U.S. patent application number 09/308034 was filed with the patent office on 2002-01-10 for stabilized aqueous nucleoside triphosphate solution.
Invention is credited to IHLENFELDT, HANS-GEORG, LEITENBERGER, VOLKER, MUHLEGGER, KLAUS, SCHMIDT, AXEL.
Application Number | 20020004230 09/308034 |
Document ID | / |
Family ID | 7811645 |
Filed Date | 2002-01-10 |
United States Patent
Application |
20020004230 |
Kind Code |
A1 |
IHLENFELDT, HANS-GEORG ; et
al. |
January 10, 2002 |
STABILIZED AQUEOUS NUCLEOSIDE TRIPHOSPHATE SOLUTION
Abstract
The invention concerns stable aqueous solutions containing one
or several nucleoside triphosphates wherein the respective solution
has a pH value of more than 7.5 and contains no additional
substances with a stabilizing effect. The nucleoside triphosphate
solutions are used in particular for DNA synthesizing reactions
such as e.g. RT-PCR, cycle sequencing, random priming and nick
translation. One of the most important applications of such
solutions containing deoxynucleoside triphosphates (d-NTP) is their
use in the polymerase chain reaction (PCR).
Inventors: |
IHLENFELDT, HANS-GEORG;
(IFFELDORF, DE) ; SCHMIDT, AXEL; (MUNICH, DE)
; MUHLEGGER, KLAUS; (POLLING, DE) ; LEITENBERGER,
VOLKER; (SEESHAUPT, DE) |
Correspondence
Address: |
MARILYN L AMICK
ROCHE DIAGNOSTICS CORPORATION
9115 HAGUE ROAD
BUILDING D
INDIANAPOLIS
IN
462500457
|
Family ID: |
7811645 |
Appl. No.: |
09/308034 |
Filed: |
November 19, 1999 |
PCT Filed: |
November 11, 1997 |
PCT NO: |
PCT/EP97/06276 |
Current U.S.
Class: |
435/91.2 ;
435/87; 435/91.5; 435/91.51; 536/23.1 |
Current CPC
Class: |
C12Q 1/68 20130101; C12Q
1/686 20130101; C12Q 1/6846 20130101; C12Q 1/6869 20130101; C12Q
1/6806 20130101; C12Q 1/6846 20130101; C12Q 2527/125 20130101; C12Q
2527/119 20130101; C12Q 1/686 20130101; C12Q 2527/119 20130101;
C12Q 1/6869 20130101; C12Q 2535/113 20130101; C12Q 2527/119
20130101 |
Class at
Publication: |
435/91.2 ;
536/23.1; 435/91.5; 435/91.51; 435/87 |
International
Class: |
C07H 021/02; C07H
021/04; C12P 019/38; C12P 019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 14, 1996 |
DE |
196 47 055.2 |
Claims
1. Stable aqueous solution containing nucleoside triphosphates,
wherein the pH value of the solution is above ca. 7.5.
2. Stable aqueous solution as claimed in claim 1, wherein the
nucleoside triphosphates are modified nucleoside triphosphates.
3. Stable aqueous solution as claimed in claim 1 or 2, wherein the
pH value is in a range between 7.5 and 11.
4. Stable aqueous solution as claimed in claim 1, 2 or 3, wherein
the concentration of the nucleoside triphosphates is ca. 2 to 200
mmol/l.
5. Stable aqueous solution as claimed in one of the claims 1 to 4,
wherein the solution contains deoxynucleoside triphosphates.
6. Stable aqueous solution as claimed in claims 1 to 5 containing a
substance which buffers at or above pH 7.5.
7. Stable aqueous solution as claimed in claims 1 to 6 which are
free of further stabilizing agents.
8. Use of a stable aqueous solution as claimed in claims 1 to 7 for
a DNA and/or RNA synthesizing reaction.
9. Use of a stable aqueous solution as claimed in claims 1 to 7 to
replicate DNA and/or RNA sequences or fragments.
10. Use of a stable aqueous solution as claimed in claims 1 to 7 to
specifically replicate nucleic acid fragments in the presence of an
enzyme with reverse transcriptase activity.
11. Use of a stable aqueous solution as claimed in claims 1 to 7
for the cycle sequencing of nucleic acids.
12. Use of a stable aqueous solution as claimed in claims 1 to 7
for the specific replication of deoxynucleic acid sequences or
fragments.
13. Use of a stable aqueous solution as claimed in claims 1 to 7
for random priming.
14. Use of a stable aqueous solution as claimed in claims 1 to 7
for nick translation.
Description
[0001] The invention concerns stable aqueous solutions containing
nucleoside triphosphates in which the solution has a pH value above
7.5.
[0002] Nucleoside triphosphates (NTP) such as ribonucleoside,
deoxynucleoside and dideoxynucleoside triphosphates have a variety
of uses in the field of biochemistry and molecular biology. Most of
the applications relate to reactions which synthesize or replicate
DNA and RNA such as the reverse transcriptase polymerase chain
reaction (RT-PCR), cycle sequencing and nick translation. In the
case of RT-PCR, DNA chains are synthesized in the 5'-3' direction
by for example reverse transcriptase whereby an RNA strand serves
as the template. Certain NTPs such as dideoxynucleoside
triphosphates (dd-NTP) can be used as chain terminators in the
sequencing of DNA. One of the most important applications of
deoxynucleoside triphosphates (d-NTP) is their use in the
polymerase chain reaction (PCR). In this application it is
absolutely essential that the NTP solutions are stable above all
during storage. The d-NTPs (d-ATP, d-CTP, d-GTP, d-TTP, d-UTP among
others) are usually stored as Na or Li salts and typically at
concentrations of 0.1 mol/l and are commercially available in this
form. As a rule the pH values are physiological pH values i.e.
between ca. 7.0 and 7.5.
[0003] A disadvantage of the current, i.e. commercially available,
NTP solutions is in particular the instability of the NTPs during
storage or thermal stress. The NTPs have a tendency to decompose
over time to form the corresponding diphosphates and
monophosphates. The triphosphate content decreases especially at
higher temperatures. The triphosphate content already decreases by
ca. 2-3% within ten days at a pH value of ca. 7.5 and a temperature
of 35.degree. C. In contrast at room temperature the triphosphate
content is observed to decrease by only ca. 1% after six weeks.
Hence the decomposition of the triphosphates in aqueous solution
limits the shelf life of the NTP solutions. Consequently the
suppliers of d-NTPs for example only guarantee a shelf life of 12
months for dNTP solutions. However, there is a need for aqueous
solutions which only contain dNTP and this at high concentrations
and which have a longer long-term stability than the presently
available solutions.
[0004] Attempts to improve the stability of triphosphates have up
to now merely related to corresponding adenosine triphosphate
solutions. The stability of the adenosine triphosphate was examined
in relation to the pH value. The presence of stabilizers was
described as being absolutely essential in accordance with the
state of the art. Thus the stability of adenosine triphosphate in
aqueous solution at a pH value of preferably 8.3 to 9.2 is
described as being optimal in the presence of EDTA (JP 64/003444).
In the presence of stabilizers such as guanidine/amino-guanidine or
creatinine a pH value of 9 to 10 (JP 71/038270 and JP 71/033592),
in the presence of methionine as a stabilizer a pH value of
preferably 9 to 10.5 (JP 67/019115), in the presence of the
stabilizers phosphate and sorbitol/mannitol/glycerol/ benzyl
alcohol/PEG a pH value of 8 to 11 (JP 67/015115) and in the
presence of glycerol/H.sub.3PO.sub.4 a pH value of 3.7 (FR4078) is
described. However, the presence of stabilizers in d-NTP solutions
can be critical for many applications or cause interference.
[0005] Hence the object of the present invention was to provide a
stabilized aqueous solution containing NTPs without the addition of
any stabilizers.
[0006] The object was achieved according to the invention by the
aqueous NTP solutions having a pH value of above approximately 7.5.
These nucleotide triphosphates include ribonucleoside
triphosphates, deoxynucleotide and dideoxynucleotide triphosphates
wherein the five naturally occurring as well as modified bases such
as isoguanine, deaza compounds and derivatives thereof come into
consideration as bases. Furthermore the nucleoside triphosphates
can be labelled with reporter groups. As a rule the solutions
according to the invention have a pH value in a range of more than
7.5 to a maximum of 11. A pH value of ca. 8 to 10 proved to be
particularly advantageous. The pH value can be set by adding a base
(e.g. NaOH, KOH, LiOH) as well as by adding a buffer (e.g Tris
buffer, Na carbonate buffer, phosphate buffer).
[0007] The concentration of the NTP solution is preferably between
ca. 2 mmol/l and 200 mmol/l. A concentration of the NTPs of ca. 100
to 150 mmol/l is particularly preferred.
[0008] Stable d-NTP solutions are a particularly important feature
of this invention. The stability of these solutions appears to be
advantageous especially with regard to an application in the
polymerase chain reaction. As a rule the pH value of the d-NTP
solution is above ca. 7.5 and below ca. pH 11. A pH value between
ca. 8 and 10 proved to be particularly advantageous. The
concentration of the stable d-NTP solution is between 2 mmol/l and
200 mmol/l. A concentration of the d-NTPs of 100 to 150 mmol/l is
particularly preferred.
[0009] It has surprisingly turned out that the stability of the
NTPs in aqueous solution at pH values of more than 7.5 and without
the addition of any stabilizers is higher than in the previously
known solutions which have a pH value of ca. 7.0 to 7.5. The
stability of the d-NTPs in aqueous solution reaches an optimum at a
pH value of ca. 8 to 10. The increase of the pH value does not
cause any additional degradation reactions i.e. the pattern of the
degradation products remains unchanged at the pH values according
to the invention. Surprisingly the degradation reactions proceed
considerably more slowly at higher pH values such as for example
8.3 than at physiological pH values such as for example 7.5.
[0010] Hence it has turned out that at increased pH values no
by-products are formed at all which could impair the use of the
d-NTPs e.g. for the PCR reaction. Even after ca. 90 days at a
temperature of 35.degree. C. the PCR function test is positive. The
higher pH value is uncritical for the PCR itself since most PCR
amplifications are carried out in any case at pH values of more
than 8.0. Hence for example aqueous d-NTP solutions which have a pH
value of more than ca. 7.5 and less than/equal to ca. 11 proved to
be stable on the one hand and advantageous for use in the PCR
reaction. In this case a pH value of the d-NTP solution between 8
and 10 proven to be particularly advantageous.
[0011] The stable NTP solutions according to the invention can be
used for all DNA and RNA synthesizing and DNA and RNA replicating
reactions. In particular the stable NTP solution according to the
invention can also be used for RT-PCR, for nick translation, random
priming and for sequencing (cycle sequencing). Furthermore the
stable NTP solutions according to the invention proved to be
advantageous with regard to a longer duration of use of the NTPs.
That means that the stable solutions according to the invention can
be stored for a considerably longer period than the previously used
d-NTP solutions.
[0012] Figure legends
[0013] FIG. 1: Decrease of the d-GTP content The decrease of the
d-GTP concentration at a temperature of 35.degree. C. was monitored
over a period of 140 days at pH values of 7.5, 7.9 and 8.4.
[0014] FIG. 2: Decrease of the d-CTP content The decrease of the
d-CTP concentration at a temperature of 35.degree. C. was monitored
over a period of 90 days at pH values of 7.5, 7.9 and 8.3.
[0015] FIG. 3: Decrease of the d-TTP content The decrease of the
d-TTP concentration at a temperature of 35.degree. C. was monitored
over a period of 90 days at pH values of 7.5, 7.9 and 8.3.
[0016] FIG. 4: Decrease of the d-UTP content The decrease of the
d-UTP concentration at a temperature of 35.degree. C. was monitored
over a period of 90 days at pH values of 7.5, 7.9 and 8.3.
[0017] FIG. 5: Decrease of the d-ATP content The decrease of the
d-ATP concentration at a temperature of 35.degree. C. was monitored
over a period of 65 days at pH values of 7.5, 7.9 and 8.4.
[0018] FIG. 6: Triphosphate content in relation to the pH value
[0019] FIG. 7: pH dependence of the stability of UTP
[0020] FIG. 8: pH dependence of the stability of UDP
[0021] FIG. 9: pH dependence of the stability of ATP
[0022] FIG. 10: pH dependence of the stability of ADP
[0023] FIG. 11: pH dependence of the stability of
7-deazadeoxy-GTP
[0024] FIG. 12: pH dependence of the stability of
7-deazadeoxy-GTP
[0025] FIG. 13: Dependence of the stability of dATP on the
concentration of the solution at pH=8.3, in which c=100 mmol/l, 10
mmol/l and 2 mmol/l
[0026] FIG. 14: Dependence of the stability of dATP on the
concentration of the solution at pH=8.3; in which c=100 mmol/l, 10
mmol/l and 2 mmol/l
[0027] FIG. 15: Dependence of the stability of dCTP on the
concentration of the solution at pH=8.3, in which c=100 mmol/l, 10
mmol/l and 2 mmol/l
[0028] FIG. 16: Dependence of the stability of dCTP on the
concentration of the solution at pH=8.3, in which c=100 mmol/l, 10
mmol/l and 2 mmol/l
[0029] The invention is further elucidated by the following
examples:
EXAMPLE 1
[0030] Production of a Stable d-NTP Solution According to the
Invention
[0031] d-NTPs were purified by anion chromatography with the aid of
a salt gradient and desalted by reverse osmosis. This is followed
by an ultrafiltration (exclusion limit 1000-5000 D) to remove
DNAses/RNAses. The concentration of the solution is then adjusted
with sterile water to typically 100 mM. The pH value is adjusted to
the corresponding pH value (>7.5) by the addition of bases
(alkali/alkaline earth/ammonium hydroxide; amines) usually
NaOH.
EXAMPLE 2
[0032] Degradation of the Triphosphate at Various pH Values
[0033] d-NTP solutions at a concentration of 100 to 110 mmol/l were
adjusted to pH values between 7.5 and 8.3 with sodium hydroxide
solution. The sample was stored at 35.degree. C., 22.degree. C.,
4.degree. C. and -20.degree. C. Aliquots were removed at various
time points and the purity was examined by means of HPLC. The
relative amount of the tri, di and monophosphate as well as of the
free base was determined by integrating the areas.
[0034] The decrease of the triphosphate content is dependent on pH.
The decrease was slowest at ca. pH 8.3 for all examined nucleotides
(FIG. 1-5).
[0035] I.e. even at higher pH values such as 8.3 no additional
peaks are seen in the HPLC chromatogram which could indicate
decomposition products.
EXAMPLE 3
[0036] Determination of the pH Optimum of the d-NTPs
[0037] d-NTP solutions (dCTP, dTTP, dUTP) at a concentration of 100
to 110 mmol/l were adjusted to pH values between 7.5 and 12 (d-ATP,
dGTP not pH 7.9 and 8.3) with sodium hydroxide solution. The sample
was stressed for 35 days at 35.degree. C. and subsequently the
purity was examined by means of HPLC. The relative amount of the
tri, di and monophosphate as well as of the free base was
determined by integrating the areas.
[0038] For all examined d-NTPs the optimum was in a range between
pH 9.0 and 11.0. Up to pH 12 there was only a slight degradation
(except for d-CTP which is deaminated at pH 12 to form d-UTP) (see
FIG. 6, 7, 8, 9, 10, 11, 12).
EXAMPLE 4
[0039] Calculation of the Stabilization of d-NTPs at pH 8.3
Compared to pH 7.5
[0040] The stabilization was estimated by the following formula
from three independent stress experiments of d-NTPs at pH 8.3 and
7.5 at 35.degree. C. in which samples were taken at intervals
between 7 and 89 days.
.DELTA. content (pH 8.3)-.DELTA. content (7.5).times.100
.DELTA.content (pH 7.5)
[0041] in which .DELTA.content (pH . . . ) =content (t=0)-content
(t)
[0042] This resulted in the following stabilizations for the
individual nucleotides in percent at a pH value of 7.5 compared to
a pH value of 8.3 (table 1):
1TABLE 1 Nucleotide average value maximum value minimum value d-ATP
19% 33% 9% d-CTP 20% 37% 10% d-GTP 21% 30% 12% d-TTP 5% 17% 26%
d-UTP 5% 15% 25%
EXAMPLE 5
[0043] Stabilization at Room Temperature
[0044] After 204 days (20.degree. C.) the difference in the pH
stabilization became apparent (in the real-time model). In the case
of D-ATP solutions the triphosphate content for example decreased
by ca. 7.6% at pH 7.5, by ca. 6.3% at pH 8.3 (difference 17%). In
the case of d-GTP solutions the triphosphate content decreased for
example by ca. 6.8% at pH 7.5 and by ca. 5.2% at pH 8.3 (difference
23%).
* * * * *