplification method, it is preferred to use, as nucleic
acid-synthesizing enzyme, an exonuclease-free DNA or RNA polymerase
and a ligase. When a nucleic acid probe for a homogeneous solution
system has hybridized to a template nucleic acid, it is indicated
that the probe has been incorporated in the nucleic acid polymer in
the present invention. If a DNA or RNA polymerase having
exonuclease activity is used, however, the probe is degraded. After
the degraded nucleotide is converted with kinase or the like into
the triphosphate, the triphosphate is incorporated in the nucleic
acid polymer in the present invention. [0166] (1) Probes
represented by the probe proposed by Morrison, et al. (Morrison et
al., Anal. Biochem., 183, 231-244, 1989). [0167] (2) Probes
represented by the probe proposed by Mergney, et al. (Mergney et
al., Nucleic Acid Res., 22,920-928, 1994). [0168] (3) Probes
represented by the molecular beacon method (Tyagi et al., Nature
Biotech., 14, 303-308, 1996; Schofield et al., Applied and Environ.
Microbiol., 63, 1143-1147, 1997). [0169] (4) Probes represented by
the probe disclosed by Livak, et al. (U.S. Pat. No. 5,538,848).
[0170] (5) Probes represented by the probe proposed by Kurata, et
al. (Kurata et al., Nucleic Acids Research, 2001, Vol. 29, No. 6,
e34,). These probes are nucleic acid probes for homogeneous
solution systems, in each of which a single-stranded
oligonucleotide is labeled with a fluorescent dye. The base labeled
with the fluorescent dye is G or C, or there is G or C at a
position 1 to 3 bases apart from a base in a target nucleic acid,
said base corresponding to a labeled base and being counted as the
1.sup.st base. [0171] (6) Probes represented by the probe proposed
by Davis et al. (Davis et al., Nucleic Acids Res., 24,702-706,
1996). [0172] (7) Probes represented by the probe proposed by Horn,
et al. (U.S. Patent Application Publication No. US2001/0009760A1,
Publn. Date: Jul. 26, 2001).
[0173] A description will hereinafter be made about the method of
the present invention as applied to a nucleic acid amplification
method instead of the above-described nucleic acid polymerization
method. The expression "nucleic acid amplification method" as used
in the present invention means a method for the in vitro
amplification of a nucleic acid no matter whether it is known or
unknown. The expression "nucleic acid amplification method" shall,
therefore, be interpreted to include all nucleic acid amplification
methods such as, for example, PCR, LCR (ligase chain reaction),
TAS, ICAN (isothermal and chimeric primer-initiated amplification
of nucleic acids), LAMP, NASRA, RCA, TAMA, and UCAN.
[0174] Further, the abbreviation "PCR" means various known PCR
techniques. Examples shall include real-time monitoring
quantitative PCR, RT-PCR, RNA-primed PCR, stretch PCR, reverse PCR,
PCR making use of an Alu sequence, multiplex PCR, PCR making use of
a mixed primer, PCR making use of PNA, and techniques for studying
or analyzing melting curves on nucleic acids amplified by PCR.
[0175] Described specifically, the method of the present invention
as applied to a nucleic acid amplification method is a method for
assaying a nucleic acid by conducting a nucleic acid amplification
reaction in the above-described nucleic acid polymerization system,
especially in a system with a nucleic acid primer or nucleic acid
probe contained therein under conventionally-known conditions
(Kurata et al., Nucleic Acids Research, 2001, Vol. 29, No. 6, e34)
while taking a hybridization reaction, a nucleic acid
polymerization reaction (nucleic acid elongation reaction) and a
denaturation reaction as 1 cycle, and then measuring a change in
fluorescence intensity or relative fluorescence intensity or the
amount of the change in each cycle successively to determine the
concentration or copy number of a template nucleic acid before the
nucleic acid amplification. By measuring the change in fluorescence
intensity or the amount of the change in each cycle successively,
it is possible to determine the number of cycles (Ct value) at
which the change in fluorescence intensity or relative fluorescence
intensity or the amount of the change begins to be observed
(visually). From a relationship between concentrations or copy
numbers of a standard template nucleic acid before the nucleic acid
amplification and their corresponding Ct values, the concentration
or copy number of the template nucleic acid in the sample before
the nucleic acid amplification can be determined. The method of the
present invention as applied to various nucleic acid amplifications
will be described specifically in Examples 4 to 11.
[0176] The above-described nucleic acid probe for a homogeneous
solution system can be suitably used as a nucleic acid primer
(reverse and/or forward primer). In this case, an unlabeled nucleic
acid primer obtained by removing the fluorescent dye and/or
quencher from the nucleic acid probe for the homogeneous solution
system can also be used suitably. As a nucleic acid polymer
amplified by the nucleic acid amplification method contains at
least one fluorescent dye, information available from a
denaturation curve of the polymer by using at least one measurement
wavelength is useful.
[0177] The nucleic acid amplification system useful in the present
invention is any one of the nucleic acid polymerization systems (1)
to (8), preferably (6), (7) and (8), more preferably (6) and
(7).
[0178] The method according to the present invention also includes
data processing methods to be described hereinafter. In the
above-described method for analyzing the data obtained by the
nucleic acid amplification method, preferred data can be obtained
when the intensity of fluorescence from a nucleic acid
polymerization system, which contains a template nucleic acid
and/or nucleic acid-synthesizing enzyme, in each cycle is corrected
by the intensity of fluorescence from a nucleic acid polymerization
system, which does not contain any template nucleic acid and/or
nucleic acid-synthesizing enzyme, in each corresponding cycle.
Further, intensities of fluorescence from a fluorescent dye or
nucleic-acid-specific fluorescent dye, which can play a role as a
donor, in the FRET phenomenon of a nucleic acid polymerization
system as measured successively may be divided by their
corresponding intensities of fluorescence from a fluorescent dye,
which can play a role as an acceptor, as measured successively or
vice versa to correct the data. The thus-corrected data are useful
as preferred data. It is to be noted that an electronic recording
medium, on which a procedure including operational processing steps
for such a correction (hereinafter called "correction processing
steps") is recorded, is also embraced by the present invention.
Needless to say, a measurement and/or data analysis system equipped
with such an electronic recording medium as well as measurements
making use of such a system shall all fall within the breadth of
the present invention.
[0179] A specific description will hereinafter be made about a
method for analyzing data obtained by real-time quantitative PCR
which makes use of the nucleic acid polymerization reaction in the
present invention. According to real-time quantitative PCR, data
are measured in a real-time manner by a system which is now
composed of a reactor for conducting PCR, an instrument for
detecting fluorescence from a fluorescent dye, a user interface,
namely, a computer-readable, recording medium with individual steps
of a data analysis method recorded in the form of a program thereon
(also called a "sequence detection software system"), and a
computer for controlling them and analyzing data. It is also
preferred to conduct the assay of the present invention by such a
system.
[0180] The PCR reactor is an apparatus for repeatedly conducting a
thermal denaturation reaction and annealing reaction of a template
nucleic acid and an elongation reaction of a nucleic acid (for
example, the temperature can be controlled to 95.degree. C.,
60.degree. C. and 72.degree. C. in cycles). The detection
instrument is composed of a fluorescence-inducing argon laser,
spectrograph and CCD camera. The computer-readable recording medium
with the individual steps of the data analysis method recorded in
the form of the program thereon is installed in the computer, is
used to control the above-described system via the computer, and
contains the program for analyzing or processing data outputted
from the detection instrument.
[0181] The data analysis program recorded on the computer-readable
recording medium comprises a step of measuring the intensity of
fluorescence in each cycle, a step of displaying the thus-measured
intensity of fluorescence as a function of its cycle, that is, a
PCR amplification plot on a display of the computer, a step of
calculating the cycle number of PCR (threshold cycle number: Ct) at
which a change in fluorescence intensity or relative fluorescence
intensity or the amount of the change begins to be detected, a step
of preparing a calibration line to permit the determination of the
copy number of a nucleic acid in a sample from a Ct value, and a
step of printing data and plot values in each of the above steps.
While PCR is exponentially proceeding, a linear relationship is
established between the logarithmic values of copy numbers of a
target nucleic acid at the time of the initiation of PCR and Ct
values. By preparing a calibration line based on known copy numbers
and detecting the Ct of a sample in which an unknown copy number of
a template nucleic acid is contained, the initial copy number of
the template nucleic acid at the time of the initiation of PCR can
be calculated accordingly.
[0182] A description will hereinafter be made about a method for
measuring or analyzing a polymorphism and/or mutation by using the
method of the present invention. This method is a method for
assaying a nucleic acid, which is characterized by conducting a
nucleic acid polymerization reaction or nucleic acid amplification
reaction in any one of the nucleic acid polymerization systems (1)
to (9), preferably (6), (7) and (8), more preferably (6) and (7)
for a nucleic acid polymerization method or nucleic acid
amplification method, measuring a change in the intensity of
fluorescence from the nucleic acid polymerization system or the
amount of the change successively or non-successively, and
assaying, studying or analyzing a polymorphism (including SNP)
and/or mutation from the measurement value. It is preferred to
combine the method with a sequence-specific extension method. This
method will be described specifically in Example 7 to Example
10.
[0183] In this case, it is preferred to conduct the nucleic acid
polymerization reaction or nucleic acid amplification reaction in a
nucleic acid polymerization system containing at least one
unlabeled or labeled nucleic acid primer prepared such that a base
at the 3' end of the primer or the 2.sup.nd base from the 3' end
(said base at the 3' end being counted as the 1.sup.st base) does
not complement to the base of a target polymorphism (including SNP)
and/or mutation in the template nucleic acid (both of the bases can
form a hydrogen bond) although the remaining bases complement. When
the nucleic acid polymerization reaction or nucleic acid
amplification reaction does not proceed by using the primer as a
precursor in the nucleic acid polymerization system in which the
primer is contained, no change takes place in the intensity of
fluorescence from the nucleic acid polymerization system. When the
nucleic acid polymerization reaction or nucleic acid amplification
reaction proceeds conversely, a change takes place in the intensity
of fluorescence. In general, the reaction temperature may
preferably be equal to of higher than the TM value of the primer,
but lower than its denaturation reaction temperature (for example,
95.degree. C.). By choosing a base, which does not complement to
the base of the template nucleic acid, as the 3.sup.rd base from
the 3' end of the primer and forming an artificial mismatch between
the template nucleic acid and the primer, any non-specific
elongation reaction can be inhibited. In other words, it becomes
possible to perform a more accurate determination of a
polymorphism. The reaction temperature in this case may preferably
range from a temperature about 5.degree. C. lower than the TM value
of the primer to a temperature lower than a denaturation reaction
temperature. When a primer having a base, which does not complement
to a base of a polymorphism or mutation in a template nucleic acid,
is called "an A-type primer" in the present invention, the primer
having a base which does complement shall be called "a B-type
premier". The combined use of the A-type primer and the B-type
primer in this assay method makes it possible to obtain surer
data.
[0184] Specifically, the method can be carried out in the following
manner.
[0185] In the following polymerization systems, a nucleic acid
polymerization reaction or nucleic acid amplification reaction is
conducted. A detailed description about this method will be made in
Examples 7 and 8.
[0186] 1) Any one of the above-described nucleic acid
polymerization systems (1) to (8), preferably (6), (7) and (8),
more preferably (6) and (7) for the nucleic acid polymerization
method or nucleic acid amplification method. This nucleic acid
polymerization system may preferably contain at least one A-type
primer as a labeled or unlabeled nucleic acid primer.
[0187] 2) The nucleic acid polymerization system described above
under 1) contains at least one B-type primer as described
above.
[0188] 3) The nucleic acid polymerization system described above
under 1) contains at least one A-type primer and at least one
B-type primer as described above (with the proviso that an A-type
primer and B-type primer labeled with the same fluorescent dye are
excluded).
[0189] When an A-type primer and B-type primer labeled with the
same fluorescent dye are used, a polymorphism (including SNP) or
mutation can be assayed by conducting a nucleic acid polymerization
reaction or nucleic acid amplification reaction in the reaction
system 1) or 2), measuring a change in fluorescence intensity or
the amount of the change successively or non-successively, and then
comparing and studying the measurement values. When an A-type
primer and B-type primer labeled with different fluorescent dyes
are used, a nucleic acid polymerization reaction or nucleic acid
amplification reaction can be suitably conducted in the reaction
system 3). It is, however, to be noted that even in this case, a
nucleic acid polymerization reaction or nucleic acid amplification
reaction can also be conducted suitably in the reaction system 1)
or 2).
[0190] As described above, the assay, study or analysis of a
polymorphism (including SNP) and/or mutation is also feasible by
using, instead of the above-described A-type primer or B-type
primer, at least one unlabeled or labeled dideoxynucleotide having
a base either complementary (capable of forming a hydrogen bond
with) or non-complementary to the base in the target polymorphism
(including SNP) and/or mutation in the template nucleic acid.
[0191] Therefore, the present invention also embraces reaction
solutions and assay kits and devices for assaying, studying or
analyzing polymorphisms (including SNP) and/or mutations, each of
which is characterized by containing at least one A-type primer
and/or at least one B-type primer as well as at least one substance
selected from the group consisting of template nucleic acids,
nucleic acid-synthesizing enzyme s, unlabeled nucleotides, labeled
nucleotides, immune-related labeled nucleotides, labeled
dideoxynucleotides and unlabeled dideoxynucleotides.
[0192] The assay method of the present invention for nucleic acids
can be used in various fields such as medicine, legal medicine,
anthropology, paleobiology, biology, genetic engineering, molecular
biology, agriculture, and plant breeding. It can also be suitably
applied to microorganism systems called "co-cultivation systems of
microorganisms" or "symbiotic systems of microorganisms", in each
of which various microorganisms exist together or at least one
microorganism exists together with other animal- or plant-derived
cells and they cannot be isolated from each other. Further, the
present invention can also be suitably applied to various nucleic
acid assay methods, for example, FISH, LCR, SD, and TAS.
[0193] The present invention can be defined as described in any one
of claims 1-18, and can also include the following preferred
embodiments:
[0194] [1] A method for assaying a nucleic acid, which is
characterized by conducting a nucleic acid polymerization reaction
in any one of the below-described nucleic acid polymerization
systems, measuring a change in the intensity of fluorescence from
the nucleic acid polymerization system or the amount of the change,
and then assaying, studying or analyzing a polymorphism (including
SNP) and/or a mutation from the measurement value. [0195] (1) A
nucleic acid polymerization system containing a template nucleic
acid, at least one labeled nucleotide, and a nucleic
acid-synthesizing enzyme. [0196] (2) A nucleic acid polymerization
system similar to the nucleic acid polymerization system (1) but
additionally containing an unlabeled nucleotide. [0197] (3) A
nucleic acid polymerization system containing at least one labeled
dideoxynucleotide and a nucleic acid-synthesizing enzyme. [0198]
(4) A nucleic acid polymerization system similar to the nucleic
acid polymerization system (3) but additionally containing at least
one nucleotide selected from the group consisting of labeled
nucleotides and unlabeled nucleotides. [0199] (5) A nucleic acid
polymerization system containing a template nucleic acid, unlabeled
dideoxynucleotide, labeled nucleotide and nucleic acid-synthesizing
enzyme. [0200] (6) A nucleic acid polymerization system similar to
any one of the nucleic acid polymerization systems (1) to (5) but
additionally containing a labeled nucleic acid primer or an
unlabeled nucleic acid primer. [0201] (7) A nucleic acid
polymerization system containing a template nucleic acid, unlabeled
nucleotide, labeled nucleic acid primer, and nucleic
acid-synthesizing enzyme. [0202] (8) A nucleic acid polymerization
system similar to any one of the nucleic acid polymerization
systems (1) to (7) but additionally containing a
nucleic-acid-specific fluorescent dye. [2] A method for assaying a
nucleic acid, which is characterized by conducting, in any one of
the below-described nucleic acid polymerization systems, a nucleic
acid amplification reaction while taking a hybridization reaction
(annealing reaction), a nucleic acid polymerization reaction
(nucleic acid elongation reaction) and a denaturation reaction as 1
cycle, measuring a change in fluorescence intensity or the amount
of the change in each cycle successively, and then determining the
concentration or copy number of a template nucleic acid before the
nucleic acid amplification. [0203] (1) A nucleic acid
polymerization system containing a template nucleic acid, at least
one labeled nucleotide, and a nucleic acid-synthesizing enzyme.
[0204] (2) A nucleic acid polymerization system similar to the
nucleic acid polymerization system (1) but additionally containing
an unlabeled nucleotide. [0205] (3) A nucleic acid polymerization
system containing at least one labeled dideoxynucleotide and a
nucleic acid-synthesizing enzyme. [0206] (4) A nucleic acid
polymerization system similar to the nucleic acid polymerization
system (3) but additionally containing at least one nucleotide
selected from the group consisting of labeled nucleotides and
unlabeled nucleotides. [0207] (5) A nucleic acid polymerization
system containing a template nucleic acid, unlabeled
dideoxynucleotide, labeled nucleotide and nucleic acid-synthesizing
enzyme. [0208] (6) A nucleic acid polymerization system similar to
any one of the nucleic acid polymerization systems (1) to (5) but
additionally containing a labeled nucleic acid primer or an
unlabeled nucleic acid primer. [0209] (7) A nucleic acid
polymerization system containing a template nucleic acid, unlabeled
nucleotide, labeled nucleic acid primer, and nucleic
acid-synthesizing enzyme. [0210] (8) A nucleic acid polymerization
system similar to any one of the nucleic acid polymerization
systems (1) to (7) but additionally containing a
nucleic-acid-specific fluorescent dye. [3] A method for assaying a
nucleic acid as described above under [2], which comprises
measuring a change in fluorescence intensity or the amount of the
change in each cycle successively to determine the number of cycles
(Ct value) at which the change in fluorescence intensity or the
amount of the change begins to be observed, and then determining
the concentration or copy number of a template nucleic acid before
the nucleic acid amplification from a relationship between
concentrations or copy numbers of the template nucleic acid before
the nucleic acid amplification and their corresponding Ct values.
[4] A method for assaying a nucleic acid as described above under
[2], which comprises conducting a nucleic acid amplification
reaction in any one of the nucleic acid polymerization systems
described above under [2], measuring a change in the intensity of
fluorescence from the nucleic acid polymerization system or the
amount of the change successively, and then assaying, studying or
analyzing a polymorphism (containing SNP) and/or mutation from the
measurement values. [5] A method for assaying a nucleic acid as
described above under [1] or [4], which comprises conducting a
nucleic acid polymerization reaction or nucleic acid amplification
reaction in a nucleic acid polymerization system, in which a base
at the 3' end of an unlabeled or labeled nucleic acid primer or the
second base from the 3' end (the base at the 3' end being counted
as the 1.sup.st base) contains an unlabeled or labeled nucleic acid
primer corresponding to the base in a target polymorphism
(including SNP) and/or mutation in a template nucleic acid,
measuring a change in the intensity of fluorescence from the
nucleic acid polymerization system or the amount of the change, and
then assaying, studying or analyzing the polymorphism (including
SNP) and/or mutation from the measurement value. [6] A method for
assaying a nucleic acid as described above under [1] or [4], which
comprises conducting a nucleic acid polymerization reaction or
nucleic acid amplification reaction in any one of the
below-described nucleic acid polymerization systems, and then
comparing and studying the resultant data to assay, study or
analyze a polymorphism (including SNP) and/or a mutation. [0211] 1)
A nucleic acid polymerization system similar to any one of the
above-described nucleic acid polymerization systems (1) to (8)
except that a base at the 3' end of an unlabeled or labeled nucleic
acid primer or the second base from the 3' end (the base at the 3'
end being counted as the 1.sup.st base) contains at least one of
primers (called "A-type primers") each having a base not
complementary to the corresponding base in the template nucleic
acid. [0212] 2) A nucleic acid polymerization system similar to the
above-described nucleic acid polymerization system 1) except that a
base at the 3' end of an unlabeled or labeled nucleic acid primer
or the second base from the 3' end (the base at the 3' end being
counted as the 1.sup.st base) contains at least one of primers
(called "B-type primers") each having a base complementary to the
corresponding base in the template nucleic acid. [0213] 3) A
nucleic acid polymerization system similar to the above-described
nucleic acid polymerization system 1) except that at least one
A-type primer and at least one B-type primer are contained (with
the proviso that an A-type primer and B-type primer labeled with
the same fluorescent dye are excluded). [7] A method for processing
or analyzing data obtained in any one of claims 1, 7, 9, 12 and 16
and the above-described method [1] and [3], which is characterized
by dividing intensities of fluorescence from a fluorescent dye or
nucleic-acid-specific fluorescent dye, which plays a role as a
donor, in the FRET phenomenon of a nucleic acid polymerization
system as measured successively or non-successively with the
corresponding intensities of fluorescence from a fluorescent dye
which plays a role as an acceptor in the FRET phenomenon of the
nucleic acid polymerization system, or vice versa. [8] A method for
processing or analyzing data obtained in any one of claims 1, 7, 9,
12 and 16 and the above-described method [1] and [3], which is
characterized by correcting the intensity of fluorescence from a
nucleic acid polymerization system, which contains a template
nucleic acid or nucleic acid-synthesizing enzyme, in each cycle
with the intensity of fluorescence from a nucleic acid
polymerization system, which does not contain any template nucleic
acid or nucleic acid-synthesizing enzyme, in the corresponding
cycle. [9] A reaction solution or an assay kit or device for
assaying, studying or analyzing a polymorphism (including SNP)
and/or a mutation, which is characterized by containing at least
one A-type primer and/or at least one B-type primer as well as at
least one substance selected from the group consisting of template
nucleic acids, nucleic acid-synthesizing enzyme s, unlabeled
nucleotides, labeled nucleotides, labeled dideoxynucleotides and
unlabeled dideoxynucleotides. [10] A method for assaying a nucleic
acid as described in any one of claims 1, 7, 9, 12 and 16 and the
above-described method [1] and [4], wherein the nucleic
acid-synthesizing enzyme is Vent(exo-)DNA polymerase deficient in
3'.fwdarw.5' exonuclease activity (derived from Thermococcus
litoralis), Tgo(exo-)DNA polymerase, "ThermoSequenase DNA
Polymerase" (product of Amersham Biosciences Corp.), AmpliTagGold,
or T7 Squenase DNA polymerase. [11] A method for assaying a nucleic
acid as described above under [4], wherein the nucleic acid
amplification method is PCR, ICAN, LAMP, NASBA, RCA, TAMA, or LCR.
[12] A method for assaying a nucleic acid as described above under
[11], wherein PCR is real-time quantitative PCR. [13] A device (DNA
chip) characterized in that with a nucleic acid primer labeled with
at least one labeled nucleic acid primer as described in claim 5
being immobilized on a surface of a solid, the method described in
any one of claims 1, 7, 9, 12 and 16 and the above-described
methods [1] and [2] can be conducted. [14] A method for assaying a
nucleic acid as described in any one of claims 1, 7, 9, 12 and 16
and the above-described methods [1] and [2], wherein the nucleic
acid polymerization reaction of the template nucleic acid is
conducted using the above-described device (DNA chip). [15] An
assay system capable of measuring fluorescence at varied
temperatures to assay a template nucleic acid or a nucleic acid
polymer or nucleic acid amplification product synthesized by using
the template nucleic acid as a template in accordance with the
method described in any one of claims 1, 7, 9, 12 and 16 and the
above-described methods [1], [2] and [4], characterized by the
incorporation of a computer-readable recording medium on which a
procedure for allowing a computer to perform the steps of the data
processing or analyzing method described in the above-described
method [7] or [8] has been recorded as a program. [16] A method for
labeling a base at a desired position in a nucleic acid probe for a
homogeneous solution system by using the method described in any
one of claims 1, 7, 9 and 12 and the above-described method
[2].
EXAMPLES
[0214] The present invention will next be described more
specifically based on examples and comparative examples, in which
certain terms will be used in shortened or abbreviated forms as
will be explained hereinafter.
[0215] 1) A "template nucleic acid" may be referred to as a
"template".
[0216] 2) A "nucleic acid primer" will be referred to as a
"primer".
[0217] 3) "dNTSs", "dATP", "dGTP", "dTTP" and "dUTP" have the same
meanings as they are currently used in molecular biology and the
like.
[0218] The templates, labeled or unlabeled nucleotides, and labeled
or unlabeled primers used in the examples were obtained by relying
upon custom synthesis services (NIHON GENE RESEARCH LABORATORIES,
INC.; http://www.ngrl.co.jp) unless otherwise specifically
indicated.
[0219] The primers used in the examples and their base sequences
are as will be described below. It is to be noted that in each base
sequence, the right end is the 3' end while the left end is the 5'
end.
[0220] (Base Sequences of Synthesized, Single-Stranded DNAs)
TABLE-US-00019 Primer 1: cagactcgac agtgtagacc cg Primer 2:
agagtttgat cctggctcag Primer 3: ttgcatgtgt taggcctg
[0221] On the other hand, Templates 1 to 9 had the following base
sequences, respectively. In each base sequence, the right side is
the 3' side while the left side is the 5' side. TABLE-US-00020
Template 1: acacacacac acacttcggg tctacactgt cgagtctg Template 2:
tatctatcta tctatctatc tatctatctt cgggtctaca ctgtcgagtc tg Template
3: ttattcttat tcttattctt attcttattc ttattcttat tcttcgggtc
tacactgtcg agtctg Template 4: ttatttcttt atttctttat ttctttattt
ctttatttct ttatttcttt atttcttcgg gtctacactg tcgagtctg Template 5:
ttattttctt tattttcttt attttcttta ttttctttat tttctttatt ttctttattt
tcttcgggtc tacactgtcg agtctg Template 6: ttattttttc ttttttattt
tttctttttt attttttctt ttttattttt tcttttttat tttttctttt ttattttttc
ttttttattt tttcttcggg tctacactgt cgagtctg Template 7: ttatttttct
tttatttttc ttttattttt cttttatttt tcttttattt ttcttttatt tttcttttat
ttttcttcgg gtctacactg tcgagtctg Template 8: ttatttttct ttttattttt
ctttttattt ttctttttat ttttcttttt atttttcttt ttatttttct ttttattttt
cttcgggtct acactgtcga gtctg Template 9: ttattttttc ttttttattt
tttctttttt attttttctt ttttattttt tcttttttat tttttctttt ttattttttc
ttttttattt tttcttcggg tctacactgt cgagtctg Template 10: gcttcgggtc
tacactgtcg agtctg Template 11: gctccgggtc tacactgtcg agtctg
Example 1 (Invention Method B(2))
[0222] Using the FRET phenomenon between a fluorescent dye specific
to a double-stranded nucleic acid and a fluorescence-labeled
nucleotide, each template nucleic acid was assayed.
(A) Various Methods
1) Synthesis of Template DNAs and Primers
[0223] Single-stranded DNAs (Templates 1 to 9), which were used as
templates in this example, and a 22-base primer (Primer 1) were
prepared by a DNA synthesizer, "ABI394" (manufactured by
PerkinElmer Inc., U.S.A.). Templates 1 to 9 had on the 3' sides
thereof a common sequence complementary to Primer 1. Those
templates were each designed such that seven labeled dUTPs would be
incorporated in the course of an elongation reaction. The
combinations of the respective templates (i.e., the respective
single-stranded DNAs) and Primer 1 have the following
characteristics: [0224] A combination of Template 1 and Primer 1:
One fluorescence-labeled nucleotide is incorporated whenever one
unlabeled nucleotide is incorporated. [0225] A combination of
Template 2 and Primer 1: One fluorescence-labeled nucleotide is
incorporated whenever three unlabeled nucleotides are incorporated.
[0226] A combination of Template 3 and Primer 1: One
fluorescence-labeled nucleotide is incorporated whenever five
unlabeled nucleotides are incorporated. [0227] A combination of
Template 4 and Primer 1: One fluorescence-labeled nucleotide is
incorporated whenever seven unlabeled nucleotides are incorporated.
[0228] A combination of Template 5 and Primer 1: One
fluorescence-labeled nucleotide is incorporated whenever eight
unlabeled nucleotides are incorporated. [0229] A combination of
Template 6 and Primer 1: One fluorescence-labeled nucleotide is
incorporated whenever ten unlabeled nucleotides are incorporated.
[0230] A combination of Template 7 and Primer 1: One
fluorescence-labeled nucleotide is incorporated whenever eleven
unlabeled nucleotides are incorporated. [0231] A combination of
Template 8 and Primer 1: One fluorescence-labeled nucleotide is
incorporated whenever thirteen unlabeled nucleotides are
incorporated. 2) Polymerization (Amplification) Reaction
Conditions
[0232] Used as a DNA polymerase was "Vent(exo-)DNA Polymerase"
derived from Thermococcus litoralis and deficient in 3'.fwdarw.5'
exonuclease activity (NEW ENGLAND BioLabs, Beverly, Mass.).
Employed as fluorescence-labeled nucleotides, on the other hand,
were Cyanine5-dUTP (650 nm/668 nm), Lissamine.TM.-5-dUTP (570
nm/588 nm) and Texas Red(r)-5-dUTP (593 nm/612 nm) (the
parenthesized values mean the maximum absorption wavelength/maximum
fluorescence wavelength; Perkin Elmer Inc., U.S.A.). As a donor dye
for inducing the FRET phenomenon on each labeled nucleotide
incorporated by the DNA polymerase, was used "SYBR(r)Green I
Nucleic Acid Gel Strain" (Molecular Probes Corporation, U.S.A.)
which binds specifically to a double-stranded nucleic acid, has a
maximum excitation wavelength at 497 nm, and emits the maximum
fluorescence around 520 nm.
[0233] A reaction solution was prepared as will be described below.
[0234] 20 mM Tris-HCl (pH 8.8); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; [0235] 2.5 mM MgSO.sub.4; 0.1% Triton
X-100; 0.25 mg/mL BSA; [0236] 200 nM dATP; 200 nM dGTP; 200 nM
dCTP; [0237] 200 nM Cyanine5-dUTP, Lissamine.TM.-5-dUTP, or Texas
Red(r)-5-dUTP; 1.times.SYBR(r)Green I; 2 nM primer; [0238] 20 nM
synthesized, single-stranded template DNA; [0239] 0.1 U (unit)
Vent(exo-)DNA Polymerase.
[0240] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were mixed into a homogeneous solution.
Subsequent to thermal denaturation at 95.degree. C. for 15 seconds,
the solution was incubated at 65.degree. C. for 15 minutes.
"LightCycler.TM. System" (Roche Diagnostics AG, Germany) was used
for the detection of fluorescence. Upon conducting the detection,
F1 (530 nm) in the system was used for the detection of
SYBR(r)Green I, F2 (640 nm) for the detection of
Lissamine.TM.-5-dUTP or Texas Red(r)-5-dUTP, and F3 (710 nm) for
the detection of Cyanine5-dUTP. Further, the excitation intensity
was fixed at 75%.
3) Details of Used Experiment Systems
[0241] Models 1 to 9: Combinations of Primer 1 and Templates 1 to
9. Cyanine5-dUTP was used as a fluorescence-labeled nucleotide.
[0242] Model 10: A combination of Primer 1 and Template 9. As a
fluorescence-labeled nucleotide, Cyanine5-dUTP was used, and
Vent(exo-)DNA polymerase was not added. [0243] Models 11 to 19:
Combinations of Primer 1 and Templates 1 to 9. As a
fluorescence-labeled nucleotide, Lissamine.TM.-5-dUTP was used, and
Vent(exo-)DNA polymerase was not added. [0244] Model 20: A
combination of Primer 1 and Template 9. As a fluorescence-labeled
nucleotide, Lissamine.TM.-5-dUTP was used, and Vent(exo-)DNA
polymerase was not added. [0245] Models 21 to 29: Combinations of
Primer 1 and Templates 1 to 9. As a fluorescence-labeled
nucleotide, Texas Red(r)-5-dUTP was used. [0246] Model 30: A
combination of Primer 1 and Template 9. As a fluorescence-labeled
nucleotide, Texas Red(r)-5-dUTP was used, and Vent(exo-)DNA
polymerase was not added. [0247] Models 31 to 39: Combinations of
Primer 1 and Templates 1 to 9. No fluorescence-labeled nucleotide
was used, and instead, dTTP was used. [0248] Model 40: A
combination of Primer 1 and Template 9. No fluorescence-labeled
nucleotide was used, and instead, dTTP was used. Vent(exo-)DNA
polymerase was not added.
[0249] The results are shown in FIG. 10 to FIG. 16. Changes in the
fluorescence intensities at F1 of Models 1 to 10 are shown in FIG.
10, and changes in the fluorescence intensities at F3 of Models 1
to 10 are shown in FIG. 11. Changes in the fluorescence intensities
at F1 of Models 11 to 20 are shown in FIG. 12, and changes in the
fluorescence intensities at F2 of Models 11 to 20 are shown in FIG.
13. Changes in the fluorescence intensities at F1 of Models 21 to
30 are shown in FIG. 14, and changes in the fluorescence
intensities at F2 of Models 21 to 30 are shown in FIG. 15. Changes
in the fluorescence intensities at F1 of Models 31 to 40 are shown
in FIG. 16.
[0250] As a result, the fluorescence intensity at F1 decreased
about 40% at the maximum in Models 1 to 9, while the fluorescence
intensity at F3 increased about 4 times at the maximum in the same
models. In the reaction of Model 10 in which no enzyme was added,
no change was observed in the fluorescence intensity at F3 although
the fluorescence intensity at F1 slightly decreased (FIG. 10, FIG.
11). In Models 11 to 19, the fluorescence intensity at F1 decreased
about 40% at the maximum, while the fluorescence intensity at F2
increased about 2.5 times at the maximum. In Model 20 in which no
enzyme was added, the fluorescence intensity at F2 did not change
(FIG. 12, FIG. 13). In Models 21 to 29, the fluorescence intensity
at F1 decreased about 40% at the maximum, while the fluorescence
intensity at F2 increased about 8 times at the maximum. In Model 30
in which no enzyme was added, the fluorescence intensity at F2 did
not change (FIG. 14, FIG. 15). In Models 31 to 39 in which
SYBR(r)Green I alone was used, the fluorescence intensity at F1
increased 3 times at the maximum (FIG. 16).
[0251] As appreciated from the foregoing, the energy transfer
phenomenon was observed between the fluorescent dye specific to
double-stranded nucleic acids (SYBR(r)Green I in this example) and
the corresponding fluorescence-labeled nucleotide, so that the
corresponding template was successfully assayed. Described
specifically, as a result of the supply of energy from the
fluorescent dye, which is specific to double-stranded nucleic acids
and served as a donor, the corresponding fluorescence nucleotide as
an acceptor, the intensity of fluorescence (F1) from the donor
decreased while the intensity of fluorescence (F2 or F3) increased.
In the systems in which no enzyme was added, no change was observed
in the intensity of fluorescence. In the system which contained
only SYBR(r)Green I without addition of any fluorescence-labeled
nucleotide, no energy transfer took place so that only the
intensity of fluorescence (F1) from SYBR(r)Green I increased. This
method made it possible to assay a template nucleic acid by
measuring either a decrease in the intensity of fluorescence on the
side of a donor or an increase in the intensity of fluorescence on
the side of an acceptor in the energy transfer phenomenon.
Example 2 (Invention Method A)
[0252] Template nucleic acids were each assayed using the energy
transfer phenomenon between its fluorescence-labeled nucleotides
themselves.
1) Synthesis of Template DNAs and Primers
[0253] The primers and single-stranded DNAs employed in Example 1
were used. The combinations of the respective templates (i.e., the
respective single-stranded DNAs) and Primer 1 have the following
characteristics: [0254] A combination of Template 1 and Primer 1:
Fluorescence-labeled nucleotides and FITC-labeled nucleotides are
alternately incorporated one by one. [0255] A combination of
Template 2 and Primer 1: Fluorescence-labeled nucleotides and
FITC-labeled nucleotides are alternately incorporated one by one
whenever one unlabeled nucleotide is incorporated. [0256] A
combination of Template 3 and Primer 1: Fluorescence-labeled
nucleotides and FITC-labeled nucleotides are alternately
incorporated one by one whenever two unlabeled nucleotides are
incorporated. [0257] A combination of Template 4 and Primer 1:
Fluorescence-labeled nucleotides and FITC-labeled nucleotides are
alternately incorporated one by one whenever three unlabeled
nucleotides are incorporated. [0258] A combination of Template 5
and Primer 1: After one FITC-labeled nucleotide is incorporated,
three unlabeled nucleotides are incorporated. Next, one
fluorescence-labeled nucleotide is incorporated, followed by the
incorporation of four unlabeled nucleotides. These incorporations
are repeated. In short, fluorescence-labeled nucleotides and
FITC-labeled nucleotides are alternately incorporated one by one
whenever four unlabeled nucleotides are incorporated. [0259] A
combination of Template 6 and Primer 1: Fluorescence-labeled
nucleotides and FITC-labeled nucleotides are alternately
incorporated one by one whenever four unlabeled nucleotides are
incorporated. [0260] A combination of Template 8 and Primer 1:
Fluorescence-labeled nucleotides and FITC-labeled nucleotides are
alternately incorporated one by one whenever five unlabeled
nucleotides are incorporated. [0261] A combination of Template 8
and Primer 1: Fluorescence-labeled nucleotides and FITC-labeled
nucleotides are alternately incorporated one by one whenever six
unlabeled nucleotides are incorporated. 2) Polymerization
(Amplification) Reaction Conditions
[0262] As the fluorescence-labeled nucleotides, similar
fluorescence-labeled nucleotides as in Example 1, specifically
Cyanine5-dUTP, Lissamine.TM.-5-dUTP and Texas Red(r)-5-dUTP were
used. As the FITC-labeled nucleotide, FITC-dGTP (PerkinElmer Inc.,
U.S.A.) was used.
[0263] A reaction solution was prepared as will be described below.
[0264] 20 mM Tris-HCl (pH 8.8); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; [0265] 2.5 mM MgSO.sub.4; 0.1% Triton
X-100; 0.25 mg/mL BSA; [0266] 200 nM FITC-dGTP (donor dyes); 200 nM
dCTP; 200 nM dATP; [0267] 200 nM Cyanine5-dUTP,
Lissamine.TM.-5-dUTP, or Texas Red(r)-5-dUTP (acceptor dye); 2 nM
primer; [0268] 20 nM synthesized, single-stranded template DNA;
[0269] 0.1 U (unit) Vent(exo-)DNA Polymerase.
[0270] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were mixed into a homogeneous solution.
Subsequent to thermal denaturation at 95.degree. C. for 15 seconds,
the solution was incubated at 65.degree. C. for 15 minutes.
"LightCycler.TM. System" was used for the detection of
fluorescence. Upon conducting the detection, F1, F2 and F3 were
used, and the excitation intensity was fixed at 75%.
3) Details of Used Experiment Systems
[0271] Models 1 to 7: With the combinations of Primer 1 and
Template 1, Primer 1 and Template 2, Primer 1 and Template 3,
Primer 1 and Template 4, Primer 1 and Template 6, Primer 1 and
Template 8, and Primer 1 and Template 9, Cyanine5-dUTP was used.
[0272] Model 8: Primer 1 and Template 6 were used, and as a
fluorescence-labeled nucleotide, Cyanine5-dUTP was used.
Vent(exo-)DNA Polymerase was not added. [0273] Models 9 to 15: With
the combinations of Primer 1 and Template 1, Primer 1 and Template
2, Primer 1 and Template 3, Primer 1 and Template 4, Primer 1 and
Template 6, Primer 1 and Template 8, and Primer 1 and Template 9,
Lissamine.TM.-5-dUTP was used. [0274] Model 16: Primer 1 and
Template 6 were used, and as a fluorescence-labeled nucleotide,
Lissamine.TM.-5-dUTP was used. Vent(exo-)DNA Polymerase was not
added. [0275] Models 17 to 23: With the combinations of Primer 1
and Template 1, Primer 1 and Template 2, Primer 1 and Template 3,
Primer 1 and Template 4, Primer 1 and Template 6, Primer 1 and
Template 8, and Primer 1 and Template 9, Texas Red(r)-5-dUTP was
used. Vent(exo-)DNA Polymerase was not added.
[0276] The results are shown in FIG. 17 to FIG. 22. Changes in the
fluorescence intensities at F1 of Models 1 to 8 are shown in FIG.
17, and changes in the fluorescence intensities at F3 of Models 1
to 8 are shown in FIG. 18. Changes in the fluorescence intensities
at F1 of Models 9 to 16 are shown in FIG. 19, and changes in the
fluorescence intensities at F2 of Models 9 to 16 are shown in FIG.
20. Changes in the fluorescence intensities at F1 of Models 17 to
24 are shown in FIG. 21, and changes in the fluorescence
intensities at F2 of Models 17 to 24 are shown in FIG. 22.
[0277] As a result, the fluorescence intensity at F1 decreased
about 50% at the maximum in Models 1 to 7, while the fluorescence
intensity at F3 increased about 5 times at the maximum in the same
models. In the reaction of Model 8 in which no enzyme was added, no
change was observed in the fluorescence intensity (FIG. 17, FIG.
18). In Models 9 to 15, the fluorescence intensity at F1 decreased
about 60% at the maximum, while the fluorescence intensity at F2
increased about 2.5 times at the maximum. In Model 16 in which no
enzyme was added, the fluorescence intensity did not change (FIG.
19, FIG. 20). In Models 17 to 23, the fluorescence intensity at F1
decreased about 50% at the maximum, while the fluorescence
intensity at F2 increased about 4.5 times at the maximum. In Model
24 in which no enzyme was added, the fluorescence intensity did not
change (FIG. 21, FIG. 22).
[0278] As readily envisaged from the above results, the template
nucleic acids were each successfully assayed based on a change in
fluorescence intensity due to the energy transfer phenomenon
between the fluorescence-labeled nucleotides synthesized by the DNA
polymerase. Described specifically, the intensity of fluorescence
(F1) from the FITC-labeled nucleotide as a donor dye decreased,
while the intensity of fluorescence (at F2 or F3) from Cyanine5-,
Lissamine.TM.- or Texas Red(r)-labeled nucleotide as an acceptor
fluorescent dye increased. In the systems in which no enzyme was
added, no change was observed in fluorescence intensity. Similar to
Example 1, it was possible to assay a template nucleic acid by
measuring either a decrease in fluorescence intensity on the side
of a donor fluorescent dye or an increase in fluorescence intensity
on the side of an acceptor fluorescent dye. Therefore, the
determination of the intensity of fluorescence from an acceptor
fluorescent dye/the intensity of fluorescence from a donor
fluorescent dye makes it possible to obtain a still higher S/N
ratio. The invention method A has, therefore, been proven to be a
high-sensitivity assay method for template nucleic acids.
Example 3 (Invention Method A(2))
[0279] Real-Time Quantitative PCR Making Use of an FITC-Labeled
Nucleotide and Cy5-Labeled Nucleotide
1) Synthesis of Template
[0280] Employed as a template was a 1,400 bp DNA fragment obtained
from Pseudomonas fluorescens DSM 50108 (RF) 16S ribosomal DNA. The
template was prepared as will be described hereinafter. Using
Primers 2 and 3, a PCR reaction was conducted with the PF genome as
a template. After the resulting amplified fragment was purified by
"MICROCON PCR(r)" (Millipore Corporation, U.S.A.), its
concentration was measured and then converted into a corresponding
copy number.
2) PCR Reaction Conditions
[0281] A reaction solution was prepared as will be described below.
[0282] 20 mM Tris-HCl (pH 8.8); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; 2.5 mM MgSO.sub.4; 0.1% Triton X-100;
0.25 mg/mL BSA; 20 .mu.M primer pair; template DNA having final
concentrations of from 1.times.10.sup.9 to 1.times.10.sup.5 copies;
0.2 U Vent(exo-)DNA polymerase; 6 .mu.M dATP-dCTP-dGTP mixture; 2.5
.mu.M dTTP; 0.25 .mu.M Cy5 5-dUTP; 0.25 .mu.M FITC-5-dUTP.
[0283] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was thermally denatured at 95.degree. C. for
30 seconds, and was then subjected to PCR 40 cycles each of which
consisted of a denaturation reaction at 95.degree. C. for 10
seconds, an annealing reaction at 56.degree. C. for 10 seconds and
an elongation reaction at 72.degree. C. for 70 seconds.
"LightCycler.TM. System" was used for the detection of
fluorescence. Upon conducting the detection, F1 and F3 were used.
Further, the excitation intensity was fixed at 75%.
[0284] Under the above-described conditions, PCR was conducted, and
the intensity of fluorescence in each cycle was actually measured.
The results are plotted in FIG. 23 and FIG. 24. Specifically, with
respect to the template ribosomal DNA of the respective copy
numbers, the intensities of fluorescence during the annealing
reactions in respective cycles were measured and plotted. It is
observed that from the 6.sup.th cycle or so, the intensity of
fluorescence at F1 decreased while the intensity of fluorescence at
F3 increased. It is also appreciated that the decrease in the
intensity of fluorescence at F1 (the donor fluorescent dye) and the
increase in the intensity of fluorescence at F3 (the acceptor
fluorescent dye) takes place in the decreasing order of copy
number. It is also observed that even in the blank of 0 copy as a
result of no addition of the template DNA, the intensity of
fluorescence at F1 progressively decreased as the cycle number
increased. In this respect, the intensities of fluorescence from
each sample were corrected. Specifically, the intensity of
fluorescence at F1 from the sample in each cycle was divided by the
intensity of fluorescence from the blank in the same cycle number.
Fn=fn(56.degree. C.)/f'(56.degree. C.) where, [0285] Fn: the
corrected value of the fluorescence intensity in each cycle, [0286]
fn(56.degree. C.): the intensity of fluorescence from the sample at
56.degree. C. in each cycle, and [0287] f'n(56.degree. C.): the
intensity of fluorescence from the blank at 56.degree. C. in each
cycle.
[0288] It is also appreciated that with concerning the individual
copy numbers of the template ribosomal DNA, their fluorescence
intensities in initial cycles are not the same. In this respect,
the intensities of fluorescence from each sample were corrected.
Specifically, assuming that the intensity of fluorescence in the
5.sup.th cycle was 1, the intensity of fluorescence from each
sample in each cycle was converted. Cn=Fn(56.degree.
C.)/F5(56.degree. C.) where, [0289] Cn: the converted value of the
fluorescence intensity in each cycle, [0290] Fn(56.degree. C.): the
intensity of fluorescence from the sample at 56.degree. C. in each
cycle, and [0291] F5(56.degree. C.): the intensity of fluorescence
from the sample at 56.degree. C. in the 5.sup.th cycle.
[0292] The above-described two correction methods can each be
applied to the fluorescence intensities either after the annealing
(at 56.degree. C. in the above-described methods) or after the
elongation reaction (at 72.degree. C.).
[0293] In accordance with the processing method, the intensity of
florescence in each cycle was converted assuming that the intensity
of fluorescence in the 5.sup.th cycle was 1. The converted values
were plotted against the corresponding cycle numbers. The data
processed in the above-described manner are shown in FIG. 25 and
FIG. 26. A threshold value was set. Cycle numbers reached the
threshold value were plotted along the X-axis, copy numbers of the
template ribosomal DNA before the initiation of the reaction were
plotted along the Y-axis, and then, a calibration line was drawn.
Actually, the threshold value was set at 0.85 when the Y-axis
represents florescence intensities at F1, and the threshold value
was set at 1.5 when the Y-axis represents fluorescence intensities
at F3. Coefficients of correlation (R2) determined through the
above-described processing were 0.9965 (FIG. 27) and 0.9931 (FIG.
28), respectively.
[0294] When PCR is performed using, as a substrate, dNTP with two
kinds of fluorescence-labeled nucleotides contained therein, the
resulting product is labeled by the two kinds of
fluorescence-labeled nucleotides under the action of DNA
polymerase. Relying upon this property, real-time quantitative PCR
was performed using the energy transfer phenomenon between the two
kinds of fluorescence-labeled nucleotides themselves labeled in the
same molecule. Comparing the fluorescent intensity in the initial
phase with that in the Plateau phase, the intensity of fluorescence
(at F1 in this case) from the donor decreased about 50%, while the
intensity of fluorescence (at F3 in this case) from the acceptor
increased to about 3 times. Using those fluorescence intensities,
corrections were made on quenching of the fluorescence dye and a
difference of fluorescence intensity in the initial phase, both of
which took place as a result of the performance of PCR in cycles.
Those corrections made it possible to perform an accurate
quantitation of the template nucleic acid. According to this
method, the quantitation was feasible no matter which one of the
donor and acceptor was chosen for the measurement of a change in
fluorescence intensity. By dividing the fluorescence intensity at
F3 with that at F1, it is also possible to improve the S/N
ratio.
Example 4
[0295] Real-Time Quantitative PCR Making Use of a Fluorescent Dye
Specific to a Double-Stranded Nucleic Acid and Cy5-Labeled
Nucleotide
[0296] A template and primers similar to those employed in Example
5 were used. The composition of a reaction solution will be shown
below. [0297] 20 mM Tris-HCl (pH 8.8); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; 2.5 mM MgSO.sub.4; 0.1% Triton X-100;
0.25 mg/mL BSA; 20 .mu.M primer pair.
[0298] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was thermally denatured at 95.degree. C. for
30 seconds, and was then subjected to PCR 40 cycles each of which
consisted of a denaturation reaction at 95.degree. C. for 10
seconds, an annealing reaction at 56.degree. C. for 10 seconds and
an elongation reaction at 72.degree. C. for 70 seconds.
"LightCycler.TM. System" was used for the detection of
fluorescence. Upon conducting the detection, F3 was used. Further,
the excitation intensity was fixed at 75%. The results are shown in
FIG. 29. In a similar manner as in Example 5, the fluorescence
intensities were corrected. When the threshold value was set at 2,
the coefficient of correlation (R2) was 0.9984 (FIG. 30).
[0299] When PCR is performed using, as a substrate, dNTP with one
kind of fluorescence-labeled nucleotide contained therein, the
resulting product is labeled by the one kind of
fluorescence-labeled nucleotide under the action of DNA polymerase.
Here, a fluorescent dye which binds to a certain kinds of template
nucleic acids was mixed in advance, and real-time quantitative PCR
was performed using the energy transfer phenomenon between the
fluorescent dye and the fluorescence-labeled nucleotide in the
molecule. Comparing the fluorescent intensity in the initial phase
with that in the Plateau phase, the intensity of fluorescence from
the acceptor was found to increase to about 4 times. Using those
fluorescence intensities, corrections were made on quenching of the
fluorescence dye and a difference of fluorescence intensity in the
initial phase, both of which took place as a result of the
performance of PCR in cycles. Those corrections made it possible to
perform a measurement (quantitation) of the template nucleic
acid.
Example 5
[0300] Assay of SNP by Single-Base Elongation Reaction Making Use
of a Labeled Nucleotide
[0301] 26-Base, single-stranded DNAs (templates 10 and 11) employed
as a template in this example were prepared by a DNA synthesizer,
"ABI394". Templates 10 and 11 contained T and C, respectively, as
the fourth base as counted from the 5' end, and their remaining
sequences were the same. Templates 10 and 11 can, therefore, be
considered to be DNA fragments containing SNPs (single nucleotide
polymorphism; hereinafter abbreviated as "SNP") which are T and C,
respectively, at particular positions. Those templates were each
designed such that it contained on its 3' side a base sequence
complementary to Primer 1 and, when hybridized to the primer, the
base at the 3' end of the primer would be located adjacent to the
base at the SNP position of the template. As the
fluorescence-labeled nucleotides, "Texas Red(r)-5-ddATP"
(PerkinElmer Inc., U.S.A.) and "Cy5.TM.-5-ddGTP" (Amersham
Biosciences Corp.) were used. Further, SRBR(r)Green 1 was added to
the nucleic acid polymerization system.
2) Single-Base Elongation Reaction
[0302] A reaction solution was prepared as will be described below.
[0303] 20 mM Tris-HCl (pH 8.8); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; 2.5 mM MgSO.sub.4; 0.1% Triton X-100;
0.25 mg/mL BSA; 200 nM Texas Red(r)-5-ddATP; 200 nM
Cyaninne5.TM.-5-ddGTP; 1.times.SYBR(r)Green I; 20 nM primer; 200 nM
synthesized, single-stranded template DNA; 0.1 U Vent(exo-)DNA
polymerase.
[0304] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was thermally denatured at 95.degree. C. for
15 seconds, and was then incubated at 65.degree. C. for 10 minutes.
A fluorometer, "PerkinElmer LS-50B", was used for the detection of
fluorescence. Measurements were conducted at 480 nm excitation
wavelength and 610 nm and 670 nm fluorescence wavelengths. The slit
width was set at 10 nm.
3) Details of Used Experiment Systems
[0305] Model 1: A combination of Primer 1 and Template 10, without
addition of Vent(exo-)DNA polymerase (Blank Control). [0306] Model
2: A combination of Primer 1 and Template 10 (a homozygote of
Template 10), with addition of Vent(exo-)DNA polymerase. [0307]
Model 3: A combination of Primer 1 and Template 11 (a homozygote of
Template 11), with addition of Vent(exo-)DNA polymerase. [0308]
Model 4: A combination of Primer 1 and a 1:1 (100 nm, each) mixture
of Templates 10 and 11 (a heterozygote of Templates 10 and 11),
with addition of Vent(exo-)DNA polymerase.
[0309] As a result, the intensity of fluorescence at 610 nm from
Model 1 was 0.60, and the intensity of fluorescence at 670 nm from
Model 1 was 0.10. The intensity of fluorescence at 610 nm from
Model 2 was 2.40, and the intensity of fluorescence at 670 nm from
Model 2 was 0.10. The intensity of fluorescence at 610 nm from
Model 3 was 0.59, and the intensity of fluorescence at 670 nm from
Model 3 was 0.31. The intensity of fluorescence at 610 nm from
Model 4 was 2.01, and the intensity of fluorescence at 670 nm from
Model 4 was 0.21.
[0310] Model 2 which assumed a homozygote of Template 10 had an
intensity of fluorescence at 610 nm about 4 times as high as the
blank. It is presumed that as a result of the incorporation of the
complementary Texas-Red(r)-labeled nucleotide to the SNP position
of Template 10, an energy transfer took place with respect to
SYBR(r)Green I and the intensity of fluorescence at 610 nm from
Texas Red(r) increased. As the non-complementary Cy5.TM.-labeled
nucleotide is not incorporated to the SNP position, the intensity
of fluorescence at 670 nm remained unchanged. Model 3 which assumed
a homozygote of Template 11 had an intensity of fluorescence at 670
nm about 3 times as high as the blank. It is presumed that as a
result of the incorporation of the complementary Cy5.TM.-labeled
nucleotide to the SNP position of Template 11, an energy transfer
took place with respect to SYBR(r)Green I and the intensity of
fluorescence at 670 nm from Cy5.TM. increased. As the
non-complementary Texas-Red(r)-labeled nucleotide is not
incorporated to the SNP position, the intensity of fluorescence at
610 nm remained unchanged. With Model 4 of the heterozygotic system
that Template 10 and Template 11 were included at 1:1, the
intensity of fluorescence increased about two times at both 610 nm
and 670 nm. Those increases took place as a result of the
incorporation of both of the Texas-Red(r)-labeled nucleotide and
Cy5.TM.-labeled nucleotide. Even when two fluorescence-labeled
nucleotides are incorporated per molecule as in the case of the
measurement of SNP, it is also possible to measure a change in
fluorescence intensity as described above. Further, the use of two
kinds of fluorescence-labeled nucleotides makes it possible to
assay two kinds of template nucleic acids within a single tube.
Example 6
[0311] Detection of a Single-Base Polymorphism at the 282-bp
Fragment of p53 Gene Codon by a Single-Base Elongation Reaction
(1) Synthesis of Template DNAs and Primers
[0312] Using Primers 14 and 15, DNAs for use as templates in this
example were prepared by PCR reaction.
(PCR Reaction Conditions)
[0313] A reaction solution was prepared as will be described
below.
[0314] 20 mM Tris-HCl (pH 8.0); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; 2.5 be located adjacent to the SNP
position.
[0315] A reaction solution was prepared as will be described below.
[0316] 1 U Thermo Sequenase I DNA Polymerase (Amersham-Pharmacia
Biotech); 10.times. Thermo Sequence I DNA Polymerase buffer; 200 nM
Texas Red-5-ddATP; 200 nM Cy5-5-ddGTP; 1.times.SYBR Green I; 200 nM
primer; template DNA.
[0317] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was thermally denatured at 95.degree. C. for
30 seconds, and was then incubated at 50.degree. C. for 1 minute.
Taking those steps as 1 cycle, PCR was conducted 40 cycles. A
fluorometer, "LS-50B" (PerkinElmer Inc.), was used for the
detection of fluorescence. Measurements were conducted at 480 nm
excitation wavelength and 610 nm and 670 nm fluorescence
wavelengths. The slit width was set at 10 nm.
[0318] As a result, the intensity of fluorescence at 610 nm from
the C-allele homozygote was 0.60, and the intensity of fluorescence
at 670 nm from the C-allele homozygote was 2.10. The intensity of
fluorescence at 610 nm from the T-allele homozygote was 2.40, and
the intensity of fluorescence at 670 nm from the T-allele
homozygote was 0.58. The intensity of fluorescence at 610 nm from
the heterozygote was 1.60, and the intensity of fluorescence at 670
nm from the heterozygote [0319] mM MgCl.sub.2; 0.1% Triton X-100;
200 nm primer pair; 50 ng human genome DNA; 1 U AmpliTaqGold
(Applied Biosystems); 200 .mu.M dNTPs.
[0320] The final volume of the reaction solution was 25 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was thermally denatured at 95.degree. C. for
10 minutes, and was then subjected to PCR 40 cycles each of which
consisted of a denaturation reaction at 95.degree. C. for 30
seconds, an annealing reaction at 60.degree. C. for 30 seconds and
an elongation reaction at 72.degree. C. for 30 seconds.
(2) Purification of PCR Product
[0321] Since the PCR primer and dNTP were each contained in a large
excess in the thus-prepared PCR product, the PCT product was
purified by a "Quiagen PCR Product Purification Kit". As an
alternative, shrimp alkaline phosphatase (usb) and exonuclease I
(usb) were added in amounts of 4 U and 20 U, respectively to the
PCR product, and subsequent incubation at 37.degree. C. for 90
minutes, the resulting mixture was heated at 85.degree. C. for 15
minutes to inactivate the enzymes. The purified or
enzymatically-treated solution was provided as a template.
(3) Single-Base Elongation Reaction
[0322] As a genotyping primer, the oligonucleotide described above
with respect to Primer 16 was used. The oligonucleotide had been
designed such that, when hybridized to the template prepared by the
PCR reaction, the base at its 3' end would was 1.23. The intensity
of fluorescence at 610 nm in a blank test conducted without
addition of any template DNA was 0.61, and the intensity of
fluorescence at 670 nm in the blank test was 0.60. It is to be
noted that the samples used in this example were those determined
beforehand to be of the genotype by another method (a restriction
fragment length polymorphism method).
[0323] The C-allele homozygote had an intensity of fluorescence at
670 nm about 5 times as high as the blank. It is presumed that as a
result of the incorporation of the CY5-labeled nucleotide, an
energy transfer took place with respect to SYBR Green I and the
intensity of fluorescence at 670 nm from CY5 increased. As the
non-complementary Texas-Red-labeled nucleotide is not incorporated
to the SNP position, the intensity of fluorescence at 610 nm
remained unchanged. When the T-allele homozygote was used as a
template, the intensity of fluorescence at 610 nm was about 4 times
as high as the blank. It is presumed that as a result of the
incorporation of the complementary Texas-Red-labeled nucleotide to
the SNP position, an energy transfer took place with respect to
SYBR Green I and the intensity of fluorescence at 610 nm from Texas
Red increased. The non-complementary CY5-labeled nucleotide was not
incorporated to the SNP position so that the intensity of
fluorescence at 670 nm remained unchanged. With the heterozygote,
the intensity of fluorescence increased about two times at both 610
nm and 670 nm. Those increases took place as a result of the
incorporation of both of the Texas-Red-labeled nucleotide and
Cy5-labeled nucleotide. Even when a PCR product is used as a
template as described above, it is also possible to detect two
kinds of nucleic acids within a single tube by using the method of
the present invention.
Example 7
[0324] Detection of a Single-Base Polymorphism in Aldehyde
Dehydrogenase 2 (ALDH2) Gene by a Sequence-Specific Elongation
Method Making Use of an Allele-Specific Primer
[0325] ALDH2 is one of the genes associated with alcohol
metabolism, and is located on the long arm of chromosome 12. The
mutation allele (ALDH2*2) frequently found on Japanese is a point
mutation in that GAA, which encodes the 487.sup.th amino acid, Glu
(glutamic acid), in ALDH2 exon 12 has changed to AAA which encodes
Lys (lysine).
(1) Synthesis of a Template
[0326] Using Primers 4 and 5, a template DNA for use in this
example was prepared by PCR reaction from a human genome DNA. A
reaction solution had the following composition. [0327] 20 mM
Tris-HCl (pH 8.0); 10 mM KCl; 10 mM (NH.sub.4).sub.2SO.sub.4; 2.5
mM MgCl.sub.2; 0.1% Triton X-100; 200 nm primer pair; 50 ng human
genome DNA; 1 U AmpliTaqGold; 200 .mu.M dNTPs mixture.
[0328] The final volume of the reaction solution was 25 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was thermally denatured at 95.degree. C. for
10 minutes, and was then subjected to PCR 40 cycles each of which
consisted of a denaturation reaction at 95.degree. C. for 30
seconds, an annealing reaction at 60.degree. C. for 30 seconds and
an elongation reaction at 72.degree. C. for 30 seconds.
(2) Purification of PCR Product
[0329] The thus-prepared PCT product was purified by a PCR product
purification kit (Quiagen), or subjected to enzymatic treatment
(shrimp alkaline phosphatase and exonuclease I were added in
amounts of 4 U and 20 U, respectively to the PCR product, and
subsequent incubation at 37.degree. C. for 90 minutes, the
resulting mixture was heated at 85.degree. C. for 15 minutes to
inactivate the enzymes. The purified or enzymatically-treated
solution was provided as a template for use in an analysis of a
single-base polymorphism.
(3) Sequence-Specific Elongation Reaction
[0330] Synthesized were allele-specific primers the 3' ends of
which were complementary to their SNPs. Primer 3 was C-allelic at
the 3' end, while Primer 7 was T-allelic at the 3' end. Except for
these 3' ends, these primers had the same sequence which was
complementary to the template. An analysis of a single-base
polymorphism by a sequence-specific elongation reaction is based on
the principle that an elongation reaction by a DNA polymerase is
inhibited if any mismatch exists at the 3' end of a primer. When
the 3' ends are complementary, FRET takes place by the two kinds of
fluorescent-dye-labeled nucleotides incorporated by an elongation
reaction so that the intensity of fluorescence changes. When the 3'
ends are not complementary, no fluorescence-labeled nucleotide is
incorporated so that the intensity of fluorescence remains
unchanged.
[0331] A reaction solution was prepared as will be described below.
[0332] 20 mM Tris-HCl (pH 8.8); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; 2.5 mM MgSO.sub.4; 2 .mu.M dATP; 2 .mu.M
dTTP; 1.2 .mu.M dCTP; 400 nM Cy5-5-dCTP; 400 nm FITC-5-dCTP; 200 nM
primer; purified PCT product.
[0333] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was thermally denatured at 95.degree. C. for
15 seconds, and was then subjected to PCR 20 cycles each of which
consisted of an annealing reaction at 60.degree. C. for 1 minute
and an elongation reaction at 72.degree. C. for 20 seconds.
"LightCycler.TM. System" was used for the detection of
fluorescence. Upon conducting the detection, F1 (530 nm) and F3
(710 nm) which the system was equipped with were used for the
detection of FITC and CY5, respectively. Further, the excitation
intensity was fixed at 75%.
[0334] Changes in the intensities of fluorescence from FITC and CY5
upon use of Primer 6 in the three kinds of gene forms (C-allele
homozygote, T-allele homozygote, and heterozygote) are shown in
FIG. 31 and FIG. 32, respectively. Changes of the intensities of
fluorescence from FITC and CY5 upon use of Primer 7 are shown in
FIG. 33 and FIG. 34, respectively. In the case of the C-allele
homozygote, the fluorescence intensity changed only when Primer 6
the 3' end of which was C-allelic was added. Specifically, the
intensity of fluorescence from FITC as a donor decreased, while the
intensity of fluorescence from CY5 as an acceptor increased. When
Primer 7 the 3' end of which was T-allelic was added, no change in
the fluorescence intensity was observed. It is presumed that an
elongation reaction took place only when Primer 6 the 3' end of
which was complementary to the template was added. In the case of
the T-allele homozygote, only when Primer 7 the 3' end of which was
T-allelic was added, the intensity of fluorescence from FITC
decreased and the intensity of fluorescence from CY5 as the
acceptor increased. In the case of the heterozygote, on the other
hand, a change in fluorescence intensity was observed no matter
which one of Primer 6 and Primer 7 was added. It is to be noted
that the samples used in this example were those determined
beforehand to be of the genotype by another method (a restriction
fragment length polymorphism method). It has, therefore, been
demonstrated that a combination of the method of the present
invention with the sequence-specific elongation method permits an
analysis of a single-base polymorphism.
Example 8
[0335] Detection of a Single-Base Polymorphism in ALDH2 Gene by a
Sequence-Specific Elongation Method Making Use of a Template
Prepared by the ICAN Method (Isothermal and Chimeric
Primer-Initiated Amplification of Nucleic Acids).
[0336] A template DNA for use in this example was prepared by an
isothermal gene amplification method, which made use of an RNA-DNA
chimeric primer, a DNA polymerase having strand displacement
activity and template exchange activity, and RNaseH. The primer
used upon preparation of the template DNA had the same base
sequence as Primers 4 and 5, and the three bases at its 3' end had
been replaced by a ribonucleotide.
(1) Synthesis of a Template DNA
(ICAN Reaction Conditions)
[0337] 35 mM Tris-HCl (pH 7.8); 10 mM MgSO.sub.4; 5% DMSO; 1 .mu.M
primer pair; 200 ng human genome DNA; 2.2 U "BcaBEST DNA
Polymerase" (TAKARA SHUZO CO., LTD.); 15 U RNase H (TAKARA SHUZO
CO., LTD.); 1 mM dNTPs.
[0338] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was reacted at 55.degree. C. for 60 minutes,
and was then heated at 90.degree. C. for 5 minutes to inactivate
the enzymes.
(2) Enzymatic Treatment of ICAN Amplification Product
[0339] Shrimp alkaline phosphatase and exonuclease I were added in
amounts of 4 U and 20 U, respectively to the ICAN amplification
product, and subsequent incubation at 37.degree. C. for 90 minutes,
the resulting mixture was heated at 85.degree. C. for 15 minutes to
inactivate the enzymes.
(3) Sequence-Specific Elongation Reaction
[0340] A reaction solution was prepared as will be described below.
[0341] 20 mM Tris-HCl (pH 8.8); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; 2.5 mM MgSO.sub.4; 2 .mu.M dATP; 2 .mu.M
dGTP; 2 .mu.M dTTP; 1.2 .mu.M dCTP; 400 nM Cy5-5-dCTP; 400 nm
FITC-5-dCTP; 100 nM Primers 6 and 7; ICAN amplification product;
0.1 U Vent(exo-)DNA polymerase.
[0342] As a result, in the case of the C-allele homozygote, the
fluorescence intensity changed only when Primer 6 the 3' end of
which was C-allelic was added. Specifically, the intensity of
fluorescence from FITC as a donor in the FRET phenomenon decreased,
while the intensity of fluorescence from CY5 as an acceptor
increased. When Primer 7 the 3' end of which was T-allelic was
added, no change was observed. In the case of the T-allele
homozygote, only when Primer 7 the 3' end of which was T-allelic
was added, the intensity of fluorescence changed. In the case of
the heterozygote, on the other hand, a change in fluorescence
intensity was observed no matter which one of Primer 6 and Primer 7
was added. It has, therefore, been confirmed that the detection of
a single-base polymorphism is feasible even when an ICAN
amplification product is used as a template.
Example 9
[0343] Detection of a Single-Base Polymorphism in a
Prostate-Specific Antigen by a Sequence-Specific Elongation Method
Making Use of a Template Prepared by an LAMP (Loop-Mediated
Isothermal Amplification) Method
[0344] A template DNA for use in this example was prepared by an
isothermal gene amplification method making use of four primers and
also, a strand-displacing DNA polymerase as an enzyme.
(1) Synthesis of a Template DNA
LAMP Reaction Conditions
[0345] 10.times. Thermopol Buffer (NEB); 2 mM MgSO.sub.4; 200 ng
human genome DNA; 8 U Bst DNA polymerase; 4 M betaine (Sigma
Chemicals Company); 10 mM dNTPs; 40 pmol Primer 8; 40 pmol Primer
9; 5 pmol Primer 10; 5 pmol Primer 11.
[0346] The final volume of the reaction solution was 25 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was reacted at 65.degree. C. for 60 minutes,
and was then heated at 80.degree. C. for 10 minutes to inactivate
the enzymes.
(2) Enzymatic Treatment of LAMP Amplification Product
[0347] Shrimp alkaline phosphatase and exonuclease I were added in
amounts of 4 U and 20 U, respectively to the LAMP amplification
product, and subsequent incubation at 37.degree. C. for 90 minutes,
the resulting mixture was heated at 85.degree. C. for 15 minutes to
inactivate the enzymes.
(3) Sequence-Specific Elongation Reaction
[0348] A reaction solution was prepared as will be described below.
[0349] 20 mM Tris-HCl (pH 8.8); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; 2.5 mM MgSO.sub.4; 400 nM Cy5-5-dCTP; 400
nm FITC-5-dCTP; 2 .mu.M dATP; 2 .mu.M dGTP; 2 .mu.M dTTP; 1.2 .mu.M
dCTP; 100 nM Primer 12 and Primer 13; LAMP amplification product;
0.1 U Vent(exo-)DNA polymerase.
[0350] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was thermally denatured at 95.degree. C. for
15 seconds, and was then subjected to a PCR reaction 20 cycles each
of which consisted of an annealing reaction at 60.degree. C. for 1
minute and an elongation reaction at 72.degree. C. for 20 seconds.
"LightCycler.TM. System" was used for the detection of
fluorescence. Upon conducting the detection, F1 (530 nm) and F3
(710 nm) which the system was equipped with were used for the
detection of FITC and CY5, respectively. Further, the excitation
intensity was fixed at 75%.
[0351] As a result, in the case of the C-allele homozygote, the
fluorescence intensity changed only when Primer 12 the 3' end of
which was C-allelic was added. Specifically, the intensity of
fluorescence from FITC decreased, while the intensity of
fluorescence from CY5 increased. In the case of the T-allele
homozygote, only when Primer 13 the 3' end of which was T-allelic
was added, the intensity of fluorescence changed. In the case of
the heterozygote, on the other hand, a change in fluorescence
intensity was observed no matter which one of Primer 12 and Primer
13 was added. It has, therefore, been demonstrated that the
detection of a single-base polymorphism is feasible even when a
product amplified by the LAMP method is used as a template.
Example 10
[0352] Analysis of a Single-Base Polymorphism by an Allele-Specific
Elongation Reaction Making Use of a Reverse Transcriptase
[0353] Single-base polymorphisms of LCHAD (long-chain 3-hydroxyacyl
coenzyme A dehydrogenase) and OAT (organic anion transporter) were
analyzed by a sequence-specific elongation method making use of a
reverse transcriptase.
(1) Synthesis of a Template RNA
[0354] An RNA for use as a template in this example was prepared as
will be described below.
[0355] Using the primers of the below-descried SEQ ID NOS: 17, 18,
21 and 22, duplex PCR was performed. One of the primers in each
pair was provided with the 5' RNA polymerase promoter sequence. As
reaction conditions for the multiplex PCR, Ampli Taq Gold 1 U (200
nM), primer pairs (50 ng), human genome DNA, Ampli Taq Gold buffer
(200 .mu.M) and dNTPs were combined into a homogeneous mixture, and
the final volume was adjusted to 20 .mu.L. The resulting reaction
solution was thermally denatured at 95.degree. C. for 10 minutes,
and was then subjected to PCR 40 cycles each of which consisted of
a denaturation reaction at 95.degree. C. for 30 seconds, an
annealing reaction at 65.degree. C. for 30 seconds and an
elongation reaction at 72.degree. C. for 30 seconds. Subsequently,
a transcription reaction was conducted using a "T7 Ampliscribe Kit"
(Epicentre Technologies).
(3) Preparation of Microarrays
[0356] For microarrays, standard microscope glass slide were used.
After the glass slides were activated at the surfaces thereof with
isothiocyanate, NH.sub.2-modified oligonucleotides (SQ ID NOS: 19,
20, 23, 24) were immobilized on the surfaces, respectively. Each
oligonucleotide was dissolved with 400 mM sodium carbonate buffer
(pH 9.0) such that its concentration was adjusted to 20 .mu.M. The
resulting oligonucleotide solutions were dropped onto their
corresponding glass slides in the form of spots of 2 mm in
diameter, exposed to vaporized ammonia, and then washed three times
with distilled water.
(3) Sequence-Specific Elongation Method
[0357] The template RNA prepared as described above was dissolved
in a mixture of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.2 M NaCl and
0.1% Triton X-100, and the resulting solution was added in an
amount of 10 .mu.L per spot to the arrays. The arrays were then
incubated at 37.degree. C. for 20 minutes to effect annealing.
After the arrays were washed with 0.1 M NaCl, a reverse
transcriptase "MMLV" (Epicenter Technologies) (6 U), dNTPs (dATP,
dGTP, FITC-dUTP, CY5-dCTP) (6 .mu.M) and a buffer furnished
together with those enzymes were added, followed by a reaction at
52.degree. C. for 1 hour.
(4) Detection of Signals
[0358] Using a "CONFOCAL SCAN ARRAY 400" (GSI Lumonics), the
microscope glass slides were scanned at 480 nm excitation
wavelength and 650 nm fluorescence wavelength. Values obtained by
subtracting background fluorescence intensities were used for the
determination of genotypes.
[0359] The slide with Primer 19, a primer specific to the C-allele
of LCHAD, immobilized thereon showed a high signal intensity of
about 900 only when the template containing the C-allele zygote of
LCHAD was added as spots, and signals were all 100 or lower when
the template free of the C-allele zygote was added as spots. The
slide with Primer 20, a primer specific to the G-allele of LCHAD,
immobilized thereon showed a high signal intensity (at around 800)
only when the template containing the G-allele zygote of LCHAD was
added as spots, and signals were all 100 or lower when the template
free of the G-allele zygote was added as spots. The slide with
Primer 23, a primer specific to the C-allele of OAT, immobilized
thereon showed a high signal intensity of about 1200 only when the
template containing the C-allele zygote of OAT was added as spots,
and signals were all 100 or lower when the template free of the
C-allele zygote was added as spots. As appreciated from the
foregoing, it has been demonstrated that the nucleic acid assay
method of the present invention permits an analysis of a
single-base polymorphism by using a reverse transcriptase.
Example 11
[0360] Using fluorescein
chlorotriazinyl-4-dC(deoxycytidine)nucleotide monomer, a nucleic
acid was detected based on the quenching phenomenon by
guanidine.
1) Template DNA and Primer
[0361] Primer 1 and Template 12 were used. [0362] Model 1: A
combination of Primer 1 and Template 12. [0363] Model 2: A
combination of Primer 1 and Template 12, without addition of
Vent(exo-)DNA Polymerase. 2) Amplification Reaction Conditions
[0364] A reaction solution was prepared as will be described below.
[0365] 20 mM Tris-HCl (pH 8.8); 10 mM KCl; 10 mM
(NH.sub.4).sub.2SO.sub.4; 2.5 mM MgSO.sub.4; 0.5% Triton X-100; 5%
DMSO; 0.25 mg/mL BSA; 200 nM fluorescein chlorotriazinyl-4-dC; 200
nM dGTP; 200 nM dATP; 200 nM dUTP; 2 nM primer; 50 nM synthesized,
single-stranded DNA; 0.1 U Vent(exo-)DNA polymerase.
[0366] The final volume of the reaction solution was 20 .mu.L. The
above reactant solutions were combined into a homogeneous mixture.
The reaction solution was thermally denatured at 95.degree. C. for
15 seconds, and was then incubated 65.degree. C. for 15 minutes.
"LightCycler.TM. System" was used for the detection of
fluorescence. F1 was used for the detection of fluorescence, and
the excitation intensity was fixed at 75%.
[0367] As a result, in Model 1, the intensity of fluorescence at F1
decreased 24% at the maximum. In the reaction of Model 2 in which
no enzyme was added, no change was observed in the intensity of
fluorescence. From the above results, it is considered that the
intensity of fluorescence decreased as a result of an interaction
between the dc-FITC-labeled nucleotide, which had been incorporated
into the strands under synthesis by the DNA polymerase, and guanine
existing in the complementary chain. TABLE-US-00021 [Base sequences
of primers] Primer 4: gtgtaaccca taacccccaa ga Primer 5: caccagcaga
ccctcaagc Primer 6: cccacactca cagttttcac ttc Primer 7: cccacactca
cagttttcac ttt Primer 8: tgttcctgat gcagtgggca gctttagtct
gcggcggtgt tctg Primer 9: tgctgggtcg gcacagcctg aagctgacct
gaaatacctg gcctg Primer 10: tgcttgtggc ctctcgtg Primer 11:
ggggtggga agctgtg Primer 12: tgatcttgct gggtcggcac agc Primer 13:
tgatcttgct gggtcggcac agt Primer 14: acctgatttc cttactgcct cttgc
Primer 15: gtcctgcttg cttacctcgc ttagt Primer 16: tgtgcctgtc
ctgggagaga c Primer 17: ttctaatacg actcactata gggagaccct tgccaggtga
ttggc Primer 18: gcggtcccaa aagggtcagt gtttctgtgg tcacgaagtc Primer
19: ctctaatagt gctggctc Primer 20: ctctaatagt gctggctg Primer 21:
ttctaatacg actcactata gggagacctt tgtagctggg aacttc Primer 22:
gcggtcccaa aagggtcagt accaaaacct ggtaaatacg g Primer 23: gagatagcag
acaacgtcc Primer 24: gagatagcag acaacgtcg Template 12: ttgttttgtt
ttgttttgtt ttgttttgtt ttgttttgtt ttgttttgtt ttgttttgtt ttgttttgtt
cgggtctaca ctgtcgagtc tg
INDUSTRIAL APPLICABILITY
[0368] Without needing such a nucleic acid probe for a homogeneous
solution system that changes in fluorescence intensity upon
hybridization, a simple, easy, fast, low-cost, high-sensitivity
assay method of a nucleic acid is realized by monitoring a change
in the intensity of fluorescence from a nucleic acid polymerization
system upon conducting a nucleic acid polymerization reaction to
incorporate a labeled nucleotide in a nucleic acid polymer. This
method has made it possible to assay all nucleic acids such as
genes existing in a single system in the nature.
Sequence CWU 1
1
36 1 22 DNA Artificial Synthetic DNA 1 cagactcgac agtgtagacc cg 22
2 20 DNA Artificial Synthetic DNA 2 agagtttgat cctggctcag 20 3 18
DNA Artificial Synthetic DNA 3 ttgcatgtgt taggcctg 18 4 38 DNA
Artificial Synthetic DNA 4 acacacacac acacttcggg tctacactgt
cgagtctg 38 5 52 DNA Artificial Synthetic DNA 5 tatctatcta
tctatctatc tatctatctt cgggtctaca ctgtcgagtc tg 52 6 66 DNA
Artificial Synthetic DNA 6 ttattcttat tcttattctt attcttattc
ttattcttat tcttcgggtc tacactgtcg 60 agtctg 66 7 79 DNA Artificial
Synthetic DNA 7 ttatttcttt atttctttat ttctttattt ctttatttct
ttatttcttt atttcttcgg 60 gtctacactg tcgagtctg 79 8 86 DNA
Artificial Synthetic DNA 8 ttattttctt tattttcttt attttcttta
ttttctttat tttctttatt ttctttattt 60 tcttcgggtc tacactgtcg agtctg 86
9 92 DNA Artificial Synthetic DNA 9 ttattttctt ttattttctt
ttattttctt ttattttctt ttattttctt ttattttctt 60 ttattttctt
cgggtctaca ctgtcgagtc tg 92 10 99 DNA Artificial Synthetic DNA 10
ttatttttct tttatttttc ttttattttt cttttatttt tcttttattt ttcttttatt
60 tttcttttat ttttcttcgg gtctacactg tcgagtctg 99 11 105 DNA
Artificial Synthetic DNA 11 ttatttttct ttttattttt ctttttattt
ttctttttat ttttcttttt atttttcttt 60 ttatttttct ttttattttt
cttcgggtct acactgtcga gtctg 105 12 118 DNA Artificial Synthetic DNA
12 ttattttttc ttttttattt tttctttttt attttttctt ttttattttt
tcttttttat 60 tttttctttt ttattttttc ttttttattt tttcttcggg
tctacactgt cgagtctg 118 13 26 DNA Artificial Synthetic DNA 13
gcttcgggtc tacactgtcg agtctg 26 14 26 DNA Artificial Synthetic DNA
14 gctccgggtc tacactgtcg agtctg 26 15 22 DNA Artificial Synthetic
DNA 15 gtgtaaccca taacccccaa ga 22 16 19 DNA Artificial Synthetic
DNA 16 caccagcaga ccctcaagc 19 17 23 DNA Artificial Synthetic DNA
17 cccacactca cagttttcac ttc 23 18 23 DNA Artificial Synthetic DNA
18 cccacactca cagttttcac ttt 23 19 44 DNA Artificial Synthetic DNA
19 tgttcctgat gcagtgggca gctttagtct gcggcggtgt tctg 44 20 45 DNA
Artificial Synthetic DNA 20 tgctgggtcg gcacagcctg aagctgacct
gaaatacctg gcctg 45 21 18 DNA Artificial Synthetic DNA 21
tgcttgtggc ctctcgtg 18 22 16 DNA Artificial Synthetic DNA 22
ggggtgggaa gctgtg 16 23 23 DNA Artificial Synthetic DNA 23
tgatcttgct gggtcggcac agc 23 24 23 DNA Artificial Synthetic DNA 24
tgatcttgct gggtcggcac agt 23 25 25 DNA Artificial Synthetic DNA 25
acctgatttc cttactgcct cttgc 25 26 25 DNA Artificial Synthetic DNA
26 gtcctgcttg cttacctcgc ttagt 25 27 21 DNA Artificial Synthetic
DNA 27 tgtgcctgtc ctgggagaga c 21 28 45 DNA Artificial Synthetic
DNA 28 ttctaatacg actcactata gggagaccct tgccaggtga ttggc 45 29 40
DNA Artificial Synthetic DNA 29 gcggtcccaa aagggtcagt gtttctgtgg
tcacgaagtc 40 30 18 DNA Artificial Synthetic DNA 30 ctctaatagt
gctggctc 18 31 18 DNA Artificial Synthetic DNA 31 ctctaatagt
gctggctg 18 32 46 DNA Artificial Synthetic DNA 32 ttctaatacg
actcactata gggagacctt tgtagctggg aacttc 46 33 41 DNA Artificial
Synthetic DNA 33 gcggtcccaa aagggtcagt accaaaacct ggtaaatacg g 41
34 19 DNA Artificial Synthetic DNA 34 gagatagcag acaacgtcc 19 35 19
DNA Artificial Synthetic DNA 35 gagatagcag acaacgtcg 19 36 92 DNA
Artificial Synthetic DNA 36 ttgttttgtt ttgttttgtt ttgttttgtt
ttgttttgtt ttgttttgtt ttgttttgtt 60 ttgttttgtt cgggtctaca
ctgtcgagtc tg 92
* * * * *
References