Method for purifying solution comprising polynucleotide

Abstract

The present invention relates to methods of purifying solutions comprising newly synthesized polynucleotides, in particular in the field of polynucleotide synthesis, such as enzymatic polynucleotide synthesis. The invention also relates to a device configured to carry out said purification process.

Description

Technical Field

[0001] The present invention relates to a method for purifying a solution containing newly synthesized polynucleotides, in particular in the field of polynucleotide synthesis (eg enzymatic polynucleotide synthesis). The present invention also relates to an apparatus configured to implement the purification method and a kit thereof.

[0002] introduction

[0003] Polynucleotide synthesis is increasingly used in research and medicine to provide therapeutic and diagnostic solutions. Examples include genomic and diagnostic sequencing, multiplex nucleic acid amplification, therapeutic antibody development, synthetic biology or nucleic acid amplification.

[0004] Polynucleotide synthesis can be accomplished chemically or enzymatically. Both are typically performed by transferring a liquid solution from filter plate to filter plate, which are themselves transferred from workstation to workstation, to separate the newly synthesized polynucleotides from the solid support used during synthesis and possibly from contaminants.

[0005] More specifically, polynucleotide synthesis is carried out on the solid support in the first dedicated filter plate. Once the synthesis step is completed, the newly synthesized polynucleotide attached to the solid support is transferred from the first filter plate to the second filter plate (also referred to as the transfer plate) to release the polynucleotide from the solid support. Finally, the liquid solution containing the released polynucleotide is transferred to the third filter plate (referred to as the desalting plate) to precipitate and purify the polynucleotide from pollutants. Therefore, those skilled in the art usually use at least three filter plates and as many transfer steps.

[0006] While this approach is effective, it is still costly because a larger number of filter plates and other consumables are used, which are more expensive, less durable, and generate more waste.

[0007] In addition, the large number of transfer steps, cleaning steps and purification steps may produce a smaller amount of purified synthetic polynucleotides. In addition, it also increases the risk of external contamination. Moreover, this requires breaking the vacuum several times during the process, resulting in delays and possible material losses.

[0008] The object of the invention is to overcome at least one of the aforementioned shortcomings, and to further produce other advantages by proposing a novel method for purifying liquid solutions, the method comprising directly newly synthesizing polynucleotides in the first filter plate, eliminating the need for any liquid solution of the transfer of newly synthesized polynucleotides from sample loading to recovery. At the same time, such a method greatly reduces the risk of external contamination in the liquid solution, and sample loss, and is a more environmentally friendly method. In addition, the method increases the amount of the polynucleotides recovered. The inventors must overcome the following fact, that is, there are many impurities in the plate, and they need to ensure that impurities are eliminated, because they cannot eliminate impurities in the process of transferring to another plate. And, must optimize and select reagents, because precipitation occurs in the same plate, and therefore need to optimize the ratio of buffer and chaotropic agent. Summary of the invention

[0009] The present invention relates to a method for purifying a solution comprising polynucleotides and contaminants, the solution being contained in a well of a filter plate, the well being provided with a filter, the method comprising:

[0010] - a step of adding a chaotropic agent to the solution contained in the wells to obtain a mixture,

[0011] - a step of incubating the mixture so as to obtain in the wells a precipitate and a supernatant, the precipitate comprising the polynucleotides and the supernatant comprising at least a portion of the contaminants,

[0012] - a step of applying a differential pressure to the wells to remove the supernatant through the filter, the precipitate being retained by the filter,

[0013] - a step of adding a low-salt eluent to dissolve the polynucleotides in the precipitate and transferring the polynucleotides dissolved in the low-salt eluent to a container,

[0014] wherein said steps of said method are performed in a well equipped with a filter,

[0015] The method is performed in a well equipped with a filter, and the method comprises a step of enzymatically synthesizing a polynucleotide prior to the step of adding a chaotropic agent, the step of enzymatically synthesizing a polynucleotide being performed in the well in which the step of adding a chaotropic agent is performed.

[0016] Here, and in everything that follows, it is understood that a filter plate means a plate comprising a plurality of wells, each well being equipped with a filter arranged at the bottom of the well.

[0017] The method for purifying polynucleotides and contaminants contained in a solution according to the present invention comprises at least four steps, all of which are carried out in the same well of a filter plate. Therefore, it is no longer necessary to transfer the solution from one filter plate to another. By eliminating this transfer, the risk of polynucleotide loss is greatly reduced.

[0018] In one or more embodiments, the mixture can be agitated during the second step of incubation. Optionally, the agitation is accomplished by shaking the filter plate.

[0019] The steps of enzymatically synthesizing polynucleotides, adding chaotropic agents, incubating the mixture, applying differential pressure, and adding low-salt eluent are performed in the same hole of the filter plate. Therefore, the method of the present invention allows the synthesis, purification, and separation of polynucleotides to be performed in the same hole of the filter plate. This allows purification to be performed directly after synthesis without any transfer.

[0020] In one or more embodiments, the step of enzymatically synthesizing a polynucleotide comprises:

[0021] a) providing an initiator having a 3'-terminal nucleotide containing a free 3'-hydroxyl group,

[0022] b) a substep of contacting the initiator having a free 3'-O-hydroxyl group with a 3'-blocked nucleotide triphosphate and a polymerase under elongation conditions such that the initiator is elongated by incorporation of the 3'-blocked nucleoside triphosphate to form a 3'-blocked extension segment

[0023] c) a substep of unblocking the extension fragment to form an extension fragment having a free 3'-hydroxyl group, and

[0024] d) repeating sub-steps b) and c) until the sub-step of forming a polynucleotide,

[0025] e) The sub-step of cleaving the polynucleotide from the initiator.

[0026] It will be appreciated that each incorporation of a nucleoside triphosphate to extend the initiator constitutes one cycle.

[0027] In one or more embodiments, the substep of cleaving the polynucleotide from the initiator is performed by enzymatic digestion, cleavage by photo-irradiation, and / or by chemical cleavage.

[0028] In one or more embodiments, sub-step d) of repeating sub-steps b) and c) is performed by contacting the extended fragment obtained in step c) under elongation conditions until a polynucleotide is formed.

[0029] Examples of enzymatic polynucleotide synthesis are described, for example, in International Patent Publication No. WO 2015 / 159023 to Ybert et al.

[0030] In one or more embodiments, the method includes the step of adding beads before the step of incubating the mixture to help precipitate the mixture. Therefore, the beads can be added to the solution before or simultaneously adding the chaotropic agent. Alternatively, the beads can be added to the mixture after adding the chaotropic agent. In any case, chaotropic agent and beads are added to the same hole to obtain a mixture.

[0031] In one or more embodiments, when the method includes a step of enzymatically synthesizing a polynucleotide, the step of adding beads is preferably performed after the synthesis step.

[0032] In one or more embodiments, the beads are glass beads, preferably silica glass beads.

[0033] In one or more embodiments, the beads are hollow glass beads, preferably silica hollow glass beads.

[0034] In one or more embodiments, the beads have a diameter of 9 microns to 13 microns.

[0035] In one or more embodiments, the pore size of the filter of the well is smaller than the diameter of the beads. Therefore, the beads cannot pass through the filter of the well. It should be understood that in the context of the present invention, when the method includes a step of enzymatically synthesizing polynucleotides before the step of adding a chaotropic agent, at least a first type of beads, preferably agarose beads, can be used as a support for performing the enzymatic synthesis; and a second type of beads, preferably glass beads, can be used to precipitate the mixture according to the present invention, both types of beads being retained by the filter of the well.

[0036] In one or more embodiments, the filter preferably has a pore size comprised between 0.45 μm and 1.2 μm.

[0037] In one or more embodiments, the filter plate preferably comprises 96 wells or 384 wells.

[0038] In one or more embodiments, the chaotropic agent is selected from the group consisting of: an isopropanol solution, a sodium iodide solution, a sodium perchlorate solution, and a combination thereof. Preferably, the chaotropic agent is an isopropanol solution.

[0039] In one or more embodiments, the step of incubating the mixture to obtain a precipitate is preferably performed at room temperature, and preferably for at least 10 minutes.

[0040] In one or more embodiments, a precipitate and a supernatant are formed in the well by incubating the mixture, the supernatant comprising at least a portion of the contaminants, and the precipitate comprising the polynucleotides and optionally another portion of the contaminants.

[0041] In one or more embodiments, the method includes the step of washing the precipitate to remove contaminants from the precipitate.

[0042] In one or more embodiments, the method comprises:

[0043] - a step of washing the precipitate with a washing solution in order to dissolve the contaminants that may be present in the precipitate, and

[0044] - a step of applying a differential pressure to the pores to remove the washing solution comprising dissolved contaminants of the precipitate through the filter,

[0045] The step of washing the precipitate and the step of applying the differential pressure are performed after the supernatant is removed by the filter, that is, after the step of applying the differential pressure.

[0046] In one or more embodiments, the wash solution is an ethanol solution, preferably comprising 70% to 80% ethanol by volume.

[0047] In one or more embodiments, the length of the at least one newly synthesized polynucleotide is at least 10 nucleotides long, preferably at least 17 nucleotides long.

[0048] The present invention also relates to any kit configured to carry out the method according to any one of the embodiments of the present invention, said kit comprising:

[0049] - Chaotropic solution

[0050] - Washing solutions and / or buffer solutions for purification

[0051] - Elution solution and / or buffer to recover the purified polynucleotide

[0052] -optionally, an instruction manual,

[0053] -Optionally, a bead solution.

[0054] In one or more embodiments, the kit is configured to perform the enzymatic synthesis and methods of the invention, wherein the kit further comprises:

[0055] - at least one reaction medium comprising nucleic acid fragments made of nucleotides,

[0056] - an enzymatic nucleotide addition reagent, a nucleotide or a combination of nucleotides suitable for addition by said enzymatic addition reagent,

[0057] - at least one washing solution and / or at least one buffer solution for use in the purification phase of the enzymatic synthesis,

[0058] - At least one amplification reaction medium comprising at least one enzymatic nucleic acid amplification reagent and natural nucleotides suitable for use by the enzymatic amplification reagent.

[0059] The invention also relates to an apparatus configured to implement a method according to any one of the preceding embodiments.

[0060] In one or more embodiments, the apparatus comprises a synthesis station and a recovery station, wherein the synthesis station comprises a filter plate located above a pressure regulator and / or a shaker station.

[0061] In one or more embodiments, the apparatus may further include a cooling or heating station configured to be located below the plate.

[0062] Further characteristics and advantages of the invention will become apparent from the following description, on the one hand, and from several embodiments given by way of indication and not limitation with reference to the accompanying schematic drawings, on the other hand.

[0063] BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This represents an embodiment of the invention which has no washing steps in the wells of the filter plate and comprises a final step of applying a differential pressure to the wells to recover the purified polynucleotide.

[0065] Figure 2 Another embodiment of the invention is represented, which comprises an additional step of adding a wash solution to the wells of the filter plate, and a final step of applying a differential pressure to the wells to recover the purified polynucleotides.

[0066] Figure 3 The yield distribution corresponding to the amount of DNA recovered in pmol per well of the filter plate is represented.

[0067] Figure 4 Represents the DNA / salt ratio distribution corresponding to the salt contamination rate in each well of the filter plate.

[0068] Figure 5 Represents the DNA / protein ratio distribution corresponding to the protein contamination rate in each well of the filter plate.

[0069] Fig. 6A This is a gel image showing the effect of purity measured by OP2.

[0070] Figure 6B The purity estimate of the %N bands used for the synthetic oligonucleotides is indicated. DETAILED DESCRIPTION OF THE INVENTION

[0072] definition

[0073] "Polynucleotide" is a polymer composed of nucleotide monomers covalently linked to form a chain or fragment of a nucleic acid chain (e.g., DNA or RNA). In the context of the present invention, "polynucleotide" and "polynucleotides" are interchangeable and have the same meaning and scope.

[0074] In the context of the present invention, "synthetic polynucleotide" means an extended polynucleotide sequence obtained by an enzymatic synthesis method (eg WO 2015 / 159023) until the target polynucleotide is obtained.

[0075] Method of the present invention

[0076] Typically, purification of a liquid solution containing a polynucleotide of interest is performed using several workstations and plates to extract and ultimately recover the purified polynucleotide of interest.

[0077] However, for ecological or economical purposes, it is desirable to reduce the number of workstations and plates and to improve the automation of high-throughput polynucleotide extraction using basic hardware modules: differential pressure devices (e.g., vacuum), stirrers, distribution and plates. Improving the automation of high-throughput polynucleotide extraction requires simplifying existing devices so that more and more polynucleotides can be extracted in a shorter period of time. However, the simplification of existing devices first requires changing the purification method and secondly requires overcoming the technical difficulties arising from the changes in the prior art methods. This is the purpose of the present invention, which aims to limit the transfer of material from one plate to another.

[0078] Furthermore, in a first aspect, the present invention provides a method for purifying a solution comprising polynucleotides and contaminants, the solution being contained in a well of a filter plate, the well being provided with a filter, the method comprising:

[0079] - a step of adding a chaotropic agent to the solution contained in the wells, so as to obtain a mixture,

[0080] - a step of incubating the mixture so as to obtain a precipitate and a supernatant in the wells, the precipitate comprising the polynucleotides and the supernatant comprising at least a portion of the contaminants,

[0081] - a step of applying a differential pressure to the wells so that the supernatant is removed through the filter and the precipitate is retained by the filter,

[0082] - a step of adding a low-salt eluent to dissolve the polynucleotides in the precipitate and transferring the polynucleotides dissolved in the low-salt eluent to a container,

[0083] Therein the method is carried out in a well equipped with a filter.

[0084] In particular, the solution is contained in the wells of a filter plate, wherein the filter plate is located above the differential pressure station and / or the shaker station.

[0085] Filter plates are well known to those skilled in the art to provide a one-step assay without the need for centrifugation or precipitation, and filter plates in particular are commonly used for PCR assays in 96 or 384 well formats.

[0086] According to the present invention, the method is carried out in a single filter plate without the need to transfer a liquid solution comprising polynucleotides. For this reason, the first step requires adding a chaotropic agent in the hole of the filter plate to obtain a mixture. The mixture preferably comprises pollutants, polynucleotides, chaotropic agents and optional pearls.

[0087] In order to improve the precipitation and thus the purification of the polynucleotide of interest, the method comprises a second step of incubating the mixture so as to obtain in the wells a precipitate and a supernatant, the precipitate comprising the polynucleotide and the supernatant comprising the contaminants.

[0088] Chaotropic agents can cause precipitation of polynucleotides present in liquid solutions, especially when salt is present in the liquid solution. Chaotropic agents can interfere with the weak intramolecular interactions of polynucleotides, allowing the destruction of their 3-dimensional conformation and thus denaturation. Denaturation causes changes in solubility, resulting in precipitation of polynucleotides.

[0089] According to the present invention, all chaotropic agents known to those skilled in the art can be used. In a preferred embodiment, the chaotropic agent is selected from the group consisting of: one of isopropanol solution, sodium iodide solution, sodium perchlorate solution and a combination thereof. More preferably, the chaotropic agent is an isopropanol solution. For example, the volume concentration of the isopropanol solution is at least 60% (v / v).

[0090] In a preferred embodiment, the step of incubating the mixture to obtain a precipitate is performed at room temperature, more preferably the incubation is performed for at least 10 minutes.

[0091] In some embodiments of the invention, the mixture results in the formation of a precipitate comprising the polynucleotides and a portion of the contaminants; and the formation of a supernatant comprising another portion of the contaminants. Thus, the mixture produces a precipitate and a supernatant in the well, the precipitate comprising the polynucleotides and optionally some contaminants, and the supernatant optionally comprising another portion of the contaminants. The contaminants may be enzymes, salts or buffers.

[0092] The third step consists of applying a differential pressure in the wells to remove the supernatant through the filter, thereby removing contaminants; the precipitate is retained by the filter. Optionally, the precipitate and beads are retained together by the filter.

[0093] The differential pressure applied to the supernatant does not solubilize the polynucleotides, but only removes contaminants, so that the pellet retained by the filter contains only the polynucleotides of interest and, optionally, the beads.

[0094] The differential pressure applied to the supernatant can be a positive pressure or a vacuum, more preferably a vacuum. For example, the differential pressure can include 5kPa to 60kPa. "Positive pressure" means a pressure higher than atmospheric pressure. The vacuum used is a device commonly used by those skilled in the art, and it is configured to be located below the filter plate so that the supernatant can be removed from the holes by the filter of the filter plate. The precipitate containing the polynucleotides and optionally some contaminants is retained by the filter.

[0095] In a specific embodiment, when the precipitate contains polynucleotides and part of the contaminants, the method of the present invention further comprises a washing step to dissolve the contaminants present in the precipitate.

[0096] Therefore, in one or more embodiments of the present invention, the method further comprises:

[0097] - a step of washing the precipitate with a washing solution in order to dissolve the contaminants that may be present in the precipitate, and

[0098] - a step of applying a differential pressure to the wells in order to remove a washing solution comprising dissolved contaminants of the precipitate through the filter,

[0099] The washing step and the step of applying a differential pressure are performed after removing the supernatant through a filter.

[0100] In a preferred embodiment, the washing step and the applying step are performed after removing the supernatant by means of a filter and before the step of adding a low-salt eluent.

[0101] The wash solution that can be used is any solution that does not dissolve the polynucleotide but only dissolves the contaminants present in the precipitate, whereby the polynucleotide can pass through the pore to remove the contaminants. In a preferred embodiment, the wash solution is an ethanol solution, more preferably a 70% to 80% ethanol solution.

[0102] In a preferred embodiment of the present invention, the differential pressure applied to the precipitate is also a positive pressure or a vacuum, more preferably a vacuum. The vacuum is also configured to be located below the filter plate so that contaminants can be removed from the pores through the filter of the filter plate.

[0103] Therefore, according to any embodiment of the present invention, the differential pressure applied to the precipitate and / or supernatant is positive pressure or vacuum, more preferably vacuum. In a specific embodiment, vacuum can be applied to the supernatant and positive pressure can be applied to the precipitate, or vice versa.

[0104] Once the supernatant is removed from the wells, the fourth step of adding a low-salt eluent aims to dissolve the precipitate retained by the filter, in particular to resuspend and dissolve the polynucleotides. Dissolution is particularly useful in order to transfer them to the desired container. "Low-salt eluent" means an eluent without most of the mineral salts present in water, preferably the low-salt eluent is purified or distilled water, more preferably ultrapure water suitable for PCR known to those skilled in the art, such as molecular biology grade water (nuclease-free and protease-free).

[0105] Therefore, the dried polynucleotide obtained after removing the supernatant can be recovered into a desired container using a low salt eluent solution.

[0106] The desired receptacles are preferably the wells of a recovery plate.

[0107] In another preferred embodiment, the low-salt eluent is selected from one of purified water, distilled water, ultrapure water and a combination thereof.

[0108] According to the present invention, the length of the newly synthesized at least one polynucleotide is at least 10 nucleotides long, preferably at least 17 nucleotides long.

[0109] In a more preferred embodiment according to the present invention, the method comprises:

[0110] - a step of adding a chaotropic agent to the solution contained in the wells so as to obtain a mixture,

[0111] - a step of incubating said mixture so as to obtain in the wells a precipitate and a supernatant, the precipitate comprising the polynucleotides and optionally contaminants, and the supernatant comprising at least a portion of the contaminants,

[0112] - a step of applying a differential pressure to the wells so as to remove the supernatant through the filter, the precipitate being retained by the filter,

[0113] - a step of washing the precipitate with a washing solution in order to dissolve the contaminants that may be present in the precipitate, and

[0114] - a step of applying a differential pressure to the wells so as to remove a washing solution comprising dissolved contaminants in the precipitate through the filter,

[0115] - a step of adding a low-salt eluent to dissolve the polynucleotide in the precipitate, and transferring the polynucleotide dissolved in the low-salt eluent to a desired container,

[0116] Each step of the method is carried out in a well equipped with a filter.

[0117] In one or more embodiments of the present invention, the method further comprises the step of adding beads before the step of incubating the mixture. The beads and the chaotropic agent are added to the hole simultaneously to obtain a mixture, which is particularly suitable for use as a precipitation surface and promotes polynucleotide precipitation. When the beads are glass beads, preferably silica glass beads, precipitation is further improved.

[0118] In a more preferred embodiment, the beads are glass beads, which are hollow beads. It is particularly suitable that the glass beads are hollow to have a density equal to or slightly higher than that of the chaotropic agent, so as to facilitate resuspension.

[0119] The beads are preferably 9 to 13 microns in diameter.

[0120] When beads are added, the method of the invention is carried out in the wells of a filter plate, wherein the pore size of the filter is smaller than the diameter of the beads. Preferably, the pore size of the filter is between 0.45 μm and 1.2 μm.

[0121] Once a supernatant containing the beads and a pellet are formed, the wells are rinsed. Due to the specific diameter of the beads, the pellet containing the beads and the polynucleotide is thus retained by the filter of the filter plate.

[0122] In a preferred embodiment, the method of the present invention is carried out under the condition of enzymatic synthesis. Therefore, the method preferably includes an additional step of enzymatic synthesis of polynucleotides before the step of adding a chaotropic agent, and the step of enzymatic synthesis of polynucleotides is carried out in the hole implementing the step of adding a chaotropic agent. Therefore, the synthesis of polynucleotides is also carried out in the hole implementing the purification method according to the present invention.

[0123] Preferably, the additional step of enzymatically synthesizing polynucleotides comprises:

[0124] a) providing an initiator having a 3'-terminal nucleotide containing a free 3'-hydroxyl group,

[0125] b) a substep of contacting the initiator having a free 3'-O-hydroxyl group with a 3'-O-blocked nucleoside triphosphate and a polymerase under elongation conditions such that the initiator is elongated by incorporation of the 3'-O-blocked nucleoside triphosphate to form a 3'-O-blocked extension segment

[0126] c) a substep of unblocking the extension fragment to form an extension fragment having a free 3'-hydroxyl group, and

[0127] d) repeating sub-steps b) and c) until the sub-step of forming a polynucleotide,

[0128] e) A sub-step of cleaving the polynucleotide from the initiator, preferably by endonuclease digestion.

[0129] In a preferred embodiment, sub-step d) of repeating sub-steps b) and c) is performed by contacting the extended fragment obtained in step c) under elongation conditions until a polynucleotide is obtained.

[0130] There are a very large number of DNA polymerases that are able to catalyze the synthesis of polynucleotides in the presence or absence of a template strand. Thus, DNA polymerases of the polX family are involved in a wide range of biological processes, in particular in DNA repair mechanisms or mechanisms for correcting errors occurring in DNA sequences. These enzymes are able to insert nucleotides into a nucleic acid strand that are excised after identification of a sequence error. DNA polymerases of the polX family include DNA polymerase β (Pol β), λ (Pol λ), μ (Pol μ), yeast IV (Pol IV) and terminal deoxyribonucleotidyl transferase (TdT). In particular, TdT is particularly useful and is therefore very widely used in methods for the enzymatic synthesis of nucleic acid molecules.

[0131] However, these DNA polymerases only allow the incorporation of natural nucleotides. In all cases, natural DNA polymerases lose their catalytic activity in the presence of non-natural nucleotides, particularly 3'-OH modified nucleotides, which exhibit greater steric hindrance than natural nucleotides. Therefore, enzymes capable of catalyzing polynucleotide synthesis by incorporating such nucleotides have been developed. In particular, DNA polymerase variants have been developed, preferably TdT variants, for example, as described in US2020 / 0002690.

[0132] In a more preferred embodiment, enzymatic synthesis of polynucleotides is described, for example, in International Patent Publication No. WO 2015 / 159023 to Ybert et al.

[0133] Kit of the present invention

[0134] The present invention also relates to any kit configured to implement the method according to any of the preceding embodiments, comprising:

[0135] - a chaotropic solution,

[0136] - washing solutions and / or buffer solutions for purification,

[0137] - an eluent solution and / or a buffer to recover the purified polynucleotide,

[0138] -optionally, an instruction manual,

[0139] -Optionally, a bead solution.

[0140] In a preferred embodiment, the kit is configured to implement the enzymatic synthesis and method of the present invention, wherein the kit further comprises:

[0141] - at least one reaction medium comprising a nucleic acid fragment comprising n nucleotides,

[0142] - an enzymatic nucleotide addition reagent, a nucleotide or a combination of nucleotides suitable for addition by said enzymatic addition reagent,

[0143] - at least one washing solution and / or buffer for use in the purification phase of the enzymatic synthesis,

[0144] - At least one amplification reaction medium comprising at least one enzymatic nucleic acid amplification reagent and natural nucleotides suitable for use by the enzymatic amplification reagent.

[0145] Different types of reagent kits can be provided according to the needs of the experimenter. Similarly, different types of reagent kits can be provided according to whether they are used automatically.

[0146] The device of the present invention

[0147] In another aspect, the present invention also relates to an apparatus configured to implement the method according to any one of any of the preceding embodiments.

[0148] In a preferred embodiment, the apparatus comprises a synthesis station and a recovery station, wherein the synthesis station comprises a filter plate (11) located above a pressure regulator and / or a shaker station.

[0149] Preferably, the device further comprises a cooling or heating station configured to be located below the plate. The cooling or heating station is particularly used to specifically control the temperature of the wells, and therefore the temperature of the solution contained in the wells, so as to, for example, heat to release the synthesized polynucleotides attached to the solid support for enzymatic synthesis, or quickly cool to room temperature to carry out the method according to the invention.

[0150] Other features and advantages of the invention will become more apparent from the following examples and results, which are of course non-limiting. Example

[0151] The examples demonstrate the effectiveness of the method of the invention, in particular by carrying out the enzymatic synthesis in the same filter plate followed by the synthesis and purification method of the invention.

[0152] Materials & Methods

[0153] As described in international patent application WO 2015 / 159023, 750 pmol of initiator DNA strands, which are small DNA strands with 3'-terminal nucleotides containing free 3'-hydroxyl groups, are used to initiate enzymatic synthesis and extend the target DNA strand. The initiator is loaded on a solid support and then dispensed into each well of a 384w filter plate. The filter pores are 1.2 μm wide and the membrane is made of polyethersulfone (PES).

[0154] The loaded plate was placed in a device sold by DNA Script under the trade name SYNTAX to perform the enzymatic synthesis method described in WO 2015 / 159023. Enzymatic synthesis was performed on a set of custom sequences. (16 DNA probes of 20 to 28 mer)

[0155] After synthesis, the following steps described in Table 1 below were performed in each well of the synthesis plate.

[0156] The first step was as follows: "25 μL of low salt eluent (MB water) was dispensed into each well of the plate, then incubated at room temperature, 1500 rpm for 5 seconds, then the liquid was evacuated during 30 seconds at -40 kPa, this step was repeated twice".

[0157]

[0158]

[0159] Table 1

[0160] SE buffer uses low salt eluent (MB water). W3 buffer is 10mM Tris, 0.25mM EDTA, 100mM NaCl, 0.5% Tween, proteinase K 0.1mg / mL. LB buffer is 10mM Tris, 500mM MgCl2, 1700mM NaCl, Endo V 2.9μM. W1 buffer is 80% EtOH, 20% Mili Q water. W6 buffer is pure isopropanol.

[0161] To demonstrate the effectiveness of the approach, the following success criteria have been identified:

[0162] - C1: Use of a single filter plate for synthesis and a method according to the invention, which proceeds in 100% automation in the installation,

[0163] - C2: For 100% of the wells, DNA recovery must reach a yield > 200 pmol (measured by absorbance at 260 nm for 750 pmol loading).

[0164] -C3: Salt contamination must be: For 100% of the wells,

[0165] -C4: Protein contamination must be: For 100% of the wells,

[0166] - C5: The method according to the invention should have no influence on the purity of the synthesized strand.

[0167] The samples were then recovered and analyzed in a 384UV quantitation plate. The inventors analyzed the recovered DNA for "yield," salt or protein contamination, and checked for purity.

[0168] The amount of DNA recovered in pmol is called the "yield", and the amount of primer DNA loaded is usually 750 pmol, which sets a maximum recovery yield of 750 pmol. In practice, we recovered 200 pmol to 400 pmol of DNA, depending on the protocol, solid support, etc. used.

[0169] The DNA was then recovered in a UV quantification plate and quantified using 260nm absorbance in an Epoch microplate spectrophotometer. The inventors also used 230nm and 280nm to estimate salt and protein contamination, respectively. For "pure" nucleic acids, the 260 / 230 value is higher than the corresponding 260 / 280 value.

[0170] Expected 260 / 230 values ​​are typically in the range of 2.0 to 2.2. If this ratio is significantly lower than expected, it may indicate the presence of contaminants that absorb at 230 nm.

[0171] Expected 260 / 280 values ​​are typically in the range of 1.8 to 2.0. If the ratio is significantly lower than expected, it may indicate the presence of contaminants that absorb at 280 nm.

[0172] The synthesized DNA was then analyzed using the Oligo Pro II (OP2) system, which utilizes UV detection and parallel capillary electrophoresis to provide single nucleotide resolution and direct assessment of oligonucleotide purity.

[0173] result

[0174] The results are shown in Figures 3 to 6B middle.

[0175] According to the first criterion C1, the method has been automated using a single filter plate with minimal hardware and software requirements and without the need for human intervention.

[0176] Before the purification method according to the present invention, 750 pmol of DNA was loaded into each well of the plate. Figure 3As shown, no well contains less than 200 pmol. Furthermore, the inventors have demonstrated that more than 50% of the wells contain at least 400 pmol of DNA, which indicates that the method of the present invention has a very good efficiency, about at least 10% to 20% efficiency, compared to the results obtained by the methods of the prior art.

[0177] The inventors have also shown that contamination in each well is very low. To check for contamination, the inventors measured the DNA / salt ratio and then the DNA / protein ratio. Figure 4 The DNA / salt ratio of all wells is shown to be at least 1.8. Figure 5 The DNA / protein ratios of all wells in the assay were at least 1.6. These results also represent an increase of approximately 20% in the recovery of polynucleotides compared to the prior art.

[0178] Finally, the inventors have demonstrated that the method of the invention has no effect on the purity of the synthesized strands for the analysis of oligonucleotides using OP2. Fig. 6A and Figure 6B In the example above, the purity of the oligonucleotide is shown to correspond to the optimal standard for the given chemistry and size of the oligonucleotide.

[0179] In summary, the method of the invention meets all requirements for a high-performance purification method. This opens up new perspectives and new functionalities for devices incorporating such methods.

Claims

1. A method for purifying a solution comprising polynucleotides and contaminants, the solution being contained in a well of a filter plate, the well being provided with a filter, the method comprising: - a step of adding a chaotropic agent to said solution contained in said wells, so as to obtain a mixture, - a step of incubating said mixture so as to obtain in said wells a precipitate and a supernatant, said precipitate comprising said polynucleotide and said supernatant comprising at least a portion of said contaminants, - a step of applying a differential pressure to said wells so as to remove said supernatant through said filter, said precipitate being retained by said filter, - a step of adding a low-salt eluent to dissolve the polynucleotides in the precipitate and transferring the polynucleotides dissolved in the low-salt eluent to a container, The method is performed in a well equipped with the filter, and the method comprises a step of enzymatically synthesizing polynucleotides before the step of adding a chaotropic agent, the step of enzymatically synthesizing polynucleotides being performed in the well in which the step of adding a chaotropic agent is performed.

2. The method according to the preceding claim, wherein the step of enzymatically synthesizing a polynucleotide comprises: a) providing an initiator having a 3'-terminal nucleotide containing a free 3'-hydroxyl group, b) a substep of contacting an initiator having a free 3'-O-hydroxyl group with a 3'-O-blocked nucleoside triphosphate and a polymerase under elongation conditions such that the initiator is elongated by incorporation of the 3'-O-blocked nucleoside triphosphate to form a 3'-O-blocked extension segment c) a sub-step of unblocking the extension segment to form an extension segment having a free 3'-hydroxyl group, and d) repeating sub-steps b) and c) until the sub-step of forming the polynucleotide, e) the sub-step of cleaving said polynucleotide from said initiator.

3. The method according to claim 2, wherein the sub-step of cleaving the polynucleotide from the initiator is performed by enzymatic digestion, by photocleavage and / or by chemical cleavage.

4. The method according to any one of the preceding claims, wherein the incubation step is performed at room temperature.

5. The method according to any one of the preceding claims, wherein the incubation step is carried out for at least 10 minutes.

6. The method according to any one of the preceding claims, wherein the filter plate preferably comprises 96 holes or 384 holes 7. The method according to any one of the preceding claims, wherein the chaotropic agent is selected from the group consisting of: an isopropanol solution, a sodium iodide solution, a sodium perchlorate solution, and combinations thereof.

8. The method according to any one of the preceding claims, wherein by incubating the mixture a precipitate and a supernatant are formed in the wells, the supernatant comprising at least a portion of the contaminants and the precipitate comprising the polynucleotides and optionally another portion of the contaminants 9. The method according to any one of the preceding claims, wherein the method comprises: - a step of washing said precipitate with a washing solution in order to dissolve contaminants that may be present in said precipitate, and - a step of applying a differential pressure to said pores in order to remove said washing solution, comprising the contaminants dissolved in said precipitate, through said filter, The step of washing the precipitate and the step of applying differential pressure are performed after removing the supernatant through the filter.

10. The method according to the preceding claim, wherein the washing solution is an ethanol solution, preferably comprising 70 to 80% ethanol by volume.

11. The method according to any one of the preceding claims, wherein the method comprises the step of adding beads before the step of incubating the mixture.

12. A method according to the preceding claim, wherein the pore size of the filter of the pores is smaller than the diameter of the beads.

13. The method according to any one of claims 11 or 12, wherein the beads are glass beads, preferably silica glass beads.

14. A method according to the preceding claim, wherein the beads are hollow beads.

15. A method according to any preceding claim, wherein the filter has a pore size of 0.45 μm to 1.2 μm.

16. The method of any one of claims 11 to 14, wherein the beads have a diameter of 9 to 13 microns.

17. The method according to any one of the preceding claims, wherein the length of the newly synthesized at least one polynucleotide is at least 10 nucleotides long, preferably at least 17 nucleotides long.

18. A kit configured to carry out the method according to any one of the preceding claims, the kit comprising: - a chaotropic solution, - washing solutions and / or buffer solutions for purification, - an eluent solution and / or a buffer to recover the purified polynucleotide, -Optionally, an instruction manual.

19. The kit according to claim 18, wherein the kit further comprises: - at least one reaction medium containing a nucleic acid fragment comprising n nucleotides, - an enzymatic nucleotide addition reagent, a nucleotide or a combination of nucleotides suitable for addition by said enzymatic addition reagent, - at least one washing solution and / or buffer for use in the purification phase of said enzymatic synthesis, - at least one amplification reaction medium comprising at least one enzymatic nucleic acid amplification reagent and natural nucleotides suitable for use by said enzymatic amplification reagent.

20. Apparatus configured to carry out the method according to any one of claims 1 to 17.

21. The apparatus according to claim 20, wherein the apparatus comprises a synthesis workstation and a recovery workstation, wherein the synthesis workstation comprises a filter plate located above a pressure regulator and / or a shaker workstation.

22. The apparatus of claim 21, wherein the apparatus further comprises a cooling or heating station configured to be located below the filter plate.

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