MXPA99004392A - Stabilized aqueous nucleoside triphosphate solution - Google Patents

Abstract

The invention relates to stable aqueous solutions containing one or more nucleoside triphosphates, wherein each solution has a pH value higher than 7.5 and no additional stabilizing substances. The nucleoside triphosphate solutions are specially used for DNA synthesizing reactions, for instance Rt-PCR, cycle sequencing, random priming and nick translation. One of the most important applications of corresponding solutions containing deoxy-nucleoside triphosphate (d-NTP) is their use in the polymerase chain reaction (PCR).

Description

STABILIZED AQUATIC SOLUTIONS OF N CLEOSIDE TRIFOSPHATE FIELD OF THE INVENTION The invention relates to stable aqueous solutions containing nucleoside triphosphates in which the solution has a pH value greater than 7.5.

BACKGROUND OF THE INVENTION Nucleoside triphosphates (NTP) such as ribonucleoside triphosphates, deoxynucleoside and dideoxynucleoside have a variety of uses in the field of biochemistry and molecular biology. Most applications refer to reactions which synthesize or replicate DNA and RNA such as reverse transcriptase polymerase chain reaction (RT-PCR), cycle sequencing and flexion translation. In the case of RT-PCR, the DNA strands are synthesized in the 5 '-3' direction in for example the reverse transcriptase wherein one strand of RNA serves as the template. Certain NTPs such as the triphosphates of REF .: 30158 dideoxynucleosides as terminator chains in the DNA sequence. One of the most important applications of deoxynucleoside triphosphates (d-NTP) is its use in the polymerase chain reaction (PCR). In this application it is absolutely essential that the previous NTP solutions are all stable during storage. The d-NTPs (d-ATP, d-CTP, d-GTP, d-TTP, d-UTP and others) are usually stored as Na (sodium) or Li (lithium) salts and are available in this form. typically at concentrations of 0.1 mol / 1. As a rule, the pH values are physiological pH values that is, between approximately 7.0 and 7.5.

DESCRIPTION OF THE INVENTION A disadvantage of the current, that is, of the commercially available NTP solutions is in particular in the instability of the NTPs during storage or thermal stress. The NTPs have a tendency to decompose with time to form the corresponding monophosphates and diphosphates. The triphosphate content decreases especially at high temperatures. The triphosphate content decreases by about 2-3% within ten days at a pH value of about 7.5 and at a temperature of 35 ° C. In contrast to room temperature, the triphosphate content is observed by decreasing only about 1% after seven weeks. Therefore, the decomposition of the triphosphates in aqueous solutions 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 the dNTP solutions. However, there is a need for aqueous solutions which only contain dNTP and are at high concentrations and which have greater long-term stability than currently available solutions.

In order to improve the stability of the triphosphates, only solutions of corresponding adenosine triphosphates have now been reported. The stability of 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 the adenosine triphosphate in the 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 / DERWENT 66-11664F). They are described in the presence of stabilizers such as • guanidine / amino-guanidine or creatinine a pH value of 9 to 10 (JP 71/038270 / DERWENT 71-722169 and JP 71/033592 / DERWENT 71-631879), in the presence of methionine as a stabilizer a pH value of preferably 9 to 10.5 (JP 67/019115 / DERWENT 66-29019F), in the presence of the stabilizing phosphate and sorbitol / anitol / glycerol / benzoyl alcohol / PEG a pH value of 8 to 11 (JP 67/015115 / DERWENT 6-28392F) and in the presence of glycerol / H3P04 a pH value of 3.7 (FR4078 / DERWENT 66-22085F). However, the presence of stabilizers in d-NTP solutions can be critical for many applications or cause interference.

A so-called, already prepared case (Pharmacia) is known for the labeling of random radioactive preparations. In this kit, all the components of the reaction are pre-mixed and already with pre-aliquots for mixtures of individual tests and stabilized in dry (crystallized). However, one disadvantage of this form is that there is no flexibility with respect to the size of the test mix, in addition, the time required to dissolve the "crystallized" components in which the process is completed, is slow and makes it less reproducible .

In European Patent EP 0 049 909, the object is to provide all the components of the reaction of a kit for the labeling of nucleic acids already mixed in a liquid form, however, the storage stability of the mixture is only made by the addition of glycerol as a stabilizer.

Accordingly, the object of the present invention is to provide an aqueous stabilized solution containing NTPs without the addition of any stabilizer.

The object was made in accordance with the invention by the aqueous solutions of NTP having a pH value of approximately above 7.5. These nucleoside triphosphates include ribonucleoside triphosphates, deoxynucleotides and dideoxynucleotide triphosphates wherein all five occur naturally as well as modified bases such as isoguanine, deacety compounds and derivatives thereof as bases. In addition, nucleoside triphosphates can be labeled with reporter groups. As a rule, the solutions according to the invention have a pH value in the range of more than 7.5 to a maximum of 11. A pH value of about 8 to 10 will be provided as being particularly advantageous. The pH value can be regulated by adding a base (for example, NaOH, KOH, LiOH) as well as by the addition of a buffer (for example Tris buffer, Na buffer, phosphate buffer).

The concentration of the NTP solution is preferably about 2 mmol / L and 200 mmol / L. Particularly preferred is a concentration of the NTPs of about 100 to 150 mmol / l.

Stable d-NTP solutions are a particularly important feature of this invention. The stability of these solutions seems to be especially advantageous with respect to an application in the reaction of the polymerase chain. As a rule, the pH value of the d-NTP solution is above about 7.5 and below about 11. A pH value between about 8 and about 10. The concentration of the stable solution is provided as being particularly advantageous. between 2 mmol / l and 200 mmol / l. A particularly preferred concentration of the d-NTPs is 100 to 150 mmol / L.

It has been surprisingly surprising that the stability of NTPs in an aqueous solution at pH values of more than 7.5 and without the addition of any stabilizer is higher than in previously known solutions which have a pH value of about 7.0 to 7.5. The stability of the d-NTPs in the aqueous solution reaches an optimum at a vapor pH of about 8 to 10. The increase in the pH value does not cause any further degradation reaction ie the model of the degradation products remains unchanged at 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 values such as for example 7.5.

Consequently, it has become that at an increased pH value no by-products or derivatives are formed in which all may deteriorate the use of the d-NTPs for example, for the PCR reaction. Even after approximately 90 days at a temperature of about 35 ° C the PCR function test is positive. The higher pH value is not critical for the same PCR test since more amplifications are performed in some cases at pH values of more than 8.0. However, for example, aqueous d-NTP solutions which have a pH value of more than about 7.5 and less than / equal to about 11 are provided to be stable in one way and advantageous for use in the PCR reaction. In this case, a pH value of the d-NTP solution between 8 and 10 is particularly advantageous.

The stable NTP solutions according to the invention can be used for all DNA and RNA synthesis and DNA and RNA replication reactions. In particular, the stable NTP solution according to the invention can be used for RT-PCR, for nick translation or translation: random preparation and for the sequences (sequence cycle). In addition, the stable NTP solutions according to the invention are provided as being advantageous with respect to a longer duration of use of the NTPs. This suggests that the stable solutions according to the invention can be stored for a considerably longer period than the previously used d-NTP solutions.

Legend of the Figures Figure 1: Decrease in D-GTP content. The decrease in d-GTP concentration at a temperature of 35 ° C was monitored over a period of 140 days at pH values of 7.5, 7.9, and 8.4.

Figure 2: Decrease in d-CTP content The decrease in d-CTP concentration at a temperature of 35 ° C was monitored over a period of 90 days at pH values of 7.5, 7.9 and 8.3.

Figure 3: Decrease in d-TTP content The decrease in d-TTP concentration at a temperature of 35 ° C was monitored over a period of 90 days at pH values of 7.5, 7.9 and 8.3.

Figure: Decrease in d-UTP content The decrease in d-UTP concentration at a temperature of 35 ° C was monitored over a period of 65 days at pH values of 7.5, 7.9 and Figure 5: Decrease in d-ATP content The decrease of the concentration d-ATP 'at a temperature of 35 ° C was monitored during a period of 65 days at pH values of 7.5, 7.9 and 8.3.

Figure 6: Triphosphate content in relation to the pH value Figure 7: pH dependence of UTP stability Figure 8: pH dependence of UDP stability Figure 9: pH dependence of ATP stability Figure 10: pH dependence of ADP stability Figure 11: pH dependence of the stability of 7- desaza-desoxy-GTP Figure 12: pH dependence of the stability of 7-desaza-deoxy-GTP Figure 13: pH dependence of the stability of dATP in the concentration of the solution at pH 8.3, in which c = 100 mmol / l, 10 mmol / l and 2 mmol / l.

Figure 14: pH dependence of the stability of dATP in the concentration of the solution at pH 8.3, in which c = 100 mmol / l, 10 mmol / l and 2 mmol / l.

Figure 15: pH dependence of the stability of dCTP in the concentration of the solution at pH 8.3, in which c = 100 mmol / l, 10 mmol / l and 2 mmol / l.

Figure 16: pH dependence of the stability of dCTP in the concentration of the solution at pH 8.3, in which c = 100 mmol / l, 10 mmol / l and 2 mmol / l.

The invention is further demonstrated by the following examples: Example 1: Production of a stable d-NTP solution according to the invention.

The d-NTPs were purified by anion chromatography with the aid of a salt gradient and desalted by reverse osmosis. This was followed by ultrafiltration (exclusion limit 1000-5000 D) to remove DNAses / RNAses. The concentration of the solution was then adjusted with sterile water to typically 100 mM. The pH value was adjusted to the corresponding pH value (> 7.5) by the addition of bases (alkali / alkaline earth metal / ammonium hydroxide; amines) usually NaOH.

Example 2: Degradation of triphosphate at several pH values The d-NTP solutions were adjusted to pH values between 7.5 and 8.3 with a sodium hydroxide solution at a concentration of 100 to 110 mmol / l. The sample was stored at 35 ° C, 22 ° C, 4 ° C and -20 ° C. The aliquots were removed at various time points and the purity was examined by means of HPLC. It was determined by the integration of the areas, the relative amount of the tri, di and monophosphates as well as the free bases.

The decrease in the triphosphate content is pH dependent. The decrease was slowly to approximately pH 8.3 for all nucleotides examined (Figure 1-5).

That is, even at high pH values such as 8.3 no additional peaks are observed in the HPLC crnatogram which may indicate decomposition products.

Example 3: Determination of the optimum pH of the d-NTPs The d-NTP solutions (dCTP, d TTP, dUTP) adjusted to pH values between 7.5 and 12 (d-ATP, d-GTO without pH 7.9 and 8.3) at a concentration of 100 to 110 mmol / l. The sample was subjected to stress for 35 days at 35 ° C and the purity was subsequently examined by means of HPLC. It was determined by the integration of areas, the relative amount of tri, di and monophosphate as well as the free base.

Pata all d-NTP examined the optimum was in the range between pH 9.0 and 11.0. Up to pH 12 there was only slight degradation (except for d-CTP which was demeaned to pH 12 to form d-UTP) (see figure 6, 7, 8, 9, 10, 11 and 12).

Example 4: Calculation of the stabilization of d-NTPs at pH 8.3 compared to a pH 7.5 The stabilization was estimated by the following formula from three independent experiments subjected to stress, from d-NTPs at pH 8.3 and 7.5 at 35 ° C in which the samples were taken at intervals between 7 and 89 days. content ? (pH 8.3) - content? (7.5) x 100 content? (pH 7.5) in which, he contended? (pH = content (t = 0) -content (t) This results 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): Table 1: Example 5: Stabilization at room temperature After 204 days (20 ° C) the difference in pH stabilization became apparent (in the real-time model). In the case of the d-ATP solutions the triphosphate content for example decreased by approximately 7.6% at pH 7.5, by approximately 6.3% at pH 8.3 (17% difference). In the case of the d-GTP solutions, the triphosphate content decreased, for example, by approximately 6.8% at pH 7.5 and by approximately 5.2% at pH 8.3 (23% difference).

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the manufacture of the objects to which it refers. Having described the invention as above, the content of the following is claimed as property.

Claims (13)

1. A st aqueous solution, containing nucleoside triphosphates, characterized in that the pH value of the solution is above about 7.5, the solution is free of additional stabilizing agents and the PCR function test is positive after about 90 days at a time. temperature of 35 ° C.

2. A st aqueous solution according to claim 1, characterized in that the nucleoside triphosphates are triphosphates of modified nucleosides.

3. St aqueous solution according to claim 1 or 2, characterized in that the pH value is in the range between 7.5 and 11.

4. A st aqueous solution according to claim 1, 2 or 3, characterized in that the concentration of the nucleoside triphosphate is from about 2 to 200 mmol / l.

5. A st aqueous solution according to one of claims 1 to 4, characterized in that the solution contains deoxynucleoside triphosphates.

6. St aqueous solution as claimed in claims 1 to 5, characterized in that it contains a substance which absorbs at or above a pH 7.5

7. Use of a st aqueous solution as claimed in claims 1 to 6, for a DNA and / or RNA synthesizing reaction.

8. Use of a st aqueous solution as claimed in claims 1 to 6, to replicate fragments or DNA and / or RNA sequences.

9. Use of a st 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.

10. Use of a st aqueous solution as claimed in claims 1 to 6 for the sequence of the nucleic acid cycle.

11. Use of a st aqueous solution as claimed in claims 1 to 6 for the specific replication of deoxynucleic acid sequences or fragments.

12. Use of a st aqueous solution as claimed in claims 1 to 6 for the random preparation.

13. Use of a st aqueous solution as claimed in claims 1 to 6 for nick translation or translation.