Method and microfluidic device for denaturing a nucleic acid

Abstract

A method for denaturing a nucleic acid, comprising the following steps: - Providing (31) a solution (21) of the nucleic acid, - Denaturing (33) the nucleic acid by adding at least one base (23) to the solution (21), and - Adjusting (34) the pH of the solution to a value of less than 7 by adding at least one acid (24) to the solution (21), wherein the acid has a pKa value in the range of 1.0 to 5.0.

Description

The present invention relates to a method for denaturing a nucleic acid. Furthermore, the present invention relates to a microfluidic device which is configured to carry out the method.Prior ArtMethylation of cytosines at the 5'C atom is an epigenetic label that fulfills important regulatory functions. It occurs virtually exclusively in CpG context. This labeling plays a role in, among other things, the regulation of gene expression, embryonic development and cell differentiation. Variations in methylation pattern are associated with a variety of diseases.An analysis of DNA methylations can be carried out by means of bisulfite conversion. Since methylated DNA sequences do not differ in their hybridization behavior from unmethylated DNA sequences and thus no array- or PCR-based methods are possible, the epigenetic information must first be converted into genetic information by chemical reaction. After purification and optional desulphonation, this is subsequently available for further molecular biological methods. For a successful bisulfite conversion, it is decisive that the DNA is denatured, since the bisulfite conversion proceeds optimally on single-stranded DNA molecules.Disclosure of the InventionThe method for denaturing a nucleic acid, in particular DNA, comprises the following steps:providing a solution of the nucleic acid,denaturing the nucleic acid by adding at least one base to the solution; andadjusting a pH of the solution to a value of less than 7 by adding at least one acid to the solution, wherein the acid has a pK S- value in the range of 1.0 to 5.0.This method is suitable in particular for implementation in a microfluidic device. In this application, the use of a base as a denaturing agent is particularly advantageous. Denaturation of a nucleic acid could also be achieved by incubation at high temperatures up to 98°C. In such a procedure, heating is usually carried out again, even during the later bisulfite conversion, to high denaturation temperatures of up to 98° C., in order to avoid formation of DNA duplexes. A great problem of these high temperatures is, however, a high loss of yield of nucleic acid during bisulfite conversion and subsequent purification, since the nucleic acid is often too severely degraded. Furthermore, bisulfite conversion always represents a compromise between the complete conversion of cytosines to avoid false positive results and the avoidance of the conversion of methylated cytosines, which would lead to false negative results. As a rule, longer bisulfite conversions at higher temperatures lead to more complete conversions of cytosines, but also to more undesired conversions of methylated cytosines.In addition to these basic problems of denaturation by means of high temperatures, their integration in microfluidic devices is problematic in that only limited temperatures can be reached in these devices. This is due firstly to the limited temperature resistance of the plastic components used and secondly to a limited heating power, in particular in large compartments in which relatively large volumes have to be treated, as is required, for example, in bisulfite conversion.However, the chemical denaturation of the nucleic acid by addition of at least one base can be carried out at lower temperatures of, for example, 37° C. In the context of this method, addition is understood to mean combining the solution of the nucleic acid with a further substance. Addition is therefore to be understood in particular not only as introducing the base into the solution of the nucleic acid, but also as introducing the solution of the nucleic acid into the base.Upon completion of denaturation, the solution has an alkaline pH greater than 7. Since bisulfite conversion proceeds under acidic conditions, the subsequent adjustment of the pH of the solution to a value of less than 7 is contemplated. In principle, a strong acid such as hydrochloric acid could be used to adjust the pH. This could be added as a concentrated aqueous acid or in the form of a salt such as ammonium chloride which releases hydrogen chloride by dissociation. However, such strong acids would damage the plastic components of a microfluidic device and are therefore not provided in the method. An acid having a pK S- value in the range of 1.0 to 5.0, preferably in the range of 3.0 to 5.0, is, on the other hand, suitable for use in the process.The base is preferably a solution of sodium hydroxide and / or sodium bicarbonate. These bases are suitable for denaturing nucleic acids. While sodium hydroxide is a strong base, by means of which a particularly rapid denaturation of the nucleic acid can be achieved, the more weakly basic sodium bicarbonate can be advantageous from a safety aspect.In principle, the acid can be an inorganic acid. This is selected in particular from the group consisting of orthophosporic acid and metaphosporic acid. In addition, the acid may be an anhydride of an inorganic acid, especially phosphorus pentoxide. Preferably, however, the acid is an organic acid.Organic acids or carboxylic acids do not completely dissociate in contact with water and therefore usually have the high pK S- values advantageous for the process. Suitable organic acids are selected in particular from the group consisting of oxalic acid, glyoxylic acid, acetic acid, pyruvic acid, malonic acid, maleic acid, succinic acid, ascorbic acid, fumaric acid, acrylic acid, citric acid and gallic acid. Toxic organic acids, such as chloroacetic acid or formic acid, are preferably not used for safety reasons.It is further preferred that the organic acid contains a carbon-carbon double bond. Such organic acids function at the same time as reducing agents. Since they remain in the solution of the nucleic acid in bisulfite conversion subsequent to the adjustment of the pH, they can prevent nucleic acid degradation therein after the bisulfite reaction. Organic acids having a carbon-carbon double bond suitable for the process are selected in particular from the group consisting of maleic acid, fumaric acid, acrylic acid and ascorbic acid.Furthermore, it is preferable that the organic acid has a melting point of at least 40° C. Since denaturation of the nucleic acid can be carried out by addition of the base at a temperature of less than 40°C, such acids are present as solids in a microfluidic device. Since bisulfite conversion is volume-dependent and depends on maintaining the volume ratios of the reagents used and the nucleic acid solution to be converted, it is advantageous if the organic acid used does not take up a large proportion by volume of the reaction mixture. A solid acid can be stored in front as a solid and thus requires little volume. In addition to some organic acids, the preferred inorganic acids orthophosphoric acid and metaphosphoric acid also have such high melting points. The bringing together of the nucleic acid solution and the acid is preferably carried out when using a solid acid by passing the solution of the nucleic acid into a reaction chamber which contains the solid acid. This makes it possible to adjust the pH of the solution without the need to add further liquid to it.If, on the other hand, the acid is to be combined with the nucleic acid solution as an aqueous solution, then it is preferred that it is passed as an aqueous solution having a concentration in the range from 0.001 mol / l to 0.100 mol / l into a reaction chamber which contains the solution of the nucleic acid. In this concentration range, it is possible to adjust the pH of the solution to a value of less than 7 without thereby increasing the volume of the solution unnecessarily much.Polyurethane (PU) and polycarbonate (PC) are common plastic components of microfluidic devices which come into contact with the chemicals circulating therein. Polyurethane has amide bonds which can be hydrolyzed at low pH values. Polycarbonate has ester linkages which can be hydrolyzed at low pH values. It is therefore preferred that the pH of the solution is adjusted to a value greater than 3.Following the adjustment of the pH, bisulfite conversion of the nucleic acid is carried out in particular. This can proceed in particular in the same microfluidic device as the denaturation of the nucleic acid.In a further aspect, the invention relates to a microfluidic device in which at least one base and at least one acid having a pK S- value in the range from 1.0 to 5.0 are arranged upstream in separate chambers. The microfluidic device, which can also be referred to as lab-on-chip, is configured to carry out the method.Brief Description of the DrawingsExemplary embodiments of the invention are illustrated in the drawings and are explained in more detail in the following description. FIG. 1 shows a partial region of a microfluidic device according to an exemplary embodiment of the invention. FIG. 2 shows a flow diagram of a method according to an exemplary embodiment of the invention. FIG. 3 shows a partial region of a microfluidic device according to another exemplary embodiment of the invention.Embodiments of the InventionA microfluidic device 10 according to a first embodiment of the invention is configured to examine a sample solution containing DNA by bisulfite conversion. In this way, for example, promoter hypermethylations of tumor suppressors can be recognized as methylation markers which indicate a cancer disease. As shown in FIG. 1, a portion of microfluidic device 10 includes five chambers 11- 15. In this partial area, a method according to an exemplary embodiment of the invention can run, the steps of which are shown in FIG. 2 :After the start 30 of the method, a solution 21 of the nucleic acid DNA is provided 31 in the first chamber 11. In order to denature the nucleic acid, the solution 21 is pumped 32 into the second chamber 12. In a third chamber 13, sodium hydroxide solution having a concentration of 3 mol / l is placed upstream as base 23. Denaturation 33 of the nucleic acid takes place by pumping the base 23 from the third chamber 13 into the second chamber 12 and reacting it there with the nucleic acid at a temperature of 37° C. The sodium hydroxide solution has a pH of 13.5, which is only insignificantly lowered in the denaturation reaction. This pH is lowered to a value of 5 in the next step 34 by pumping an acid 24 into the second chamber 12. This acid 24 is 96% acetic acid, which is upstream of the fourth chamber 14. After adjusting 34 the pH, the reaction mixture is pumped from the second chamber 12 into the fifth chamber 15. In this fifth chamber 15, a mixture of reagents 25 for carrying out bisulfite conversion 35 is placed upstream. The chemical process of bisulfite conversion is shown below: In reaction step I, a cytosine of the denatured DNA strand is sulfonated at the C6 position. In reaction step II, irreversible hydrolytic deamination occurs at the C4 position, resulting in a 6-sulfonyluracil. Subsequently, a further base, not shown, is introduced into the fifth chamber 15, so that in a third reaction step III, an alkaline desulphonation takes place. Unmethylated cytosine is converted to uracil in this manner. Methylation at the C5 position of the cytosine, on the other hand, prevents sulfonation at the C6 position in reaction step I, so that the methylcytosine remains unchanged in bisulfite conversion. In this way, bisulfite conversion makes it possible to distinguish cytosine and methylcytosine from one another in one DNA strand.After carrying out the bisulfite conversion, the method is ended 36. Further purification steps and analysis steps can follow in other parts of the microfluidic device, not shown.In a second exemplary embodiment of the microfluidic device 10, which is illustrated in FIG. 3, a different arrangement of the five chambers 11- 15 is present. The arrangement of the first three chambers 11- 13 corresponds to the arrangement in the first exemplary embodiment of the microfluidic device, and the method steps 31 to 33 proceed in these chambers in the same manner as in the first exemplary embodiment. However, unlike in the first exemplary embodiment, the fourth chamber 14 is arranged between the second chamber 12 and the fifth chamber 15. The acid 24 is stored therein in the form of solid ascorbic acid. The pH is adjusted 34 in this exemplary embodiment of the invention by pumping the solution 21 of denatured nucleic acid from the second chamber 12 into the fourth chamber 14. There, the solid acid 24 dissolves in the solution 21, whereby the desired pH value of 5 is established. Subsequently, the solution 21 is pumped further into the fifth chamber 15. As in the first exemplary embodiment of the invention, reagents 25 for carrying out bisulfite conversion 35 are arranged upstream in fifth chamber 15, and the further steps of the method proceed there in the same manner as in the first exemplary embodiment. The ascorbic acid contained in the solution 21, however, participates as a reducing agent in bisulfite conversion and prevents there DNA degradation as an undesirable side reaction after the bisulfite reaction.

Claims

A method for denaturing a nucleic acid, comprising the following steps: - providing (31) a solution (21) of the nucleic acid, - denaturing (33) the nucleic acid by adding at least one base (23) to the solution (21), and - adjusting (34) a pH value of the solution to a value of less than 7 by adding at least one acid (24) to the solution (21), wherein the acid has a pK S- value in the range from 1.0 to 5.0.Method according to claim 1, characterized in that the base (23) is a solution of sodium hydroxide and / or sodium hydrogen carbonate.The method of claim 1 or 2, characterized in that the acid (24) has a pK S- value in the range of 3.0 to 5.0.Method according to any one of claims 1 to 3, characterized in that the acid (24) is an organic acid.Method according to claim 4, characterised in that the acid (24) contains a carbon-carbon double bond.Method according to any one of claims 1 to 5, characterized in that the acid (24) has a melting point of at least 40°C.Method according to claim 6, characterised in that the pH is adjusted (34) by passing the solution (21) of the nucleic acid into a reaction chamber (14) containing the solid acid (24).Method according to one of Claims 1 to 5, characterized in that the acid (24) is fed as an aqueous solution having a concentration in the range from 0.001 mol / l to 0.100 mol / l into a reaction chamber (12) which contains the solution (21) of the nucleic acid.Process according to any one of claims 1 to 8, characterised in that the pH of the solution is adjusted to a value of more than 3.Method according to one of Claims 1 to 9, characterized in that, following the adjustment (34) of the pH, bisulfite conversion (35) of the nucleic acid is carried out.Microfluidic device (10), in which at least one base (23) and at least one acid (24) having a pK S- value in the range from 1.0 to 5.0 are arranged upstream in separate chambers (11-15), and which is configured to carry out a method according to one of Claims 1 to 10.

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