CROSS-REFERENCE TO PRIOR APPLICATIONS

This application is the U.S. National Phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2016/082733, filed on Dec. 27, 2016, which claims the benefit of European Patent Application No. 15202803.1, filed on Dec. 28, 2015. These applications are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to methods or kits for purifying nucleic acids using a single wash and elution buffer solution, and more specifically in a miniaturized setting. The present invention further relates to the use of a buffer solution having a low salt concentration and relatively high pH for purifying nucleic acids.

BACKGROUND OF THE INVENTION

Nucleic acids play an important role in many different fields of research. In industrial settings nucleic acids may be detected within product quality management and in clinical settings nucleic acids may be used for diagnosis and/or for therapy. Prior to their use in many of the afore-mentioned applications, nucleic acids need to be purified.

Nucleic acid purification usually comprises several steps such as exposing a sample containing nucleic acids and selectively binding the nucleic acids to a nucleic acid binding phase, purifying the bound nucleic acids from impurities and obtaining the purified nucleic acids. Different buffers are commonly used during sample preparation, nucleic acid binding, washing and elution steps.

The most commonly used purification methods achieve binding/washing and subsequent elution of nucleic acids either based on a change of pH or based on a change of salt concentrations by using different buffers.

Qiagen's ChargeSwitch® technology is for example based on binding and washing negatively charged nucleic acids on a nucleic acid binding phase, which is positively charged when immersed in buffer solution of pH<6.5. For elution of the nucleic acids a buffer solution of pH 8.5 is used, leading to an increase in pH and the neutralization of the positive charge of the nucleic acid binding phase, thus, resulting in release of the bound nucleic acids.

Another commonly used method, which was developed by Boom et al. (EP 0 389 063) is based on lysis and binding of nucleic acids to a nucleic acid binding phase, e.g. diatoms, glass or silica in the presence of high concentrations of a chaotropic salt, e.g. guanidinium thiocyanate, which allow positively charged ions to form salt bridges between the negatively charged nucleic acids and the negatively charged binding phase. Bound nucleic acids are initially washed in the presence of high salt buffer solutions, then with alcohol-water solutions and eluted by switching to a low salt non-alcohol buffer solution such as TE buffer.

As outlined above, current nucleic acid purification methods comprise multiple washing steps followed by an elution step using a multitude of buffers characterized by different salt concentrations and/or a different pH, which render these methods time-consuming and costly.

Furthermore, remains of the binding and washing buffer solutions are sometimes difficult to remove during the final elution step and may subsequently interfere with downstream procedures such as PCR analysis or sequencing reactions.

SUMMARY OF THE INVENTION

There is hence a need for improved means and methods for simplified, fast, low-cost and efficient purification of nucleic acids.

Existing methods for nucleic acid purification are laborious and time-consuming since they comprise multiple washing steps, often using different washing buffers, followed by an ultimate step of eluting the bound nucleic acids using elution buffer.

The present invention solves this problem by providing a method for purifying nucleic acids comprising the steps of

(i) exposing a sample comprising nucleic acids to a nucleic acid binding phase and allowing the nucleic acids to bind to the nucleic acid binding phase,

(ii) washing the nucleic acids bound to the nucleic acid binding phase at least once with a buffer solution comprising a buffering agent,

(iii) eluting the nucleic acids from the nucleic acid binding phase using the buffer solution of step (ii).

An object of the present invention is to achieve simplified and fast purification of nucleic acids in high amounts. In particular, these high amounts generally improves existing nucleic acid purification methods which are generally designed to be carried out in large volumes (elution volumes of ≥50 μl) which might be a disadvantage for certain applications.

According to the present invention, this can be achieved by a method employing a single buffer solution for washing nucleic acids bound to a nucleic acid binding phase and subsequently eluting these nucleic acids from the nucleic acid binding phase.

An advantage of the method according to the present invention is that only one buffer needs to be provided in order to carry out the method, as opposed to different buffers for washing and for eluting the sample, as described in the prior art. This in turns results into a more simplified method as opposed to the prior art, and hence more economical and more reproducible.

According to the present invention, this can also be achieved by using a single buffer solution for washing and eluting nucleic acids, which is characterized by a relatively low salt concentration of ≤100 mM, and a relatively high pH of ≥7.5.

An advantage of the single buffer according to the present invention is that it is possible to achieve high elution efficiency by employing very small volumes of said buffer; moreover, by using one single buffer it is possible to achieve a faster processing of nucleic acids, which is particularly advantageous in a miniaturized environment.

According to the present invention, this simplified and fast purification of nucleic acids can further be achieved by a kit comprising

a nucleic acid binding phase,

a buffer solution comprising a buffering agent for washing and eluting of the nucleic acids or a dry form of said buffer solution,

a means for detecting the purified nucleic acids.

According to the present invention, this simplified and fast purification of nucleic acids can further be achieved by a cartridge comprising

a first chamber comprising a nucleic acid binding phase,

a second chamber, connected to the first chamber, comprising a buffer solution comprising a buffering agent for washing and eluting of the nucleic acids or a dry form of said buffer solution,

a third chamber connected to the second chamber, comprising a buffer solution comprising the buffer solution of the second chamber, or a dry form of said buffer solution.

A kit and a cartridge according to the present invention are advantageous in comparison to the prior art in that they are easier to prepare, and hence more economical, in particular for large scale productions. Moreover, by reducing the number of different ingredients comprised in the kit or in the cartridge, intervariability is also sensibly reduced.

An object of the present invention is to achieve nucleic acid wash and elution from a nucleic acid binding phase using a buffer solution of the present invention, which will allow direct and immediate further processing of the purified nucleic acids after elution, e.g. in amplification or sequencing reactions.

Existing methods for nucleic acid purification are difficult to scale down to be carried out in miniaturized environments such as microfluidic devices.

Therefore, an object of the present invention is to achieve efficient nucleic acid purification in miniaturized environments, e.g. with microfluidic devices or with magneto-capillary valves, wherein the wash and elution steps are performed in ≤500 μl or ≤50μ, respectively.

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiment(s) described herein after.

