U.S. patent application number 09/345761 was filed with the patent office on 2001-12-20 for method of assay of target nucleic acid.
Invention is credited to ISHIGURO, TAKAHIKO, ISHIZUKA, TETSUYA, SAITOH, JUICHI.
Application Number | 20010053518 09/345761 |
Document ID | / |
Family ID | 16188383 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053518 |
Kind Code |
A1 |
ISHIGURO, TAKAHIKO ; et
al. |
December 20, 2001 |
METHOD OF ASSAY OF TARGET NUCLEIC ACID
Abstract
A simple and accurate method for assay of a single-stranded RNA
containing a specific nucleic acids sequence in a sample at almost
constant temperature by using at least the following reagents (A)
to (I), which comprises a step of adding the reagents (A) to (I)
one by one (in any order), in combinations of at least two or all
at once and a step of measuring a fluorescent signal in the
presence of the reagent (I) at least once after addition of at
least the reagents (A) to (H); (A) a first single-stranded
oligonucleic acid complementary to a sequence neighboring the 5'
end of the specific nucleic acids sequence in the single-stranded
RNA, (B) a second single-stranded oligo DNA complementary to a
3'-end sequence within the specific nucleic acids sequence, (C) an
RNA-dependent DNA polymerase, (D) deoxyribonucleoside
triphosphates, (E) a third single-stranded oligo DNA having (1) a
promoter sequence for a DNA-dependent RNA polymerase, (2) an
enhancer sequence for the promoter and (3) a 5'-end sequence within
the specific nucleic acids sequence, in this order from the 5' end,
(F) a DNA-dependent DNA polymerase, (G) a DNA-dependent RNA
polymerase, (H) ribonucleoside triphosphates, and (I) a fourth
single-stranded oligo DNA complementary to the specific nucleic
acids sequence which is labeled so that it gives off a measurable
fluorescent signal on hybridization with a nucleic acid containing
the specific nucleic acids sequence.
Inventors: |
ISHIGURO, TAKAHIKO;
(KANAGAWA, JP) ; SAITOH, JUICHI; (KANAGAWA,
JP) ; ISHIZUKA, TETSUYA; (KANAGAWA, JP) |
Correspondence
Address: |
SUGHRUE MION ZINN
MACPEAK & SEAS PLLC
2100 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
200373202
|
Family ID: |
16188383 |
Appl. No.: |
09/345761 |
Filed: |
July 1, 1999 |
Current U.S.
Class: |
435/6.12 ;
435/6.1; 435/91.1; 435/91.2; 536/23.1 |
Current CPC
Class: |
C12Q 2521/119 20130101;
C12Q 2521/107 20130101; C12Q 2563/107 20130101; C12Q 2527/125
20130101; C12Q 2563/173 20130101; C12Q 2527/125 20130101; C12Q
2521/107 20130101; C12Q 2563/173 20130101; C12Q 2521/107 20130101;
C12Q 1/686 20130101; C12Q 1/6865 20130101; C12Q 1/686 20130101;
C12Q 1/6865 20130101; C12Q 1/6865 20130101 |
Class at
Publication: |
435/6 ; 435/91.1;
435/91.2; 536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; C12P 019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 1, 1998 |
JP |
JP10-186434 |
Claims
What is claimed is:
1. A simple and accurate method for assay of a single-stranded RNA
containing a specific nucleic acids sequence in a sample at almost
constant temperature by using at least the following reagents (A)
to (I), which comprises a step of adding the reagents (A) to (I)
one by one (in any order), in combinations of at least two or all
at once and a step of measuring a fluorescent signal in the
presence of the reagent (I) at least once after addition of at
least the reagents (A) to (H); (A) a first single-stranded
oligonucleic acid complementary to a sequence neighboring the 5'
end of the specific nucleic acids sequence in the single-stranded
RNA, (B) a second single-stranded oligo DNA complementary to a
3'-end sequence within the specific nucleic acids sequence, (C) an
RNA-dependent DNA polymerase, (D) deoxyribonucleoside
triphosphates, (E) a third single-stranded oligo DNA having (1) a
promoter sequence for a DNA-dependent RNA polymerase, (2) an
enhancer sequence for the promoter and (3) a 5'-end sequence within
the specific nucleic acids sequence, in this order from the 5' end,
(F) a DNA-dependent DNA polymerase, (G) a DNA-dependent RNA
polymerase, (H) ribonucleoside triphosphates, and (I) a fourth
single-stranded oligo DNA complementary to the specific nucleic
acids sequence which is labeled so that it gives off a measurable
fluorescent signal on hybridization with a nucleic acid containing
the specific nucleic acids sequence.
2. The method according to claim 1, wherein the temperature is
selected from the range of from 35 to 60.degree. C.
3. The method according to claim 1, wherein the first oligonucleic
acid as the reagent (A) is a DNA, and the method further comprises
a step of adding an RNaseH and a subsequent step of deactivating
the RNaseH by heating or by addition of an inhibitor prior to
addition of the reagent (B).
4. The method according to claim 3, wherein addition of the reagent
(A) is followed by simultaneous addition of the reagents (B) to
(H), and further by addition of the reagent (I).
5. The method according to claim 3, wherein addition of the reagent
(A) is followed by simultaneous addition of the reagents (B) to
(I).
6. The method according to claim 1, wherein the first oligonucleic
acid as the reagent (A) is a ribozyme or a DNAzyme.
7. The method according to claim 1, which further uses dimethyl
sulfoxide and/or an enzyme which degrades RNA in a DNA-RNA double
strand.
8. The method according to claim 7, which uses dimethyl sulfoxide
at a concentration of from 5 to 20%.
9. The method according to claim 7, wherein the enzyme which
degrades RNA in a DNA-RNA double strand is the RNA-dependent DNA
polymerase as the reagent (C).
10. The method according to claim 1, wherein an enzyme having both
an RNA-dependent DNA polymerase activity and a DNA-dependent DNA
polymerase activity is used as the reagents (C) and (F) to
virtually omit addition of the reagent (C) or the reagent (F).
11. The method according to claim 10, wherein the enzyme is avian
myoblastome virus polymerase.
12. The method according to claim 1, wherein the second and third
oligo DNAs as the reagents (B) and (E) are used at concentrations
of from 0.02 to 1 .mu.M.
13. The method according to claim 1, wherein the DNA-dependent RNA
polymerase as the reagent (G) is at least one enzyme selected from
the group consisting of phage SP6 polymerase, phage T3 polymerase
and phase T7 polymerase.
14. The method according to claim 1, wherein the fourth oligo DNA
as the reagent (I) is a DNA which is linked to a fluorescent
intercalative dye so that the fluorescent intercalative dye changes
its fluorescence characteristic on hybridization of the DNA with
another nucleic acid by intercalating into the resulting double
strand.
15. The method according to claim 1 or 14, wherein the fourth oligo
DNA as the reagent (I) is a DNA which has a 3'-end sequence
uncomplementary to the specific nucleic acids sequence or has a
modified 3' end.
16. The method according to claim 1, which further comprises a step
of detecting or quantifying the single-stranded RNA in the sample
based on the measured fluorescent signal or change in the measured
fluorescent signal.
17. The method according to claim 1, wherein all the reagents are
chloride-free.
18. The method according to claim 1, which further uses an
acetate.
19. The method according to claim 18, wherein the acetate is
magnesium acetate at a concentration of from 5 to 20 mM or
potassium acetate at a concentration of from 50 to 200 mM.
20. The method according to claim 1, which further uses
sorbitol.
21. A simple method for producing a nucleic acid having a specific
nucleic acids sequence at almost constant temperature by using at
least the following reagents (A) to (H), which comprises a step of
adding the reagents (A) to (G) one by one (in any order), in
combinations of at least two or all at once to a single-stranded
DNA having (1) a promoter sequence for a DNA-dependent RNA
polymerase, (2) an enhancer sequence for the promoter and (3) the
specific nucleic acids sequence, in this order from the 5' end or
to a double-stranded DNA consisting of the single-stranded DNA and
a complementary DNA strand and a step of measuring a fluorescent
signal from the reagent (H) at least once after addition of at
least the reagents (A) to (G); (A) a single-stranded oligo DNA
complementary to a 3'-end sequence within the specific nucleic
acids sequence, (B) an RNA-dependent DNA polymerase, (C) a
DNA-dependent DNA polymerase, (D) deoxyribonucleoside
triphosphates, (E) a DNA-dependent RNA polymerase, (F)
ribonucleoside triphosphates, (G) a single-stranded DNA having (1)
a promoter sequence for a DNA-dependent RNA polymerase, (2) an
enhancer sequence for the promoter and (3) a 5'-end sequence within
the specific nucleic acids sequence, in this order from the 5' end,
(H) a fourth single-stranded labeled oligo DNA complementary to the
specific nucleic acids sequence which gives a measurable
fluorescent signal on hybridization with a nucleic acid containing
the specific nucleic acids sequence.
22. The method for producing a single-stranded RNA having a
specific nucleic acids sequence according to claim 21, wherein a
DNase is added when the measured fluorescent signal or change in
the measured fluorescent signal indicates production of a
predetermined amount of the specific nucleic acids sequence.
23. The method for producing a double-stranded DNA consisting of a
DNA strand having a specific nucleic acids sequence and a
complementary DNA strand according to claim 21, wherein an RNase is
added when the measured fluorescent signal or change in the
measured fluorescent signal indicates production of a predetermined
amount of the specific nucleic acids sequence.
24. A reagent set for performing the method according to claim 1 or
21, which comprises at least a first reagent containing the first
single-stranded oligonucleic acid, a second reagent containing
tris-acetate, magnesium acetate, potassium acetate, sorbitol and
dimethyl sulfoxide, a third reagent containing dithiothreitol,
deoxyribonucleoside triphosphates, ribonucleoside triphosphates,
bovine serum albumin, the second single-stranded oligo DNA and the
third single-stranded oligo DNA, a fourth reagent containing an
RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a
DNA-dependent RNA polymerase and an RNase inhibitor and a fifth
reagent containing the fourth single-stranded oligo DNA.
25. A reagent set for performing the method according to claim 1 or
21, which comprises at least a first reagent containing the first
single-stranded oligonucleic acid, a second reagent containing
tris-acetate, magnesium acetate, potassium acetate, sorbitol and
dimethyl sulfoxide, a third reagent containing dithiothreitol,
deoxyribonucleoside triphosphates, ribonucleoside triphosphates,
bovine serum albumin, the second single-stranded oligo DNA, the
third single-stranded oligo DNA and the fourth single-stranded
oligo DNA and a fourth reagent containing an RNA-dependent DNA
polymerase, a DNA-dependent DNA polymerase, a DNA-dependent RNA
polymerase and an RNase inhibitor.
26. A reagent set for performing the method according to claim 1 or
21, which comprises at least a first reagent containing the first
single-stranded oligonucleic acid, a second reagent containing
tris-acetate, magnesium acetate, potassium acetate, sorbitol and
dimethyl sulfoxide, a third reagent containing dithiothreitol,
deoxyribonucleoside triphosphates, ribonucleoside triphosphates,
bovine serum albumin, the second single-stranded oligo DNA and the
third single-stranded oligo DNA, a fourth reagent containing the
fourth single-stranded oligo DNA, an RNA-dependent DNA polymerase,
a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and
an RNase inhibitor.
27. A reagent for performing the method according to claim 1 or 21,
which comprises at least the first single-stranded oligonucleic
acid, the second single-stranded oligo DNA, the third
single-stranded oligo DNA, the fourth single-stranded oligo DNA, an
RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a
DNA-dependent RNA polymerase, deoxyribonucleoside triphosphates,
ribonucleoside triphosphates, tris-acetate, magnesium acetate,
potassium acetate, sorbitol, dimethyl sulfoxide, dithiothreitol,
bovine serum albumin and an RNase inhibitor.
28. The reagent set or reagent according to any one of claims 24 to
27, wherein an enzyme having both an RNA-dependent DNA polymerase
activity and a DNA-dependent DNA polymerase activity is used at
least as the RNA-dependent DNA polymerase and as the DNA-dependent
DNA polymerase.
Description
[0001] The present invention relates to a method for assay of a
single-stranded RNA containing a specific nucleic acids sequence in
a sample, which enables detection and quantification of viral RNA
and bacterial mRNA and is effective in diagnosis of infectious
diseases and in judging the effects of therapeutic agents for
infectious diseases. The present invention also related to a method
for producing large amounts of DNA and RNA containing a specific
nucleic acids sequence, which is useful for cloning useful genes
and exploring unknown genes. The present invention further relates
to a reagent set for use in these methods.
[0002] Assays of biogenic components require high specificity and
sensitivity. Sequence-specific hybridizability of a nucleic acid
with a complementary nucleic acid (a nucleic acid probe) is
utilized in assays of a specific nucleic acid.
[0003] Measurable signaling corresponding to the amount to the
hybridization product is essential for quantification of a target
nucleic acids sequence. In clinical diagnosis, high-sensitive
signaling is required because samples usually contain traces of
target nucleic acids.
[0004] For quantification of a target nucleic acid in a sample, a
method which involves solid phase hybridization of the target
nucleic acid with a labeled nucleic acid probe which gives off
measurable signals on membranes, beads or gels, called sandwich
assay, has been employed. In practice, two nucleic acid probes
specific for different sequences within the target nucleic acid are
used: a first nucleic acid probe labeled with a dye of a visible
color, a fluorescent substance or an enzyme which catalyzes
production of such a dye or a fluorescent substance, and a second
probe immobilized on a solid phase. These probes are added to a
sample and hybridize with the specific nucleic acid in the sample,
forming a complex on the solid phase. Then, the reaction mixture is
separated into the supernatant and the solid phase to remove the
unhybridized first probe (B/F separation). The specific nucleic
acid in the sample can be determined by measurement of the label on
the solid phase. When the label is an enzyme which catalyzes
production of a dye of a visible color or a fluorescent substance,
a precursor of the dye or fluorescent substance is added as the
substrate of the enzyme to the sample solution after the B/F
separation, and the resulting dye or fluorescent substance is
measured.
[0005] Especially, in diagnosis of virus infections, because
clinical samples usually contains the target nucleic acid (viral
nucleic acid) in trace amounts, sandwich assay using a
chemiluminescent substance as the substrate of the enzyme or
preceded by preamplification of the target nucleic acid in samples
by polymerase chain reaction (PCR) has been attempted to increase
the signal intensity and sensitivity for the purpose of sensitive
and reproducible assay.
[0006] Another PCR-based method known for assay of a target nucleic
acid is competitive PCR. In the method, PCR is performed in the
presence of a given concentration of a nucleic acid having a base
sequence similar to that of the target nucleic acid (a competitor)
in the sample, the concentration of the target nucleic acid is
estimated from the amplification level of the target nucleic acid.
In practice, a sample is subjected to PCR in the presence of
various concentrations of a nucleic acid which has a terminal
sequence complementary to a primer and is distinguishable from the
amplification product from the target nucleic acid (for example, by
the length) by some separation means such as electrophoresis
simultaneously.
[0007] Homogeneous support-free assay for PCR-based quantification
of a target nucleic acid has also been proposed. For example, the
present inventors reported an assay method wherein the initial
amount of the target nucleic acid is determined by fluorescence
measurement of the reaction solution after each PCR cycle during
PCR in the presence of a fluorescent intercalative dye
(JP-A-5237000; Igaku-no-Ayumi, 173(12), 959-963 (1995); Analytical
Biochemistry, 229, 207-213 (1995)). In the assay method, as the PCR
amplification products are double-stranded DNA, a fluorescent
intercalative dye which changes its fluorescence characteristic for
example, by increasing the fluorescence intensity, upon
intercalation into double-stranded nucleic acid, is added to sample
solutions prior to PCR amplification, and the fluorescence
intensity of the reaction solution is monitored to determine the
initial amount of the target nucleic acid from the pattern of the
fluorescence enhancement. Further, this method makes it possible to
keep track of amplification of the target nucleic acid by
fluorometric measurement of the reaction solution in a closed
reaction vessel and therefore can obviate the problem of false
positive results attributable to carryover of the amplification
products because sampling of the reaction solution from the
reaction vessel is unnecessary.
[0008] At present, the detection or quantification limit of the
sandwich assay of a target nucleic acid is at most about 10.sup.5
copies even if the fist probe labeled with multiple enzyme
molecules is used to produce a large amount of a luminescent
substance in an enzymatic chemiluminescence system known for a
relatively high sensitivity, because the first probe
non-specifically adsorbed on the solid support gives off a
considerable background signal (background) and therefore produces
errors in the measurement of the solid phase hybridization on the
surface.
[0009] In order to prevent non-specific adsorption of a first probe
on a solid support, hydrophilic surface treatment of the support,
blocking of the adsorptive sites on the support surface with
protein, through washing of the solid support after the B/F
separation and use of a high detergency cleaning solution
containing a surfactant have been attempted.
[0010] However, chemical hydrophilic surface treatment is not
applicable to some kinds of supports depending to the material of
the support and can be technically difficult. Also, protein coating
of the support surface for blocking of the adsorption sites on the
support can lead to a different type of non-specific adsorption due
to interaction between the protein coat and the nucleic acid
segment or the label of the first probe. Washing operations after
the B/F separation can not be increased indefinitely due to
operational limitations, and the surfactant added to a cleaning
solution can induce decomposition of the hybrid formed on the
support.
[0011] In competitive PCR assay, it is necessary to perform PCR on
sample solutions containing a competitor at various concentrations
ranging over the predicted target nucleic acid concentration for
analysis of one sample. Besides, post-PCR separation of sample
solutions withdrawn from the reaction vessels, for example, by
electrophoresis is necessary. Therefore, competitive PCR assay is
difficult to automate and inappropriate for clinical diagnosis
which requires speedy handling of a great number of samples.
Further, due to the need to withdraw sample solutions from reaction
vessels, the false positive problem attributable to carryover of
the amplification products in practical application of the PCR
assay remais to be solved.
[0012] PCR-based assays in the presence of a fluorescent
intercalative dye have a problem that when the sample contains a
large amount of other double-stranded DNA such as genomic DNA in
addition to the specific nucleic acid, the intercalative dye
intercalates into other double-stranded DNA producing a significant
background, because it is based on the ability of a fluorescent
intercalative dye to intercalate into double-stranded nucleic acid.
Further, in PCR-based assays using a pair of oligo DNAs
complementary to the specific nucleic acid sequence as the primers
for chain elongation, the primers can hybridize each other,
depending on their base sequences, and elongate by using each other
as templates to produce a primer dimer. Because the intercalation
of a fluorescent intercalative dye into double-stranded nucleic
acid is not specific, production of such a primer dimer creates a
problem of a high background.
[0013] The demand for automation in clinical diagnostics likely
continues to increase to realize speedy and reproducible analyses
of a great number of samples. PCR involves repetitious rapid
heating and cooling of reaction solutions and entails strict
temperature control during heating and cooling because accuracy and
reproducibility of these operations can affect the results of PCR.
However, it is not easy to provide a full-automatic instrument
having enough incubation ability to satisfy these requirements and
a sufficient throughput capacity.
[0014] Further, in the case of RNA as viral nucleic acids in most
viruses, PCR is preceded by synthesis of cDNA by reverse
transcriptase using RNA as the template, and therefore virtually
two steps are involved.
[0015] For amplification of a target nucleotide at constant
temperature, a so-called NASBA method is known. The NASBA method
seems easy to automate because heating or cooling is unnecessary,
but requires sandwich assay or electrophoretic separation of the
amplified RNA and therefore can not solve the problems attributable
to these operations.
[0016] Accordingly, the object of the present invention is to
provide a simple and accurate method for assay of a single-stranded
RNA containing a specific nucleic acids sequence in a sample at
almost constant temperature without repetitious rapid heating and
cooling of reaction solutions in PCR or using a support in
measurement of the amplified RNA, wherein all the operations are
preferably done in a closed vessel. Another object of the present
invention is to provide a simple method for producing a nucleic
acid having a specific nucleic acids sequence at almost constant
temperature.
[0017] The present inventors developed a nucleic acid probe which
is complementary to a specific nucleic acids sequence in the target
nucleic acid and labeled with a fluorescent intercalative dye so as
to give a measurable fluorescent signal on binding to the target
nucleic acid (JP-A-7-185599/EP-A-714986/Nucleic Acid Research,
24(24), 4992-4997 (1996)). The nucleic acid probe gives a
measurable fluorescent signal on hybridization with the target
nucleic acid and therefore enables detection of hybridization and
quantification of the hybridization product without separation of
the unhybridized probe. Further, the present inventors have
established synthesis of an RNA having the specific acid sequence
by using a nucleic acid polymerase and nucleic acid primers in the
presence of the nucleic acid probe at constant temperature, namely
amplification and assay of the target RNA at constant temperature
without using a support preferably in a closed system, and have
accomplished the present invention.
[0018] According to claim 1 of the present application, the present
invention provides a simple and accurate method for assay of a
single-stranded RNA containing a specific nucleic acids sequence in
a sample at almost constant temperature by using at least the
following reagents (A) to (I), which comprises a step of adding the
reagents (A) to (I) one by one (in any order), in combinations of
at least two or all at once and
[0019] a step of measuring a fluorescent signal in the presence of
the reagent (I) at least once after addition of at least the
reagents (A) to (H);
[0020] (A) a first single-stranded oligonucleic acid complementary
to a sequence neighboring the 5' end of the specific nucleic acids
sequence in the single-stranded RNA,
[0021] (B) a second single-stranded oligo DNA complementary to a
3'-end sequence within the specific nucleic acids sequence,
[0022] (C) an RNA-dependent DNA polymerase,
[0023] (D) deoxyribonucleoside triphosphates,
[0024] (E) a third single-stranded oligo DNA having (1) a promoter
sequence for a DNA-dependent RNA polymerase, (2) an enhancer
sequence for the promoter and (3) a 5'-end sequence within the
specific nucleic acids sequence, in this order from the 5' end,
[0025] (F) a DNA-dependent DNA polymerase,
[0026] (G) a DNA-dependent RNA polymerase,
[0027] (H) ribonucleoside triphosphates, and
[0028] (I) a fourth single-stranded oligo DNA complementary to the
specific nucleic acids sequence which is labeled so that it gives
off a measurable fluorescent signal on hybridization with a nucleic
acid containing the specific nucleic acids sequence.
[0029] According to claim 21 in the present application, the
present invention provides a simple method for producing a nucleic
acid having a specific nucleic acids sequence at almost constant
temperature by using at least the following reagents (A) to (H),
which comprises a step of adding the reagents (A) to (G) one by one
(in any order), in combinations of at least two or all at once to a
single-stranded DNA having (1) a promoter sequence for a
DNA-dependent RNA polymerase, (2) an enhancer sequence for the
promoter and (3) the specific nucleic acids sequence, in this order
from the 5' end or to a double-stranded DNA consisting of the
single-stranded DNA and a complementary DNA strand and a step of
measuring a fluorescent signal from the reagent (H) at least once
after addition of at least the reagents (A) to (G);
[0030] (A) a single-stranded oligo DNA complementary to a 3'-end
sequence within the specific nucleic acids sequence,
[0031] (B) an RNA-dependent DNA polymerase,
[0032] (C) a DNA-dependent DNA polymerase,
[0033] (D) deoxyribonucleoside triphosphates,
[0034] (E) a DNA-dependent RNA polymerase,
[0035] (F) ribonucleoside triphosphates,
[0036] (G) a single-stranded DNA having (1) a promoter sequence for
a DNA-dependent RNA polymerase, (2) an enhancer sequence for the
promoter and (3) a 5'-end sequence within the specific nucleic
acids sequence, in this order from the 5' end,
[0037] (H) a fourth single-stranded labeled oligo DNA complementary
to the specific nucleic acids sequence which gives a measurable
fluorescent signal on hybridization with a nucleic acid containing
the specific nucleic acids sequence.
[0038] According to claims 24 to 28 in the present application, the
present invention also provides a reagent or reagent set for
performing the above-mentioned method, and specifically according
to claim 24, the present invention provides a reagent set for
performing the method according to claim 1 or 21, which comprises
at least
[0039] a first reagent containing the first single-stranded
oligonucleic acid,
[0040] a second reagent containing tris-acetate, magnesium acetate,
potassium acetate, sorbitol and dimethyl sulfoxide,
[0041] a third reagent containing dithiothreitol,
deoxyribonucleoside triphosphates, ribonucleoside triphosphates,
bovine serum albumin, the second single-stranded oligo DNA and the
third single-stranded oligo DNA,
[0042] a fourth reagent containing an RNA-dependent DNA polymerase,
a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and
an RNase inhibitor and a fifth reagent containing the fourth
single-stranded oligo DNA.
[0043] Specifically according to claim 25, the present invention
provides a reagent set for performing the method according to claim
1 or 21, which comprises at least
[0044] a first reagent containing the first single-stranded
oligonucleic acid,
[0045] a second reagent containing tris-acetate, magnesium acetate,
potassium acetate, sorbitol and dimethyl sulfoxide,
[0046] a third reagent containing dithiothreitol,
deoxyribonucleoside triphosphates, ribonucleoside triphosphates,
bovine serum albumin, the second single-stranded oligo DNA, the
third single-stranded oligo DNA and the fourth single-stranded
oligo DNA and
[0047] a fourth reagent containing an RNA-dependent DNA polymerase,
a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and
an RNase inhibitor.
[0048] According to claim 26, the present invention further
provides a reagent set for performing the method according to claim
1 or 21, which comprises at least
[0049] a first reagent containing the first single-stranded
oligonucleic acid,
[0050] a second reagent containing tris-acetate, magnesium acetate,
potassium acetate, sorbitol and dimethyl sulfoxide,
[0051] a third reagent containing dithiothreitol,
deoxyribonucleoside triphosphates, ribonucleoside triphosphates,
bovine serum albumin, the second single-stranded oligo DNA and the
third single-stranded oligo DNA,
[0052] a fourth reagent containing the fourth single-stranded oligo
DNA, an RNA-dependent DNA polymerase, a DNA-dependent DNA
polymerase, a DNA-dependent RNA polymerase and an RNase
inhibitor.
[0053] Specifically according to claim 27, the present invention
provides a reagent for performing the method according to claim 1
or 21, which comprises at least the first single-stranded
oligonucleic acid, the second single-stranded oligo DNA, the third
single-stranded oligo DNA, the fourth single-stranded oligo DNA, an
RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a
DNA-dependent RNA polymerase, deoxyribonucleoside triphosphates,
ribonucleoside triphosphates, tris-acetate, magnesium acetate,
potassium acetate, sorbitol, dimethyl sulfoxide, dithiothreitol,
bovine serum albumin and an RNase inhibitor, namely a single
reagent obtained by mixing them all.
[0054] FIG. 1 shows the results of the 2% agarose gel
electrophoresis of the product of the PCR using the second and
third single-stranded oligo DNAs in Example 1. Lanes 2 to 4: known
concentrations of the standard DNA, lanes 5 to 7: 0.2 to 5 .mu.l of
the PCR product.
[0055] FIG. 2 shows the results of the 2% agarose gel
electrophoresis after the reactions at various magnesium acetate
concentrations in Example 2. The arrow indicates the specific
product (about 300 bp). The concentrations of magnesium acetate are
expressed in terms of final concentration.
[0056] FIG. 3 shows the results of the 2% agarose gel
electrophoresis after the reactions at various potassium acetate
concentrations in Example 3. The arrow indicates the specific
product (about 300 bp). The concentrations of potassium acetate are
expressed in terms of final concentration.
[0057] FIG. 4 shows the results of the 2% agarose gel
electrophoresis after the reactions at various sorbitol
concentrations in Example 4. The arrow indicates the specific
product (about 300 bp).
[0058] FIG. 5 shows the results of the 2% agarose gel
electrophoresis after the reactions in the presence of various
concentrations of the standard DNA in Example 5. The arrow
indicates the specific product (about 300 bp).
[0059] FIG. 6 shows the results of the electrophoresis on a
polyacryl amide gel containing 12% urea after the reaction of the
133mer RNA, the first single-stranded oligo DNA complementary to a
neighboring sequence at the 5' end of the specific nucleic acids
sequence within the 133mer RNA and various concentrations of RnaseH
in Example 6. The arrows indicate the 133mer and the 72mer.
[0060] FIG. 7 shows the results of the 2% agarose gel
electrophoresis after the reactions in the presence of various
concentrations of the standard RNA in Example 7. The arrow
indicates the specific product (about 300 bp).
[0061] FIG. 8 shows the results of the 2% agarose electrophoresis
of the products of the reactions using various concentrations of
the standard RNA (10.sup.6 copies/5 .mu.l) after RNaseA or DNaseI
treatment in Example 8. The DNA product and the RNA product are
pointed.
[0062] FIG. 9 shows the results of densitometric quantification of
the products from the standard RNA (10.sup.6 copies/5 .mu.l) at
various reaction times after RNaseA or DNaseI treatment followed by
2% agarose gel electrophoresis in Example 9.
[0063] FIG. 10 shows the results of the 2% agarose gel
electrophoresis of the products from the standard RNA (10.sup.6
copies/5 .mu.l) at various reaction times in Example 10.
[0064] FIG. 11 shows the results of fluorescence measurement after
addition of the fourth single-stranded oligo DNA to the products
shown in FIG. 10 obtained in Example 10.
[0065] FIG. 12 shows the results of the fluorescence measurement
after various times of reactions using the standard RNA (10.sup.6
copies/5 .mu.l) in the presence of the fourth single-stranded oligo
DNA in Example 11.
[0066] FIG. 13 shows the results of the fluorescence measurement
after various times of reactions using the standard RNA (10.sup.6
copies/5 .mu.l) in the presence of the fourth single-stranded oligo
DNA having a modified 3' end (having ddTTP at the 3' end) in
Example 11.
[0067] FIG. 14 shows the results of the 2% agarose gel
electrophoresis after after various times of reactions using the
standard RNA (10.sup.6 copies/5 .mu.l) in the presence of the
fourth single-stranded oligo DNA having a modified 3' end (having
ddTTP at the 3' end) in Example 12.
[0068] FIG. 15 shows the results of the densitometric quantitative
analysis of the electrophoretogram shown in FIG. 14 in Example
12.
[0069] FIG. 16 shows the results of the fluorescence measurement
after various times of reactions using the standard RNA (10.sup.6
copies/5 .mu.l) in the presence of the fourth single-stranded oligo
DNA having a modified 3' end (having ddTTP at the 3' end) in
Example 13.
[0070] FIG. 17 shows the results of the fluorescence measurement
after various times of reactions using the standard RNA (10.sup.4,
10.sup.5 and 10.sup.6 copies/5 .mu.l) in the presence of the fourth
single-stranded oligo DNA having a modified 3' end (having ddTTP at
the 3' end) in Example 14.
[0071] FIG. 18 shows the plot of the fluorescence enhancement at a
reaction time of 3 hours against the initial concentration of the
standard RNA based on the amplification profiles shown FIG. 17
obtained in Example 14.
[0072] FIG. 19 shows the structure of the fourth single-stranded
oligo DNA used in Examples, YO-271. The DNA moiety on the left and
the fluorescent intercalative dye, oxazole yellow on the right are
linked via the linker shown in this figure so that the fluorescent
dye can intercalates into a double strand upon formation of the
double strand by the DNA moiety.
[0073] Now, the present invention will be described in detail.
[0074] According to claim 1 of the present application, the present
invention provides a method for assay of a single-stranded RNA
containing a specific nucleic acids sequence in a sample (a target
RNA). Herein, assay means both qualitative assay of the RNA in a
sample and quantitative assay of the RNA in a sample.
[0075] The specific nucleic acids sequence means a base sequence
within the single-stranded RNA which starts at the 5' end with the
sequence (3) in the after-mentioned third single-stranded oligo DNA
and end at the 3' end with a sequence complementary to the
after-mentioned second single-stranded oligo DNA. The specific
nucleic acids sequence can be defined arbitrarily but must contain
a sequence specific enough to distinguish the target RNA from other
nucleic acids. In particular, when a sequence sufficiently
distinguishable from nucleic acids other than the target nucleic
acid by its 5' and 3' ends is selected as the specific nucleic
acids sequence, the specificity of the synthesis of a
single-stranded RNA having the specific nucleic acids sequence
according to the present invention improves.
[0076] Further, when the specific nucleic acids sequence further
contains an internal sequence which sufficiently distinguishes the
target nucleic acid from other nucleic acids, the specificity of
the assay according to the present invention further improves.
[0077] In the present invention according to claim 1, when the
target RNA is present in the sample, a large amount of an RNA
having the specific nucleic acids sequence is synthesized
eventually.
[0078] The reagent (A) is a first oligonucleic acid complementary
to a neighboring sequence at the 5' end of the specific nucleic
acids sequence within the target RNA. The reagent allows the target
RNA to be cut at the 5' end of the specific nucleic acids sequence
so that on binding of a second single-stranded oligo DNA to the
target RNA, an RNA-dependent DNA polymerase synthesizes cDNA from
the resulting RNA fragment having the specific nucleic acids
sequence at the 5' end as the template in the presence of the
after-mentioned reagents (B) to (D) to give a cDNA having a 3'-end
sequence complementary to the specific nucleic acids sequence. For
example, when a DNA as the first oligonucleic acid is added
together with RNase H, the target RNA is cut where the first
oligonucleic acid binds to the target RNA, so as to have the first
base of the specific nucleic acids sequence at the 5' end.
Otherwise, a DNAzyme or ribozyme which catalytically cuts a
single-stranded nucleic acid at a specific site (Proc. Natl. Acad.
Sci. USA, 94, 4262-4266(1997)) may be used as the first
oligonucleic acid.
[0079] RNase H non-specifically cleaves an RNA in an RNA-DNA
hetero-duplex. Therefore, when RNase H is used, the RNase H is
virtually deactivated, for example, by heating or by addition of a
known RNase H inhibitor prior to addition of the reagent (B). In
the case of heating, it is satisfactory to raise the temperature to
60-70.degree. C. and keep that temperature for quite a short time.
Accordingly, when RNase H is used, after the reagent (A) and RNase
H are added separately or simultaneously, an appropriate time of
incubation precedes addition of the reagents (B) to (I). However, a
DNAzyme or a ribozyme as the reagent (A) can be added alone or at
the same time as other reagents without the need of heating or
addition of an inhibitor.
[0080] The reagent (B) is a second single-stranded oligo DNA
complementary to a 3'-end sequence within the specific nucleic
acids sequence. The reagent (B) hybridizes with the target RNA so
that an RNA-dependent DNA polymerase synthesizes cDNA from the RNA
as the template in the presence of the after-mentioned reagents (C)
to (D) to give a cDNA having a 5'-end sequence complementary to a
3'-end sequence within the specific nucleic acids sequence. The
second oligonucleic acid is added in an amount of 0.02 to 1 .mu.M
in the presence of the target nucleic acid.
[0081] The reagent (C) is an RNA-dependent DNA polymerase, and the
reagent (D) is the substrate of the reagent (C),
deoxyribonucleoside triphosphates. In the presence of the reagents
(B) to (D), a cDNA having a 5'-end sequence complementary to the 3'
end of the specific nucleic acids sequence within the target RNA is
synthesized.
[0082] To the cDNA in the form of a DNA-RNA double strand with the
template RNA, the ability to hybridize with a third single-stranded
oligo DNA as the after-mentioned reagent (E) is imparted. For this
purpose, for example, 5 to 20% of dimethyl sulfoxide (hereinafter
referred to as DMSO) is added to make the complementary binding
between the DNA and RNA less tight. DMSO does not interfere with
the actions of the other reagents and acts satisfactory only if it
is added so as to coexist with the DNA-RNA double strand at least
prior the DNA synthesis by the reagents (D) to (F). Alternatively,
because some RNA-dependent DNA polymerases represented by avian
myoblastoma virus polymerase (hereinafter AMV reverse
transcriptase) cut RNA in a DNA-RNA double strand, though with low
activities, such an enzyme with an RNA degrading action can be used
as the above-mentioned reagent (C). It is particularly preferable
to use the action of DNSO and the action of an RNA-dependent DNA
polymerase such as CMV reverse transcriptase, although it is
satisfactory to use the action of either one. By the actions of
DMSO and/or an RNA-dependent DNA polymerase, the RNA in the RNA-DNA
double strand is degraded and/or separated, leaving a
single-stranded DNA.
[0083] The reagent (E) is a third single-stranded oligo DNA having
(1) a promoter sequence for a DNA-dependent RNA polymerase, (2) an
enhancer sequence for the promoter and (3) a 5'-end sequence within
the specific nucleic acids sequence in this order from the 5' end.
The third oligo DNA is added in an amount of 0.02 to 1 .mu.M in the
presence of the target nucleic acid. The region (3) in the third
oligo DNA binds to the 3' end of the DNA synthesized in the
presence of the reagents (B) to (D). Therefore, in the presence of
the reagents (D) and (F), a DNA-dependent DNA polymerase
complementarily elongates the 3' end of the third DNA using the DNA
as the template and the 3' end of the DNA by using the third oligo
DNA as the template to give a complete double-stranded DNA.
[0084] The reagent (F) is a DNA-dependent DNA polymerase. In the
present invention, it is particularly preferred to use fewer kinds
of reagents by using a single enzyme which acts both as the
RNA-dependent DNA polymerase as the reagent (C) and as the
DNA-dependent DNA polymerase. For example, AMV reverse
transcriptase, which has the activities of the two polymerases, is
particularly preferable for use in the present invention because it
also degrades the RNA chain in a DNA-RNA double strand as described
above and is also commercially available.
[0085] The reagent (G) is a DNA-dependent RNA polymerase, and the
reagent (H) is the substrate of the reagent (G), ribonucleoside
triphosphates. The double-stranded DNA synthesized in the presence
of the reagents (D) to (F) has a promoter region for the
DNA-dependent RNA polymerase at one end. Therefore, in the presence
of the reagents (G) and (H), the synthesis of the DNA is
immediately followed by synthesis of a single-stranded RNA having
the specific nucleic acids sequence. Specific examples of the
DNA-dependent RNA polymerase as the reagent (G) include T7 RNA
polymerase, T3 polymerase and SP6 RNA polymerase.
[0086] The single-stranded RNA synthesized in the presence of the
reagents (G) to (H) has the specific nucleic acids sequence.
Therefore, coexistence of the synthesized single-stranded RNA with
the reagents (B) to (F) sets off the above-mentioned series of
reactions again from the start. Thus, in the present invention, it
is possible to synthesize above-mentioned double-stranded DNA
having a promoter region at one end from a trace of the target RNA
in the sample only by adding the respective reagents to the sample
without any hardly automatable operations such as heating, and the
double-stranded DNA gives rise to synthesis of a single-stranded
RNA having the specific nucleic acids sequence. The synthesized
single-stranded RNA participates in the next round of synthesis of
the double-stranded DNA. Consequently, the single-stranded RNA
having the specific nucleic acids sequence drastically increases
with the elapse of time.
[0087] The rate of synthesis of the single-stranded RNA having the
specific nucleic acids sequence and the final amount of the
synthesized single-stranded RNA depend on the amount of the target
RNA in the sample. Accordingly, the use of the reagent (I) in the
present invention enables determination of the target RNA in the
sample.
[0088] The reagent (I) is a fourth single-stranded labeled oligo
DNA containing the sequence complementary to the specific nucleic
acids sequence which gives a measurable fluorescent signal on
hybridization with a nucleic acid containing the specific nucleic
acids sequence. The fourth oligo DNA may be, for example, a
fluorescent intercalative dye-linked DNA. The DNA moiety is from 6
to 100 nucleotides long, preferably from 10 to 30 nucleotides long,
to secure the specificity for the specific nucleic acids sequence
in the assay. Of course, the DNA moiety must be complementary to a
sequence within the specific nucleic acids sequence which
sufficiently distinguishes the target nucleic acid from other
nucleic acids.
[0089] The DNA moiety preferably has a 3'-end sequence which is
uncomplementary to the specific nucleic acids sequence or has a
chemically modified 3' end so that the 3' end does not elongate by
the action of the already existing RNA-dependent DNA polymerase on
hybridization with the synthesized single-stranded RNA having the
specific nucleic acids sequence.
[0090] If the DNA moiety is hybridized with another nucleic acid,
the fluorescent intercalative dye intercalates into the resulting
double strand and changes its fluorescence characteristic. For this
purpose, the fluorescent intercalative dye may be linked to the DNA
moiety via a linker of an appropriate length. Any linker that does
not hinder the fluorescent intercalative dye from intercalating
into the double strand may be used without any particular
restriction. A bifunctional hydrocarbon linker having functional
groups at both ends is particularly preferable for the easiness of
its use in modification of oligonucleotides. Alternatively, a
commercial kit (C6-Thiolmodifier, tradename, Clonntech) may be
used.
[0091] The fluorescent intercalative dye is not particularly
limited as long as it changes the fluorescent characteristic, for
example emits fluorescence having a different peak wavelength, on
intercalation into a double strand. However, those which enhance
the fluorescence on intercalation are particularly preferable in
view of the easiness of fluorescence measurement. More
specifically, particularly preferable fluorescent intercalative
dyes are, for example, thiazole orange, oxazole yellow and their
derivatives, because they show radical change in the
fluorescence.
[0092] The fluorescent intercalative dye may be linked to any sites
of the DNA moiety, including the 5' end, the 3' end and the middle,
as long as the linkage neither hinders the fluorescent
intercalative dye from intercalating into a double strand nor
hinders the DNA moiety from hybridizing with RNA.
[0093] The reagent (I) gives off a measurable fluorescent signal in
the presence of the single-stranded RNA having the specific nucleic
acids sequence synthesized in the presence of the double-stranded
DNA, the reagents (G) and (H). Surprisingly, the synthesized
single-stranded RNA has been found to serve as a template for DNA
synthesis in the presence of the reagents (B) to (D) even when the
hybrid of the RNA and the fourth oligo DNA is giving off a
fluorescent signal. In other words, in the present invention, a
series of events, synthesis of DNA from RNA, synthesis of
double-stranded DNA and synthesis of RNA from double-stranded DNA
in the presence of the respective reagents, take place in the
presence of the fourth oligo DNA.
[0094] Therefore, the target nucleic acid in a sample can be
determined by measuring the fluorescent signal from the reagent (I)
which is added after addition of the reagents (A) to (H) at least
once. The reagent (I) may be added at the same time as the other
reagents because the presence of the fourth oligonucleic acid is
not an obstacle to synthesis of a single-stranded RNA having the
specific nucleic acids sequence after all.
[0095] When the fluorescent signal is measured only once, prior to
the measurement, addition of the reagents (A) to (H) is followed by
sufficient time of incubation for synthesis of the single-stranded
RNA having the specific nucleic acids sequence. The reagent (I)
must be added before the measurement, for example, at the same time
as the other reagents. This style of measurement is called an end
point assay, and the target nucleic acid in a sample can be
determined, for example, from comparison with the results obtained
by performing the same procedure on solutions containing the known
amounts of the target nucleic acid. In the present invention, the
addition of the reagents (A) to (H) is preferably followed by
durational measurement of fluorescent signals immediately or after
a certain time lag. Though during synthesis of the single-stranded
RNA, the fourth DNA binds to and separates from the synthesized RNA
repeatedly, but it is possible to monitor the increase of the
single-stranded RNA because the measured fluorescent signals from
the bound fourth DNA correlate with the amounts of the RNA at the
moments of the measurements. The measurement may be done
continuously or intermittently at constant intervals. Thus, the
time course of the fluorescent signal can be traced by durational
measurement, and the amount (initial amount) of the target RNA in a
sample can be determined, for example, from the time required to
obtain a stable fluorescent signal after addition of the reagents
(A) to (H) or the time lag between the addition of the reagents (A)
to (H) and drastic increase of the fluorescent signal.
[0096] As will be demonstrated later in Examples, in the present
invention, all the above-mentioned reagents (A) to (I) are
preferred to be free from chlorides. Further, it is preferred to
use an acetate such as magnesium acetate or potassium acetate in
addition to the reagents (A) to (H). An acetate is added at latest
at the time of synthesis of single-stranded DNA in the presence of
the reagents (B) to (D) preferably at a concentration of from 5 to
20 mM for magnesium acetate, from 50 to 200 mM for potassium
acetate. In the present invention, it is preferred to further use
sorbitol. Sorbitol is added at latest at the time of synthesis of
single-stranded DNA in the presence of the reagents (B) to (D).
Further, use of protein such as bovine serum albumin and a reducing
agent such as dithiothreitol, use of an RNase inhibitor with a view
to inhibiting degradation of the synthesized single-stranded RNA by
RNase are also preferred. Still further, a buffering agent is also
preferably used so as to keep the reaction solution within an
active pH range for the respective enzymes to be used. As the
buffering agent, tris-acetate is particularly preferable. These
reagents are added at latest at the time of synthesis of
single-stranded DNA in the presence of the reagents (B) to (D). Use
of these optional reagents leads to increase of the amount of the
single-stranded RNA synthesized as the product.
[0097] The method of the present invention enables assay of a
target RNA in a sample at constant temperature without heating. The
constant temperature is may be any temperature at which the
respective oligonucleic acids used as the reagents (A), (B), (E)
and (I) in the present invention can hybridize, and the enzymes as
the reagents (C), (F) and (F) are active, without any restriction.
Specifically speaking, the temperature is selected from the range
of from 35 to 60.degree. C. The temperature may not be exactly
constant, and it is sufficient to keep the temperature almost
constant.
[0098] As is evident from the above explanation, in the assay
method according to claim 1, the reagents to be used may be added
one by one, in combination of at least two or all at once. In
particular, when all the reagents are added to a sample all at
once, assay of the target RNA in a closed vessel can be realized to
solve the problem of carryover of the synthesized single-stranded
RNA which can happen when the vessel is opened or closed.
[0099] According to claim 21 of the present application, the
present invention provides a simple method for producing a nucleic
acid having a specific nucleic acids sequence at almost constant
temperature by using at least the following reagents (A) to (G),
which comprises a step of adding the reagents (A) to (G) one by one
(in any order), in combinations of at least two or all at once to a
single-stranded DNA having (1) a promoter sequence for a
DNA-dependent RNA polymerase, (2) an enhancer sequence for the
promoter and (3) the specific nucleic acids sequence in this order
from the 5' end or to a double-stranded DNA consisting of the
single-stranded DNA and a complementary DNA and a step of measuring
a fluorescent signal from the reagent (H) at least once in the
presence of at least the reagents (A) to (G);
[0100] (A) a single-stranded oligo DNA complementary to a 3'-end
sequence in the specific nucleic acids sequence,
[0101] (B) an RNA-dependent DNA polymerase,
[0102] (C) a DNA-dependent DNA polymerase,
[0103] (D) a deoxyribonucleoside triphosphate,
[0104] (E) a DNA-dependent RNA polymerase,
[0105] (F) a ribonucleoside triphosphate,
[0106] (G) a single-stranded DNA having (1) a promoter sequence for
a DNA-dependent RNA polymerase, (2) an enhancer sequence for the
promoter and (3) a 5'-end sequence in the specific nucleic acids
sequence in this order from the 5' end,
[0107] (H) a fourth single-stranded labeled oligo DNA containing
the sequence complementary to the specific nucleic acids sequence
which gives a measurable fluorescent signal on hybridization with a
nucleic acid containing the specific nucleic acids sequence.
[0108] As is understandable from the explanation already made, in
the method according to claim 23, a nucleic acid having a specific
nucleic acids sequence is produced by using the double-stranded DNA
from which the single-stranded RNA is synthesized in the method
according to claim 1, as the starting material. The specific
nucleic acids sequence used herein does not have to be attributed
to the target RNA, unlike the specific nucleic acids sequence in
claim 1.
[0109] The double-stranded DNA as the starting material can be
prepared by known methods using PCR or a DNA synthesizer. The first
oligo DNA as the reagent (A) hybridizes with the single-stranded
DNA having (1) a promoter sequence for a DNA-dependent RNA
polymerase, (2) an enhancer sequence for the promoter and (3) the
specific nucleic acids sequence and elongates using the DNA as the
template in the presence of the reagents (A), (C) and (D) to give
the double-stranded DNA, which serves as the starting material. The
double-stranded DNA as the starting material can also be prepared
by performing the respective operations mentioned for the method
according to claim 1 on a single-stranded RNA containing the
specific nucleic acids sequence. The single-stranded DNA as the
reagent (A), the single-stranded oligo DNA as the reagent (G) and
the single-stranded oligo DNA as the reagent (H) correspond to the
second oligo DNA, the third oligo DNA and the fourth oligo DNA in
the method according to claim 1. Therefore, the modes of addition
and actions of these reagents, preferable examples of the
respective polymerases, optional reagents other than (A) to (H) and
the procedure can be easily understandable by referring to the
previous explanation. In short, a single-stranded RNA having the
specific nucleic acids sequence is synthesized from the
double-stranded DNA as the starting material in the presence of the
reagents (E) and (F), then from the single-stranded RNA a
single-stranded DNA complementary to the specific nucleic acids
sequence is synthesized in the presence of the reagents (E) and
(F), and from the single-stranded DNA the double-stranded DNA as
the starting material is synthesized in the presence of the
reagents (C) and (D). In this method, the reagent (A) used in the
method according to claim 1 and the operations associated with the
reagent (A) such as heating are unnecessary in this method unless
the double-stranded DNA as the starting material is prepared from a
single-stranded RNA which containing the specific nucleic acids
sequence somewhere other than the 5' end.
[0110] When the fluorescent signal is measured only at once, the
measurement is done in the presence of the reagent (I) after
sufficient time of incubation following the addition of the
reagents (A) to (G). When the fluorescent signal is measured over a
period of time, the durational measurement follows addition of the
reagents (A) to (G) immediately or after a certain time lag. Once
production of a predetermined amount of a nucleic acid having the
specific nucleic acids sequence is indicated by the measured
fluorescent signal or the time course of the fluorescent signal,
then the produced nucleic acid is extracted.
[0111] To obtain a single-stranded RNA having the specific nucleic
acids sequence as the final product, RNA extraction is preceded by
addition of an enzyme which degrades DNA such as DNase. To obtain a
single-stranded DNA containing the specific nucleic acids sequence
or a double-stranded DNA consisting of such a DNA single strand and
a complementary strand as the final product, DNA extraction is
preceded by addition of an enzyme which degrades RNA such as RNase.
Of course, a mixture of such an RNA and such a DNA can be obtained
by extraction without addition of a nuclease. DNA and/or RNA can be
easily extracted by known techniques such as ethanol
precipitation.
[0112] The present invention according to claim 1 or 21 described
above can be performed, for example, by using a reagent set which
comprises at least a first reagent containing a first
single-stranded oligonucleic acid, a second reagent containing
tris-acetate, magnesium acetate, potassium acetate, sorbitol and
dimethyl sulfoxide, a third reagent containing dithiothreitol,
deoxyribonucleoside triphosphates, ribonucleoside triphosphates,
bovine serum albumin, a second single-stranded oligo DNA and a
third single-stranded oligo DNA, a fourth reagent containing an
RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a
DNA-dependent RNA polymerase and an RNase inhibitor and a fifth
reagent containing a fourth single-stranded oligo DNA, and by
adding these reagents in numerical order to a sample. When the
present invention according to claim 21 does not require the first
single-stranded oligonucleic acid, the first reagent can be
omitted.
[0113] The present invention according to claim 1 or 21 described
above can also be performed, for example, by using a reagent set
which comprises at least a first reagent containing a first
single-stranded oligonucleic acid, a second reagent containing
tris-acetate, magnesium acetate, potassium acetate, sorbitol and
dimethyl sulfoxide, a third reagent containing dithiothreitol,
deoxyribonucleoside triphosphates, ribonucleoside triphosphates,
bovine serum albumin, a second single-stranded oligo DNA, a third
single-stranded oligo DNA and a fourth single-stranded oligo DNA
and a fourth reagent containing an RNA-dependent DNA polymerase, a
DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and an
RNase inhibitor, and by adding these reagents in numerical order to
a sample. When the present invention according to claim 21 does not
require the first single-stranded oligonucleic acid, the first
reagent can be omitted.
[0114] The present invention according to claim 1 or 21 described
above can also be performed, for example, by using a reagent set
which comprises at least a first reagent containing a first
single-stranded oligonucleic acid, a second reagent containing
tris-acetate, magnesium acetate, potassium acetate, sorbitol and
dimethyl sulfoxide, a third reagent containing dithiothreitol,
deoxyribonucleoside triphosphates, ribonucleoside triphosphates,
bovine serum albumin, a second single-stranded oligo DNA and a
third single-stranded oligo DNA, a fourth reagent containing a
fourth single-stranded oligo DNA, an RNA-dependent DNA polymerase,
a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and
an RNase inhibitor, and by adding these reagents in numerical order
to a sample. When the present invention according to claim 21 does
not require the first single-stranded oligonucleic acid, the first
reagent can be omitted.
[0115] The present invention according to claim 1 or 21 described
above can also be performed, for example, by using a reagent which
comprises at least a first single-stranded oligonucleic acid, a
second single-stranded oligo DNA, a third single-stranded oligo
DNA, a fourth single-stranded oligo DNA, an RNA-dependent DNA
polymerase, a DNA-dependent DNA polymerase, a DNA-dependent RNA
polymerase, deoxyribonucleoside triphosphates, ribonucleoside
triphosphates, tris-acetate, magnesium acetate, potassium acetate,
sorbitol, dimethyl sulfoxide, dithiothreitol, bovine serum albumin
and an RNase inhibitor, and by adding the reagent to a sample. When
the present invention according to claim 21 does not require the
first single-stranded oligonucleic acid, the first single-stranded
oligo DNA can be omitted. The reagent is a single reagent
containing all the required constituents, unlike the other reagent
sets. Therefore, it works satisfactory when added to a sample only
once.
[0116] The concentration of each constituent in the above-mentioned
reagent sets or reagent should be adjusted so that each constituent
is present in an required amount in a sample when added to the
sample. As described previously, a single enzyme having the actions
of both RNA-dependent DNA polymerase and DNA-dependent DNA
polymerase may be used as the RNA-dependent DNA polymerase and
DNA-dependent DNA polymerase.
[0117] Now, the present invention will be described in further
detail by referring to Examples. However, the present invention
should be by no means is restricted to these specific Examples.
EXAMPLE 1
[0118] Preparation of Double-stranded DNA
[0119] A double-stranded DNA consisting of a DNA single strand
having the SP6 promoter sequence at the 5' end and a complementary
DNA single strand, was prepared
[0120] 70 .mu.l of a reaction solution having the following
composition was pored into a PCR tube.
[0121] 10.7 mM tris-HCl buffer (pH 8.3)
[0122] 53.6 mM potassium chloride
[0123] 2.36 mM magnesium chloride
[0124] 0.268 mM each of dATP, dGTP, dCTP and dTTP
[0125] 0.257 .mu.M third single-stranded oligo DNA =p1 (sequence)
5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGA TCA CTC CCC
TG 3'
[0126] 0.257 .mu.M second single-stranded oligo DNA
[0127] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0128] 0.032 U/.mu.l commercially available DNA-dependent DNA
polymerase (Ampli Taq, tradename, Perkin Elmer)
[0129] Then, 5 .mu.l of a 1,865-bp double-stranded DNA (10.sup.-6
copies) containing bases 1 to 1,863 in human hepatitis C virus
(HCV) cDNA was added as a standard DNA. For the base numbers,
literature (Kato et al,. Proc. Natl. Sci., USA, 1990, 87,
9524-9528) should be referred to.
[0130] Then, the reaction solution was heated and incubated at
95.degree. C. for 9 minutes, and an incubation cycle of the
following steps (1) to (3) were repeated 40 times.
[0131] (1) Incubation at 95.degree. C. for 30 seconds,
[0132] (2) Incubation at 65.degree. C. for 30 seconds and
[0133] (3) Incubation at 72.degree. C. for 1 minute.
[0134] After 40 cycles of incubation, the reaction solution was
withdrawn and subjected to 2% agarose electrophoresis followed by
ethidium bromide staining.
[0135] FIG. 1 shows the results on the gel stained with ethidium
bromide. FIG. 1 clearly shows single bands of DNA of about 320 bp.,
indicating that preparation of a DNA having the following base
sequence containing the SP6 promoter sequence (underlined) at the
5' end and a complementary strand by the above-mentioned
procedure.
[0136] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to
300 in HCV cDNA) 3'
EXAMPLE 2
[0137] Effects of Magnesium Acetate Concentration
[0138] The optimum concentration of magnesium acetate for the
present invention was studied. Reaction solutions having the
following composition were prepared, and 14 .mu.l of the reaction
solutions were pored into PCR tubes.
[0139] 85.7 mM tris-acetate (pH 8.1)
[0140] 16.1, 32.1 or 48.2 mM magnesium acetate
[0141] 214.3 mM potassium acetate
[0142] 21.4% DMSO
[0143] 32.1% sorbitol
[0144] 2.1 mM each of ATP, GTP, CTP and UTP
[0145] 2.1 mM each of dATP, dGTP, dCTP and dTTP
[0146] 214 .mu.g/ml BSA
[0147] 0.12 mM second single-stranded oligo DNA (sequence) 5' ACT
CGC AAG CAC CCT ATC A 3'
[0148] Then, 10 .mu.l of a standard DNA (10.sup.4 copies, 10.sup.5
copies, 10.sup.6 copies and 10.sup.7 copies/10 .mu.l) or 10 .mu.l
of DNA-free TE buffer (10 mM tris-HCl (pH 8.0) containing 0.1 mM
EDTA) as a negative control was added. The standard DNA was a
double-stranded DNA consisting of a DNA strand having the following
sequence and a complementary DNA strand.
[0149] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to
300 in HCV cDNA) 3'
[0150] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 45.degree. C. for 5 minutes. Subsequently, 5
.mu.l of a mixed solution of the following enzymes was added, and
reaction was carried out at 45.degree. C. for 1 hour.
[0151] 30 U/.mu.l commercially available SP6 RNA polymerase (Takara
Shuzo Co., Ltd.)
[0152] 12 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0153] Then, 0.6 .mu.l of a third single-stranded oilgo DNA (2.75
.mu.M) was added.
[0154] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0155] Subsequently 0.4 .mu.l of AMV reverse transcriptase (37
U/.mu.l, Takara Shuzo Co., Ltd.), as an RNA dependent DNA
polymerase and as a DNA-dependent DNA polymerase, was added. After
2 hours of incubation at 45.degree. C., 5 .mu.l of the reaction
solutions were subjected 2% agarose gel electrophoresis. After the
electrophoresis, the agarose gel was stained with 10,000-fold
diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30 minutes.
[0156] FIG. 2 shows the pattern on the stained gel. A maximal
amount of the product was obtained in the presence of magnesium
acetate at a final concentration of 15 mM.
EXAMPLE 3
[0157] Effects of Potassium Acetate
[0158] The optimum concentration of magnesium acetate for the
present invention was studied. Reaction solutions having the
following composition were prepared, and 14 .mu.l of the reaction
solutions were pored into PCR tubes.
[0159] 85.7 mM tris-acetate (pH 8.1)
[0160] 28.9 mM magnesium acetate
[0161] 214.3, 235.7, 257.1 or 278.6 mM potassium acetate
[0162] 21.4% DMSO
[0163] 32.1% sorbitol
[0164] 2.1 mM each of ATP, GTP, CTP and UTP
[0165] 2.1 mM each of dATP, dGTP, dCTP and dTTP
[0166] 214 .mu.g/ml BSA
[0167] 0.12 .mu.M second single-stranded oligo DNA
[0168] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0169] Then, 10 .mu.l of a standard DNA (10.sup.4 copies, 10.sup.5
copies, 10.sup.5 copies and 10.sup.6 copies/10 .mu.l) or 10 .mu.l
of DNA-free TE buffer (10 mM tris-HCl (pH 8.0) containing 0.1 mM
EDTA) as a negative control was added. The standard DNA was a
double-stranded DNA consisting of a DNA strand having the following
sequence and a complementary DNA strand.
[0170] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to
300 in HCV cDNA) 3'
[0171] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 45.degree. C. for 5 minutes. Subsequently, 5
.mu.l of a mixed solution of the following enzymes was added, and
reaction was carried out at 45.degree. C. for 1 hour.
[0172] 30 U/.mu.l commercially available SP6 RNA polymerase (Takara
Shuzo Co., Ltd.)
[0173] 12 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0174] Then, 0.6 .mu.l of a third single-stranded oilgo DNA (2.75
.mu.M) was added.
[0175] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0176] Subsequently 0.4 .mu.l of AMV reverse transcriptase (37
U/.mu.l, Takara Shuzo Co., Ltd.), as an RNA dependent DNA
polymerase and as a DNA-dependent DNA polymerase, was added. After
2 hours of incubation at 45.degree. C., 5 .mu.l of the reaction
solutions were subjected 2% agarose gel electrophoresis. After the
electrophoresis, the agarose gel was stained with 10,000-fold
diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30 minutes.
[0177] FIG. 3 shows the pattern on the stained gel. Even when the
initial copy numbers of the target nucleic acid was 10.sup.3, the
product was obtained in the presence of potassium acetate at a
final concentration of 120 mM. Thus, a maximal reaction efficiency
was obtained in the presence of potassium acetate at a final
concentration of from 110 to 130 mM.
EXAMPLE 4
[0178] Effects of Sorbitol
[0179] The optimum sorbitol concentration for the present invention
was studied. 20 .mu.l of reaction solutions having the following
composition were pored into PCR tubes.
[0180] 60 mM tris-acetate (pH 8.1)
[0181] 20.3 mM magnesium acetate
[0182] 187.5 mM potassium acetate
[0183] 15% DMSO
[0184] 22.5, 16.8, 13.5 or 11.3% sorbitol
[0185] (final concentration: 15, 11.3, 9 or 7.5%)
[0186] 1.5 mMl each of ATP, GTP, CTP and UTP
[0187] 1.5 mM each of ATP, dGTP, dCTP and dTTP
[0188] 150 .mu.g/ml BSA
[0189] 0.3 .mu.M second single-stranded oligo DNA
[0190] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0191] 0.3 .mu.M third single-stranded oligo DNA (sequence) 5' ATT
TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGA TCA CTC CCC TG
3'
[0192] Then, 5 .mu.l of a standard RNA (10.sup.3 copies, 10.sup.4
copies, 10.sup.5 copies or 10.sup.6 copies/5 .mu.l) or 5 .mu.l of
RNA-free TE buffer as a negative control was added. The standard
RNA had the following sequence.
[0193] (sequence) 5' GAA UCA AA-(bases 11 to 300 in HCV RNA) 3'
[0194] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 65.degree. C. for 10 minutes, and then the
tubes were allowed to stand still in ice-cold water for 5 minutes.
Then, 5 .mu.l of a mixed solution of the following enzymes was
added, and reaction was conducted at 45.degree. C. for 4 hours.
[0195] 36 U/.mu.l commercially available SP6 RNA polymerase (Takara
Shuzo Co., Ltd.)
[0196] 12 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0197] 9 U/.mu.l AMV reverse transcriptase (Takara Shuzo Co.,
Ltd.), as an RNA-dependent DNA polymerase and as a DNA-dependent
DNA polymerase
[0198] 5 .mu.l of the reaction solutions were subjected to agarose
gel electrophoresis. After the electrophoresis, the gel was stained
with 10,000-fold diluted SYBR Green II (Takara Shuzo Co., Ltd.) for
30 minutes.
[0199] FIG. 3 shows the pattern on the stained gel. The clear bands
of 300 bp were obtained in the presence of sorbitol at a final
concentration of 7.5 to 11.3% even when the initial amount of the
target nucleic acid was 10.sup.3 copies. Thus, the maximal reaction
efficiency was obtained when the final sorbitol concentration was
from 7.5 to 11.3%.
EXAMPLE 5
[0200] Amplification of Double-stranded DNA as Target Nucleic
Acid
[0201] The amount of the amplification product from various
concentrations of a double-stranded DNA as the target nucleic acid
was studied. A reaction solution having the following composition
was prepared, and 19 .mu.l of the reaction solution was poured into
PCR tubes.
[0202] 63.2 mM tris-acetate (pH 8.1)
[0203] 21.3 mM magnesium acetate
[0204] 197.4 mM potassium acetate
[0205] 22.5% DMSO
[0206] 22.5% sorbitol
[0207] 1.6 mM each of ATP, GTP, CTP and UTP
[0208] 1.6 mM each of dATP, dGTP, dCTP and dTTP
[0209] 157.9 .mu.g/ml BSA
[0210] 0.055 .mu.M second single-stranded oligo DNA
[0211] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0212] Then, 5 .mu.l of a standard DNA (10.sup.3 copies, 10.sup.4
copies, 10.sup.5 copies or 10.sup.6 copies/5 .mu.l) or 5 .mu.l of
RNA-free TE buffer as a negative control was added. The standard
DNA was a double-stranded DNA consisting of a DNA strand having the
following sequence and a complementary DNA strand.
[0213] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to
300 in HCV cDNA) 3'
[0214] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 45.degree. C. for 5 minutes. Subsequently, 5
.mu.l of a mixed solution of the following enzymes was added, and
reaction was conducted at 45.degree. C. for 1 hour.
[0215] 30 U/.mu.l commercially available SP6 RNA polymerase (Takara
Shuzo Co., Ltd.)
[0216] 12 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0217] Then, 2.75 .mu.l of a third single-stranded oligo DNA (2.75
.mu.M) was added.
[0218] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0219] Subsequently, 0.4 .mu.l of AMV reverse transcriptase (37
U/.mu.l, Takara Shuzo Co., Ltd.), as an RNA-dependent DNA
polymerase and as a DNA-dependent DNA polymerase, was added. After
2 hours of incubation at 45.degree. C., 5 .mu.l of the reaction
solutions were subjected to 2% agarose gel electrophoresis. After
the electrophoresis, the agarose gel was stained with 10,000 fold
diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30 minutes.
[0220] FIG. 5 shows the pattern on the stained gel. Whenever the
initial amount of the target nucleic acid was 10.sup.3 copies/5
.mu.l or more, bands of about 300 bp were detected, and the band
density was dependent on the initial amount of the nucleic acid,
which indicates the possibility of high sensitivity assay of a
double-stranded DNA as the target nucleic acid.
EXAMPLE 6
[0221] Specific Cutting of RNA Using First Single-stranded
Oligonucleic Acid and RNaseH
[0222] RNA was cleaved at the 5' end of a specific nucleic acids
sequence by using a first single-stranded oligonucleic acid and
RNaseH.
[0223] A reaction solution having the following composition was
prepared, and 7.2 .mu.l of the reaction solution was pored into PCR
tubes.
[0224] 40 mM tris-HCl buffer (pH 8.0)
[0225] 4 mM magnesium chloride
[0226] 1 mM dithiothreitol
[0227] 1 .mu.M first single-stranded oligo DNA (11 mer)
[0228] (sequence) 5' GTC GGG GGG AA 3'
[0229] Then, 1.8 .mu.l of RNA (133 mer, 5.7 .mu.M) was added, 10
minutes of heating at 65.degree. C. was followed by sudden cooling
in ice-cold water. The 133 mer RNA had the following sequence.
[0230] (sequence) 5' (vector sequence) GGG AAA GCU UGC AUG CCU GCA
GGU CGA CUC UAG AGG AUC CCC GGG UAC CGA GCU CGA AUU CC (sequence
from HCV) U UGG GGG CGA CAC UCC ACC AUA GAU CAC UCC CCU GUG AGG AAC
UAC UGU CUU CAC GCA GAA AGC GUC UAG C 3'
[0231] (The sequence complementary to the 11 mer DNA is
underlined.)
[0232] After 5 minutes of incubation at 37.degree. C., 1 .mu.l of
RNaseH (0.01, 0.001, 0.0001 or 0.00001 U/.mu.l, Takara Shuzo Co.,
Ltd.) was added, and reaction was conducted at 37.degree. C. for 1
hour. To the reaction solutions, 2 .mu.l gel loading buffer (0.1 M
tris-HCl (pH 8.0) containing 60 mM EDTA, 0.25% Bromophenol Blue and
40% sucrose) and then 12 .mu.l formamide were added. After 5
minutes of incubation at 65.degree. C., 12 .mu.l of the reaction
solutions were subjected to electrophoresis on a polyacrylamide gel
containing 12% urea.
[0233] The acrylamide gel was washed with water three times for 5
minutes each time and stained with 10,000-fold diluted SYBR Green
II (Takara Shuzo Co., Ltd.) for 30 minutes.
[0234] Separately, a reaction solution having the following
composition was prepared, and 10.8 .mu.l of the reaction solution
was pored into PCR tubes.
[0235] 40 mM tris-acetate buffer (pH 8.1)
[0236] 37 mM magnesium acetate
[0237] 347 mM potassium acetate
[0238] 22% sorbitol
[0239] 1 mM dithiothreitol
[0240] 0.9 .mu.M first single-stranded oligonucleic acid (11
mer)
[0241] (sequence) 5' GTC CGG GGG AA 3'
[0242] Then, 1.8 .mu.l of RNA (133 mer, 5.7 .mu.M) was added, and
10 minutes of heating at 65.degree. C. was followed by sudden
cooling in ice-cold water.
[0243] After 5 minutes of incubation at 37.degree. C., 1 .mu.l of
RNaseH (0.01, 0.001, 0.0001 or 0.00001 U/.mu.l, Takara Shuzo Co.,
Ltd.) was added, and reaction was conducted at 37.degree. C. for 1
hour. To the reaction solutions, 2 .mu.l gel loading buffer and
then 10 .mu.l formamide were added. After 5 minutes of incubation
at 65.degree. C., 12 .mu.l of the reaction solutions were subjected
to electrophoresis on a polyacrylamide gel containing 12% urea.
[0244] The acrylamide gel was washed with water three times for 5
minutes each time and stained with 10,000-fold diluted SYBR Green
II (Takara Shuzo Co., Ltd.) for 30 minutes.
[0245] FIG. 6 shows the results obtained in the respective reaction
solutions having different compositions. When the tris-HCl buffer
was used in the presence of RNaseH at final concentrations of
0.000001 and 0.00001 U/.mu.l, the band of the 133 mer disappeared,
and bands of 60 to 70 mers were detected. The still higher final
concentration of RNaseH resulted in further degradation of the 133
mer. On the other hand, when the tris-acetate buffer was used in
the presence of RNaseH at a final concentration of 0.0007 U/.mu.l,
the band of the 133 mer disappeared, and a band of a 60 to 70 mer
was detected. This band agrees with the RNA fragment obtained when
the 133 mer RNA is cut where the first single-stranded oligonucleic
acid binds to the 133 mer in terms of mobility.
[0246] Thus it was possible to cut the target RNA at the 5' end of
the specific nucleic acids sequence by using the first
single-stranded oligonucleic acid and RNaseH.
EXAMPLE 7
[0247] Amplification of RNA as Target Nucleic Acid
[0248] The present invention was performed by using various
concentrations of a target RNA, and the amplification products were
examined. A reaction solution having the following composition was
prepared, and 20 .mu.l of the reaction solution was pored into PCR
tubes.
[0249] 60 mM tris-acetate (pH 8.1)
[0250] 20.3 mM magnesium acetate
[0251] 187.5 mM potassium acetate
[0252] 22.5% DMSO
[0253] 22.5% sorbitol
[0254] 1.5 mM each of ATP, GTP, CTP and UTP
[0255] 1.5 mM each of dATP, dGTP, dCTP and dTTP
[0256] 150 .mu.g/ml BSA
[0257] 0.3 .mu.M third single-stranded oligo DNA
[0258] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0259] 0.3 .mu.M second single-stranded oligo DNA
[0260] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0261] Then, 5 .mu.l of a standard RNA (10.sup.2 copies, 10.sup.3
copies, 10.sup.4 copies, 10.sup.5 copies or 10.sup.6 copies/5
.mu.l) or 5 .mu.l of RNA-free TE buffer as a negative control was
added. The standard RNA had the following sequence.
[0262] (sequence) 5' GAA UAC AA-(bases 11 to 300 in HCV RNA) 3'
[0263] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 65.degree. C. for 10 minutes, and then the
tubes were allowed to stand still in ice-cold water for 5 minutes.
Then, 5 .mu.l of a mixed solution of the following enzymes was
added, and reaction was conducted at 45.degree. C. for 4 hours.
[0264] 36 U/.mu.l commercially available SP6 RNA polymerase (Takara
Shuzo Co., Ltd.)
[0265] 12 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0266] 9 U/.mu.l AMV reverse transcriptase (Takara Shuzo Co.,
Ltd.), as an RNA-dependent DNA polymerase and as a DNA-dependent
DNA polymerase
[0267] 5 .mu.l of the reaction solutions were subjected to agarose
gel electrophoresis. After the electrophoresis, the agarose gel was
stained with 10,000-fold diluted SYBR Green II (Takara Shuzo Co.,
Ltd.) for 30 minutes.
[0268] FIG. 7 shows the pattern on the stained gel. As shown in
FIG. 7, when the initial concentration of the target RNA was
10.sup.3 copies/5 .mu.l or more, bands of about 300 bp were
detected, and the band density was dependent on the initial amount
of the RNA. Thus, it was possible to detect the target RNA with
high sensitivity by the present invention. The amount of the
amplification product increased with the elapse of time and was
dependent on the initial amount of the target RNA.
EXAMPLE 8
[0269] Identification of Amplification Product
[0270] Amplification products was identified after treatment with
DNase or RNase. The procedure in Example 7 was followed until the
use of the enzymes in 5 .mu.l of TE buffer as a negative control
and in 5 .mu.l of a standard RNA (10.sup.5 copies or 10.sup.6
copies/5 .mu.l). The standard RNA was as follows.
[0271] (sequence) 5' GAA UAC AA-(bases 11 to 300 in HCV RNA) 3'
[0272] As the standard DNA, a double stranded-DNA consisting of a
DNA strand having the following sequence and a complementary strand
was used.
[0273] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to
300 in HCV cDNA)-3'
[0274] After the procedure, each reaction solution was incubated at
65.degree. C. for 30 minutes. 6 .mu.l of the three kinds of
reaction solutions, the standard RNA (69 ng/.mu.l) and the standard
DNA (6 ng/.mu.l) were pored into three tubes each. To one of them,
0.6 .mu.l of RNaseA (0.5 mg/ml) was added, to another one, 0.6
.mu.l of DNaseI (1 mg/ml) was added, and to the remaining one,
nothing was added. The three tubes of each set were incubated at
37.degree. C. for 1 hour, and 5 .mu.l of each reaction solution was
subjected to 2% agarose gel electrophoresis. After the
electrophoresis, the agarose gel was stained with 10,000-fold
diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30 minutes. The
compositions of the reaction solutions with the standard RNA (69
ng/.mu.l) and the standard DNA (6 ng/.mu.l) were the same as the
composition of the other reaction solutions obtained by the
procedure in Example 7 except that the enzymes were omitted.
[0275] FIG. 8 shows that the RNA product obtained by DNase I
treatment of the DNA product from 10.sup.6 copies of the target
standard RNA corresponded to 345 ng of the standard RNA in terms of
band density. The band of the RNA product obtained by the DNase I
treatment agreed with band of the standard RNA in terms of
mobility.
[0276] On the other hand, the band obtained by amplification of
10.sup.5 copies of the target standard RNA followed by RNaseA
treatment of the RNA product almost agreed with the standard DNA in
terms of mobility.
[0277] Thus, both the RNA product and the DNA product were
synthesized from the target RNA simultaneously.
EXAMPLE 9
[0278] Time Courses of RNA Production and DNA Production
[0279] The time courses of DNA production and RNA production were
followed. A reaction solution having the following composition was
prepared, and 33 .mu.l of the reaction solution was pored into 10
PCR tubes
[0280] 61 mM tris-acetate (pH 8.1)
[0281] 20.5 mM magnesium acetate
[0282] 189.2 mM potassium acetate
[0283] 21.7% DMSO
[0284] 12% sorbitol
[0285] 15 mM dithiothreitol
[0286] 1.5 mM each of ATP, GTP, CTP and UTP
[0287] 1.5 mM each of dATP, dGTP, dCTP and dTTP
[0288] 151 .mu.g/ml BSA
[0289] 0.3 .mu.M third single-stranded oligo DNA
[0290] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0291] 0.3 .mu.M second single-stranded oligo DNA
[0292] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0293] Then, 8.3 .mu.l of a standard RNA (10.sup.6 copies/5 .mu.l)
or TE buffer as the negative control was pored into five tubes. The
standard RNA had the following sequence.
[0294] (sequence) 5' GAA UAC AA-(bases from 11 to 300 in HCV RNA)
3'
[0295] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 65.degree. C. for 10 minutes, and then the
tubes were allowed to stand still in ice-cold water for 5 minutes.
After 10 minutes of incubation at 44.degree. C., 8.6 .mu.l of a
mixed solution of the following enzymes was added. After 0, 1, 2, 3
and 4 hours of reaction at 44.degree. C., one tube for each of the
standard RNA and the negative control was withdrawn and transferred
into ice.
[0296] 32.8 U/.mu.l commercially available SP6 RNA polymerase
(Takara Shuzo Co., Ltd.)
[0297] 11.8 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0298] 8.2 U/.mu.l AMV reverse transcriptase (Takara Shuzo Co.,
Ltd.), as an RNA-dependent DNA polymerase and as a DNA-dependent
DNA polymerase
[0299] 5 .mu.l of the reaction solution in each tube was subjected
to 2% agarose electrophoresis. After the electrophoresis, the
agarose gel was stained with 10,000fold diluted SYBR Green II
(Takara Shuzo Co., Ltd.) for 30 minutes and photographed. Then, the
band densities (OD) in the photograph were measured with a
densitometer.
[0300] The rest of each reaction solution was heated at 65.degree.
C. for 20 minutes, and 10 .mu.l of each reaction solution were
pored into three tubes. To one of them, 1 .mu.l of RNaseA (0.5
mg/ml) was added, to another one, 1 .mu.l of DNaseI (1 mg/ml) was
added, and to the remaining one, 1 .mu.l of TE buffer was added.
All the three tubes of each set were incubated at 37.degree. C. for
1 hour, and then 5 .mu.l of each reaction solution was subjected to
2% agarose gel electrophoresis. After the electrophoresis, the gel
was stained with 10,000-fold diluted SYBR Green II (Takara Shuzo
Co., Ltd.) for 30 minutes and photographed. Then the band densities
(OD) in the photograph were measured with a densitometer.
[0301] FIG. 9 shows the time chart of DNA production and RNA
production determined from the band densities in the
electrophoretogram. The RNA production was about 40 times greater
than the DNA production. Further, both the RNA production and the
DNA production suddenly increased 2 hours after the initiation of
the reaction. Thus, both RNA production and DNA production by the
method of the present invention showed similar amplification
curves.
EXAMPLE 10
[0302] Assay of Amplification Product Using Fourth Single-stranded
Oligo DNA
[0303] An RNA product in reaction solutions was assayed by
measuring the fluorescent signals by using a fourth single-stranded
oligo RNA. Firstly, 20 .mu.l of a reaction solution having the
following composition was pored into 30 PCR tubes.
[0304] 60 mM tris-acetate (pH 8.1)
[0305] 20.3 mM magnesium acetate
[0306] 187.5 mM potassium acetate
[0307] 22.5% DMSO
[0308] 12% sorbitol
[0309] 1.5 mM each of ATP, GTP, CTP and UTP
[0310] 1.5 mM each of dATP, dGTP, dCTP and dTTP
[0311] 150 .mu.g/ml BSA
[0312] 0.3 .mu.M third single-stranded oligo DNA
[0313] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0314] 0.3 .mu.M second single-stranded oligo DNA
[0315] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0316] Then, 5 .mu.l of a standard RNA (10.sup.6 copies/5 .mu.l)
and TE buffer as a negative control were added to 15 tubes,
respectively. The standard RNA had the following sequence.
[0317] (sequence) 5' GAA UAC AA-(bases 11 to 300 in HCV RNA) 3'
[0318] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 65.degree. C. for 10 minutes, and then the
tubes were allowed to stand still in ice-cold water for 5 minutes.
After 10 minutes of incubation at 44.degree. C., 5 .mu.l of a mixed
solution of the following enzymes was added. After 0, 1, 2, 3 and 4
hours of reaction at 44.degree. C., three tube for each of the
standard RNA and the negative control were withdrawn and
transferred into ice.
[0319] 36 U/.mu.l commercially available SP6 RNA polymerase (Takara
Shuzo Co., Ltd.)
[0320] 12 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0321] 9 U/.mu.l AMV reverse transcriptase (Takara Shuzo Co.,
Ltd.), as an RNA-dependent DNA polymerase and as a DNA-dependent
DNA polymerase
[0322] 5 .mu.l of the reaction solution in each tube was subjected
to 2% agarose electrophoresis. After the electrophoresis, the
reaction solutions in the three tubes withdrawn at each reaction
time were mixed, and 5 .mu.l of each mixture was subjected to 2%
agarose gel electrophoresis. After the electrophoresis, the agarose
gel was stained with 10,000-fold diluted SYBR Green II (Takara
Shuzo Co., Ltd.) for 30 minutes and photographed.
[0323] To 50 .mu.l of each mixture, 100 .mu.l of measuring buffer
having the following composition was added.
[0324] 40 mM tris-acetate (pH 8.1)
[0325] 13 mM magnesium acetate
[0326] 125 mM potassium acetate
[0327] 10 mM dithiothreitol
[0328] 2 U/.mu.l RNase inhibitor
[0329] 37.5 nM fourth single-stranded oligo DNA (hereinafter
referred to as YO-271)
[0330] (sequence) 5.degree. CTC GC*G GGG GCT G 3'
[0331] (* indicates the site labeled with the fluorescent
intercalative dye, oxazole yellow. The sequence of bases 1 to 11 in
the DNA moiety is complementary to the sequence of bases 223 to 233
in HCV cDNA. The fluorescent dye was linked to the DNA moiety as
described in Nucleic Acids Research, 24(24), 4992-4997(1996) for
YO-YPF-271. For the structure of YO-271, FIG. 19 should be referred
to).
[0332] The reaction solutions were incubated at 65.degree. C. for
15 minutes and suddenly cooled in ice-cold water for 5 minutes.
Then the reaction solutions were incubated at 37.degree. C. for 10
minutes and poured into fluorometric cuvettes preheated to
37.degree. C., and the fluorescent intensities were measured at an
excitation wavelength of 490 nm and an emission wavelength of 510
nm.
[0333] FIG. 10 shows the results of the electrophoresis, and FIG.
11 shows the results of the measurements of the fluorescent signals
from the fourth single-stranded oligo DNA (YO-271). As is evident
from FIG. 11, the fluorescent signal from YO-271 increased suddenly
in 2 hours for the standard RNA, whereas there was no increase in
the fluorescence intensity with a negative control. Thus, use of
the fourth single-stranded oligo DNA enabled to specific assay of
the amplification product.
EXAMPLE 11
[0334] Modification of the 3' End of Fourth Single-stranded Oligo
DNA
[0335] During synthesis of a single-stranded RNA in the presence of
a fourth single-stranded oligo DNA, elongation of the DNA from the
3' end by the action of a coexisting RNA-dependent RNA polymerase
can results in increase in a non-specific fluorescent signal.
Therefore, the 3' end of the oligo DNA was modified by treatment
with terminal transferase (TdT) (addition of ddTTP), and its effect
was examined.
[0336] TdT treatment was conducted in a reaction solution (total
volume 50 .mu.l) having the following composition at 37.degree. C.
for 1 hour.
[0337] 100 mM sodium cacodylate buffer (pH 7.2)
[0338] 1 mM cobalt chloride
[0339] 0.1 mM dithiothreitol
[0340] 0.5 mM ddTTP
[0341] 0.6 U/.mu.l TdT (Takara Shuzo Co., Ltd.)
[0342] 37.3 .mu.M fourth single-stranded oligo DNA (YO-271)
[0343] The modification product was recovered by phenol-chloroform
extraction followed by purification from the aqueous layer using a
commercial column (chroma spin-10, tradename, Toyobo Co., Ltd.) and
determined by OD260 measurement.
[0344] The resulting TdT-treated YO-271 and non-treated YO-271 were
used for measurement of fluorescent signals in the present
invention. 50 .mu.l of reaction solutions having the following
composition (containing non-treated YO-271 or TdT-treated YO-271)
were pored into 10 tubes, respectively.
[0345] 69 mM tris-acetate (pH 8.1)
[0346] 20.3 mM magnesium acetate
[0347] 187.5 mM potassium acetate
[0348] 21.5% DMSO
[0349] 12% sorbitol
[0350] 15 mM dithiothreitol
[0351] 1.5 mM each of ATP, GTP, CTP and UTP
[0352] 1.5 mM each of dATP, dGTP, dCTP and dTTP
[0353] 150 .mu.g/ml BSA
[0354] 0.3 .mu.M third single-stranded oligo DNA
[0355] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0356] 0.3 .mu.M second single-stranded oligo DNA
[0357] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0358] 112.5 nM TdT-treated YO-271 or non-treated YO-271
[0359] Then, 12.5 .mu.l of a standard RNA (10.sup.6 copies/5 .mu.l)
or TE buffer as a negative control was added to five tubes,
respectively. The standard RNA had the following sequence.
[0360] (sequence) 5' GAA UAC AA-(bases 11 to 300 in HCV RNA) 3'
[0361] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 65.degree. C. for 10 minutes, and then the
tubes were allowed to stand still in ice-cold water for 5 minutes.
After 10 minutes of incubation at 44.degree. C., 12.5 .mu.l of a
mixed solution of the following enzymes was added. After reaction
at 40.degree. C. for 0, 1, 2, 3 and 4 hours, tubes were withdrawn
and transferred into ice-cold water for the standard RNA and
negative control.
[0362] 34.2 U/.mu.l commercially available SP6 RNA polymerase
(Takara Shuzo Co., Ltd.)
[0363] 12 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0364] 8.4 U/.mu.l AMV reverse transcriptase (Takara Shuzo Co.,
Ltd.) as an RNA-dependent DNA polymerase and as a DNA-dependent DNA
polymerase
[0365] A 50 .mu.l aliquot of each reaction solution was mixed with
100 .mu.l of dilution buffer having the following composition,
incubated at 44.degree. C. for 5 minutes and transferred into a
fluorometric cuvette preheated at 44.degree. C., and the
fluorescence intensity was measured at an excitation wavelength of
490 nm and an emission wavelength of 510 nm.
[0366] 40 mM tris-acetate (pH 8.1)
[0367] 13 mM magnesium acetate
[0368] 125 mM potassium acetate
[0369] 10 mM dithiothreitol
[0370] 2 U/.mu.l RNase inhibitor
[0371] FIG. 12 and FIG. 13 show the results with the non-treated
and TdT-treated YO-271, respectively.
[0372] In the case of the non-treated YO-271, there was an increase
in the fluorescent signal in the presence of the negative control,
and the fluorescent signal in the presence of the negative control
was not significantly different from that in the presence of the
standard RNA. On the other hand, in the case of the ddTTP-treated
YO-271, there was a significant difference between the fluorescent
signal in the presence of the negative control and in the presence
of the standard RNA. Thus, for RNA synthesis in the presence of the
fourth single-stranded oligo DNA in the present invention, it is
preferred to modify the 3' end of the DNA so as to prevent
elongation of the DNA from the 3' end by the action of the
RNA-dependent DNA polymerase
EXAMPLE 12
[0373] RNA or DNA Synthesis in the Presence of Fourth
Single-stranded Oligo DNA
[0374] The effects of the ddTTP-treated YO-271 prepared in Example
11 on RNA or DNA synthesis in the presence of a fourth
single-stranded oligo DNA were examined. 50 .mu.l of a reaction
solution having the following composition was pored into 10
tubes.
[0375] 60 mM tri-acetate (pH 8.1)
[0376] 2.3 mM magnesium acetate
[0377] 187.5 mM potassium acetate
[0378] 21.5% DMSO
[0379] 12% sorbitol
[0380] 15 mM dithiothreitol
[0381] 1.5 mM each of ATP, GTP, CTP and UTP
[0382] 1.5 mM each of dATP, dGTP, dCTP and dTTP
[0383] 150 .mu.g/ml BSA
[0384] 0.3 .mu.M third single-stranded oligo DNA
[0385] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0386] 0.3 .mu.M second single-stranded oligo DNA
[0387] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0388] 112.5 nM TdT-treated YO-271
[0389] Then, 12.5 .mu.l of a standard RNA (10.sup.6 copies/5 .mu.l)
or TE buffer as a negative control was added to five tubes,
respectively. The standard RNA had the following sequence.
[0390] (sequence) 5' GAA UAC AA-(bases from 11 to 300 in HCV RNA)
3'
[0391] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 65.degree. C. for 10 minutes, and then the
tubes were allowed to stand still in ice-cold water for 5 minutes.
After 10 minutes of incubation at 44.degree. C., 12.5 .mu.l of a
mixed solution of the following enzymes was added. After reaction
at 40.degree. C. for 0, 1, 2, 3 and 4 hours, tubes were withdrawn
and transferred into ice-cold water for the standard RNA and
negative control.
[0392] 34.2 U/.mu.l commercially available SP6 RNA polymerase
(Takara Shuzo Co., Ltd.)
[0393] 12 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0394] 8.4 U/.mu.l AMV reverse transcriptase (Takara Shuzo Co.,
Ltd.) as an RNA-dependent DNA polymerase and as a DNA-dependent DNA
polymerase
[0395] 5 .mu.l of the reaction solution in each tube was subjected
to 2% agarose gel electrophoresis. After the electrophoresis, the
agarose gel was stained with 10,000fold diluted SYBR Green II
(Takara Shuzo Co., Ltd.) for 30 minutes and photographed. The band
densities (OD) in the photograph was measured with a
densitometer.
[0396] FIG. 14 shows the results of the electrophoresis, and FIG.
15 shows the results of the OD measurement of the
electrophoretogram. In the presence of the standard RNA, the
amplification product increased with time, whereas in the presence
of the negative control, there was no increase of the amplification
product. Thus, the presence of the fourth single-stranded oligo DNA
did not inhibit RNA synthesis in the present invention.
EXAMPLE 13
[0397] Analysis of the Time Course of the Amount of Amplification
Product by Using Fourth Single-stranded Oligo DNA
[0398] RNA production was monitored by using the ddTTP-treated
YO-271 prepared in Example 11 as the fourth single-stranded oligo
DNA over a period of time. Firstly, 50 .mu.l of a reaction solution
having the following composition was pored into 10 tubes.
[0399] 60 nM tris-acetate (pH 8.1)
[0400] 20.3 mM magnesium acetate
[0401] 187.5 mM potassium acetate
[0402] 21.5% DMSO
[0403] 12% sorbitol
[0404] 15 mM dithiothreitol
[0405] 1.5 mM each of ATP, GTP, CTP and UTP
[0406] 1.5 mM each of dATP, dGTP, dCTP and dTTP
[0407] 150 .mu.g/ml BSA
[0408] 0.3 .mu.M third single-stranded oligo DNA
[0409] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0410] 0.3 .mu.M second single-stranded oligo DNA
[0411] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0412] 112.5 nM TdT-treated YO-271
[0413] Then, 12.5 .mu.l of a standard RNA (10.sup.6 copies/5 .mu.l)
or TE buffer as a negative control was added to five tubes,
respectively. The standard RNA had the following sequence.
[0414] (sequence) 5' GAA UAC AA-(bases 11 to 300 in HCV RNA) 3'
[0415] The reaction solutions were overlaid with 100 .mu.l of
mineral oil and incubated at 65.degree. C. for 10 minutes, and then
the tubes were allowed to stand still in ice-cold water for 5
minutes. After 10 minutes of incubation at 44.degree. C., 12.5
.mu.l of a mixed solution of the following enzymes was added. After
reaction at 40.degree. C. for 0, 1, 2, 3 and 4 hours, tubes were
withdrawn and transferred into ice-cold water for the standard RNA
and negative control.
[0416] 34.2 U/.mu.l commercially available SP6 RNA polymerase
(Takara Shuzo Co., Ltd.)
[0417] 12 U/.mu.l commercially available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0418] 8.4 U/.mu.l AMV reverse transcriptase (Takara Shuzo Co.,
Ltd.) as an RNA-dependent DNA polymerase and as a DNA-dependent DNA
polymerase
[0419] A 50 .mu.l aliquot of each reaction solution was mixed with
100 .mu.l of dilution buffer having the following composition,
incubated at 44.degree. C. for 5 minutes and transferred into a
fluorometric cuvette preheated at 44.degree. C., and the
fluorescence intensity was measured at an excitation wavelength of
490 nm and an emission wavelength of 510 nm.
[0420] 40 mM tris-acetate (pH 8.1)
[0421] 13 mM magnesium acetate
[0422] 125 mM potassium acetate
[0423] 10 mM dithiothreitol
[0424] 2 U/.mu.l RNase inhibitor
[0425] FIG. 16 shows the results of the fluorescence measurement.
In the presence of the standard RNA, the fluorescence intensity
increased with the elapse of time, whereas in the presence of the
negative control, the fluorescence intensity did not increase. The
fluorescence profile almost agreed with the results of the
electrophoretic quantification in Example 12. Thus, use of the
fourth single-stranded oligo DNA made it possible to follow the
time course of specific amplification (RNA synthesis).
EXAMPLE 14
[0426] Monitoring of a Fluorescent Signal Using Fourth
Single-stranded Oligo DNA
[0427] The fluorescence intensity of the ddTTP-treated YO-271
prepared in Example 11 in the presence of known concentrations of a
target RNA was monitored. Firstly, 50 .mu.l of a reaction solution
having the following composition were pored into 17 PCR tubes.
[0428] 60 mM tris-acetate (pH 8.1)
[0429] 20.3 mM magnesium acetate
[0430] 187.5 mM potassium acetate
[0431] 21.5% DMSO
[0432] 12% sorbitol
[0433] 15 mM dithiothreitol
[0434] 1.5 mM each of ATP, GTP, CTP and UTP
[0435] 1.5 mM each of dATP, dGTP, dCTP and dTTP
[0436] 150 .mu.g/ml BSA
[0437] 0.3 .mu.M third single-stranded oligo DNA
[0438] (sequence) 5' ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC
CAT AGA TCA CTC CCC TG 3'
[0439] 0.3 .mu.M second single-stranded oligo DNA
[0440] (sequence) 5' ACT CGC AAG CAC CCT ATC A 3'
[0441] 112.5 nM TdT-treated YO-271
[0442] Then, 12.5 .mu.l of a standard RNA (10.sup.4 copies or
10.sup.5 copies/5 .mu.l) and TE buffer as a negative control were
pored into four tubes each. Separately, 12.5 al of the standard RNA
(10.sup.6 copies/5 .mu.l) was pored into five tubes.
[0443] The standard RNA had the following sequence.
[0444] (sequence) 5' GAA UAC AA-(bases 11 to 300 in HCV RNA) 3'
[0445] The reaction solutions were overlaid with 100 .mu.l mineral
oil and incubated at 65.degree. C. for 10 minutes, and then the
tubes were allowed to stand dtill in ice-cold water for 5 minutes.
After 10 minutes of incubation at 44.degree. C., 12.5 .mu.l of a
mixed solution of the following enzymes was added. After reaction
at 44.degree. C. for 0, 1 (only for 10.sup.6 copies/5 .mu.l of the
standard RNA), 2, 3 and 4 hours, one tube was withdrawn and
transferred into ice-cold water for the standard RNA and the
negative control, respectively.
[0446] 34.2 U/.mu.l commercially available SP6 RNA polymerase
(Takara Shuzo Co., Ltd.)
[0447] 12 U/.mu.l commercial available RNase inhibitor (Takara
Shuzo Co., Ltd.)
[0448] 8.4 U/.mu.l AMV reverse transcriptase (Takara Shuzo Co.,
Ltd.) as an RNA-dependent DNA polymerase and as a DNA-dependent DNA
polymerase
[0449] A 50 .mu.l aliquot of each reaction solution was mixed with
100 .mu.l of dilution buffer having the following composition,
incubated at 44.degree. C. for 5 minutes and transferred into a
fluorometric cuvette preheated at 44.degree. C., and the
fluorescence intensity was measured at an excitation wavelength of
490 nm and an emission wavelength of 510 nm.
[0450] 40 mM tris-acetate (pH 8.1)
[0451] 13 mM magnesium acetate
[0452] 125 mM potassium acetate
[0453] 10 mM dithiothreitol
[0454] 1 U/.mu.l RNase inhibitor
[0455] FIG. 17 shows fluorescence intensities measured at the
respective reaction times after subtraction of the background
fluorescence intensity. The plot of the fluorescence intensities at
the reaction time of 3 hours against the initial concentrations of
the target RNA based on the fluorescence profiles (FIG. 18) shows
concentration-dependent fluorescence enhancement. Thus, a
calibration curve which shows the correlation between the initial
concentration of the target RNA and fluorescence enhancement was
obtained on the base of the amplification curve obtained by
monitoring the fluorescence intensity. Thus, by measuring
fluorescence intensity for a sample containing the target RNA at an
unknown concentration, the initial amount of the target RNA could
be determined.
[0456] As described so far, the present invention enables assay of
a single-stranded RNA containing a specific nucleic acids sequence
in a sample at almost constant temperature without repetitious
rapid heating and cooling of reaction solutions. Further, the
method of the present invention enables high sensitive specific
homogeneous assay of a target nucleic acid without using a
support.
[0457] In the present invention, from a trace amount of a
single-stranded RNA in a sample, a double-stranded DNA having a
promoter region for a DNA-dependent RNA polymerase at one end is
synthesized, and the double-stranded DNA gives rise to multiple RNA
single strands. The synthesized single-stranded RNA participates in
the next round of synthesis of the double-stranded DNA.
Consequently, the single-stranded RNA synthesized in the series of
reactions as an intermediate drastically increases with the elapse
of time. However, because the rate of the RNA synthesis and the
final amount of the synthesized RNA depend on the amount of the
target RNA in the sample, assay of the target RNA is possible by
measurement of the amount of the synthesized RNA.
[0458] The fourth single-stranded oligo DNA in the present
invention enhances the fluorescence intensity on hybridization with
the single-stranded RNA in the reaction solution, and thereby
enables determination of the initial amount of the target RNA in
the sample by measuring the fluorescent intensity of the reaction
solution.
[0459] Thus, the assay method of the present invention can be
conducted at constant temperature because the reactions proceed
through binding of primers to the single-stranded RNA and DNA
synthesized as intermediates, and can be automated easily because
it does not require repetitious rapid heating and cooling of
reaction solutions for priming unlike PCR. Further, in the present
invention, the use of the fourth single-stranded oligo DNA makes is
possible to monitor RNA synthesis by measuring the fluorescent
intensity during the single-stranded RNA. Therefore, it is possible
to provide a simple one-step method for accurate speedy homogeneous
qualitative and quantitative assay of a target RNA useful for
clinical diagnosis without need of electrophoresis or sandwich
assay of the reaction product.
[0460] On the basis of the multiplication of a specific RNA
sequence at constant temperature, the present invention also
provides a simple method for producing RNA which can be conducted
under milder conditions than conventional chemical synthesis and
RT-PCR.
* * * * *