Method for synthesizing nucleic acids, in particular long nucleic acids, use of said method and kit for implementing said method

Abstract

The invention relates to a method for synthesising long nucleic acids, including at least one cycle of elongating initial fragments of nucleic acids, including a) a phase comprising the enzymatic addition of nucleotides to said fragments, b) a phase comprising the purification of the fragments having a correct sequence, c) an optional phase of enzymatic amplification, each cycle being performed in a reaction medium which is compatible with enzymatic addition and amplification, such as an aqueous medium, the synthesis method also comprising, at the end of all the elongation cycles, a last step of final amplification. The invention also relates to the use of the method for the production of genes, or sequences of synthetic nucleic acids, DNA or RNA. The invention further relates to a kit for implementing said method.

Claims

1. A method for the synthesis of a nucleic acid sequence, comprising: a) attaching initial nucleic acid fragments or elongated fragments to a first support; b) contacting the attached initial nucleic acid fragments or elongated fragments with a modified nucleoside triphosphate and a template-independent DNA polymerase so that initial nucleic acid fragments or elongated fragments are elongated by a modified nucleotide, wherein the modified nucleotide comprises a protective group that prevents multiple additions of nucleotides; c) deprotecting the elongated fragments; d) repeating steps b) and c) until a nucleic acid sequence is obtained; e) contacting the elongated fragments with a modified nucleoside triphosphate and a template-independent DNA polymerase so that elongated fragments are elongated by a modified nucleotide, wherein the modified nucleotide comprises a protective group; f) detaching the elongated fragments from the first support; g) purifying elongated fragments; and h) deprotecting the protection groups.

2. The method of claim 1, further comprising amplifying said released elongated fragments.

3. The method of claim 1, wherein said template-independent DNA polymerase is a terminal deoxynucleotidyl transferase.

4. The method of claim 1, wherein said modified nucleoside triphosphate is a 3′-O-protected nucleoside triphosphate.

5. The method of claim 1, wherein said step of purifying comprises specifically binding a 3′-O-protection group to said second support.

6. The method of claim 1, wherein said modified nucleoside triphosphate has a 3′-O-protection group and a protected nitrogenous base.

7. The method of claim 1, wherein said protective group in step e specifically binds to a second support and prevents multiple additions of nucleotides.

8. The method of claim 1, wherein said protective group in step e is a first protective group that specifically binds to a second support and said modified nucleotide further comprises a second protective group that prevents multiple additions of nucleotides.

9. The method of claim 1, wherein said protective group in step e is a first protective group that prevents multiple additions of nucleotides and said modified nucleotide further comprises a second protective group that specifically binds to a second support.

Description

DESCRIPTION OF THE FIGURES

(1) The description of an exemplary embodiment of the preferred form of the invention with use of attaching supports for immobilizing the nucleic acid fragments during certain stages of the synthesis process will be described below with reference to the figures, in which FIGS. 1 to 7 diagrammatically represent the different stages of the process:

(2) FIG. 1 the immobilization of the starting fragments;

(3) FIG. 2 the addition of a nucleotide to the immobilized fragments;

(4) FIG. 3 a washing stage and the detaching of the nucleic acid fragments which have been added to;

(5) FIG. 4 the fixing, to a second support, of the nucleic acid fragments which have been added to;

(6) FIG. 5 the deprotection of the nucleic acid fragments which have been added to;

(7) FIG. 6 an amplification stage;

(8) FIG. 7 the initiation of a new cycle using the fragments which have been added to from the preceding cycle;

(9) and FIG. 8 exhibits a diagram of the possible arrangement of the “reactors” for carrying out the phase of enzymatic addition and the phase of purification of the fragments of correct sequence.

EXAMPLE

(10) With reference to the figures, a cycle of elongation of nucleic acid fragments in the synthesis process according to the invention is described below.

(11) Strands 4 comprising at least three nucleotides are fixed to a first solid support 1, such as a glass sheet. Primers or fragments in the course of elongation 3 of nucleic acids including Xi+n nucleotides are bonded to the strands 4 fixed to the support 1 via hydrogen bonds between their respective bases. The nucleic acid fragment in the course of elongation 3 comprises a free part 33 and an immobilizable part 34 comprising at least three nucleotides complementary to those of the fixed strands 4 and representing the initiator. Nucleic acid fragments 30 immobilized on the first solid support 1 are then obtained in the reaction medium, as represented diagrammatically on the right-hand side of FIG. 1.

(12) A stage of enzymatic addition by the addition of a reaction medium including addition enzymes 5 and reagents 6 comprising at least one nucleotide 7, one end of which is blocked by a protective group 8, is subsequently carried out.

(13) This results, as represented diagrammatically on the right-hand part of FIG. 2, in the addition of the reagents 6 to the free end 33 of the fragments 30 immobilized on the first solid support, to give nucleic acid fragments which have been added to (that is to say, having received at least one nucleotide) comprising a protected end, which are immobilized on the support 1. These protected and immobilized nucleic acid fragments are referenced 36.

(14) There remain, in the reaction medium, either on the support or close to said support 1, residues of enzymes, reagents 6 and possible buffer solutions, which are then removed by washing according to the arrow 3A. After this first washing operation 3A, the fragments 36 are detached, according to the arrow 3B, from the support 1, which fragments 36 will give free protected fragments which have been added to, referenced 38 in FIG. 3.

(15) However, this detaching stage unfastens, from the support 1, both the fragments which have been added to 38 and the initial fragments 3 which have not been added to. A second support 2, in this instance magnetic beads, covered with a coating 9 formed of proteins, such as antibodies, dihydrofolate reductase (DHFR), avidin, streptavidin, glutathione S-transferase (GST), phosphopeptides (serine, tyrosine or threonine oligomers) or histidine oligomers, then intercedes, as represented diagrammatically in FIG. 4, in the reaction medium including the nucleic acid fragments of correct sequence and the fragments of incorrect sequence.

(16) This results in a fixing, by the terminal protective group 8, an attaching, to the surface of the beads 2, of the protected fragments of correct sequence 38. Initial fragments 3 which have not undergone the enzymatic addition and which thus do not comprise an end with the protective group which can become bonded to the proteins of the coating 9 of the beads 2 are then easily removed by washing.

(17) After this stage of effective selection of the fragments which have been added to of correct sequence, the magnetic beads 2 are separated by modifying the conditions of the reaction mixture. For example, modifying the pH, increasing the temperature or the action of a reagent or of an electromagnetic field makes it possible to detach, from the beads 2, the fragments which have been added to in order to obtain free unprotected fragments 37 which have been added to in the reaction medium (see FIG. 5).

(18) The beads 2 covered with the protective groups which have remained bonded to the coating 9 are then removed from the reaction medium, for example under the action of a magnetic field.

(19) FIG. 6 subsequently diagrammatically represents a stage of amplification of the unprotected fragments 37 which have been added to which have undergone the elongation cycle described up till now, under the action of an amplification reaction medium 11 comprising the amplification enzymes and also the nucleotides, for example the natural nucleotides. The number of nucleic acid fragments 37 is then greatly amplified.

(20) This amplification stage is entirely effective as it amplifies only the fragments 37 which have undergone the elongation cycle. In addition, this minimizes the use of amplification reagents.

(21) No subsequent stage of purification is then necessary, before terminating the synthesis or carrying out a new elongation cycle i+1.

(22) A new elongation cycle can then occur via the same first solid support 1 to which are fixed the strands 4 used to immobilize the fragments 37, as represented diagrammatically in FIG. 7.

(23) FIG. 8 presents the diagram of an example of an elongation chamber, preferably comprising two separate compartments, in which compartments is carried out each elongation cycle according to the present invention.

(24) The first reactor 10, a compartment in which the enzymatic addition phase is carried out, includes the first solid support 1 to which the strands 4 are fixed and is connected to a device 70 for feeding with addition nucleotides 7 and to a device 50 for feeding with addition enzyme 5.

(25) The second reactor 12, a compartment in which the phase of purification of the fragments of correct synthesis is carried out, is equipped in this instance with an electromagnetic device (electromagnet 21) and is connected to a feed 20 of supports of bead type.

(26) The two reactors 10 and 12, which are separated in order to make possible better purification of the fragments, are nevertheless connected together and connected to inlets 13, 14 for washing solution(s) and also to outlets 15, 16 for wastes to be discharged.

(27) The amplification stage(s) is (are) also carried out within the reactor 12.

(28) The nucleic acids thus synthesized are recovered at the outlet 17 of the second reactor after the final amplification stage.

(29) An example illustrative of the synthesis of a DNA fragment is described below.

(30) The enzyme chosen for carrying out the enzymatic synthesis stages is terminal deoxynucleotidyl transferase or TdT, which is commercially available (Thermo Scientific). It is also possible to produce large amounts of TdT recombinantly.

(31) The primer used to initiate the synthesis is given below:

(32) TABLE-US-00001 Seq. No. 1 5′-GGGGGGGGGGGGGGCTGCA.sup.*G-3′

(33) This primer has a restriction site involving a cleavage of the sequence by the restriction enzyme Pst1 at the arrow present in the above sequence. In the case where the sequence to be synthesized would also comprise a Pst1 site, the initial primer would be modified as required.

(34) The nucleotides used have, at their 5′ end, a triphosphate group which promotes their reactivity.

(35) They have one of the four nitrogenous bases naturally present in the DNA molecule, namely A Adenine, T Thymine, C Cytosine or G Guanine, and have, at their 3′ end, a group different from the hydroxyl group naturally present and which has the ability to block any subsequent addition of nucleotide by TdT and the ability to interact with other molecules or proteins.

(36) The synthesis conditions used originate from the description of the protocol associated with the enzyme TdT: 50 U of TdT, 200 mM sodium cacodylate, 25 mM Tris-HCl pH 7.2, 8 mM MgCl.sub.2, 0.33 mM ZnSO.sub.4, 0.2 mM dATP, 2 pmol of primer (Seq No. 1) and 100 μM of protected nucleotides are mixed for a total of 50 μL of reaction volume. The mixture is incubated at 37° C. for 5 min.

(37) Depending on the nature of the protective group chosen, the nucleotides added are deprotected by the action of a mild acid, such as 50 mM sodium acetate pH 5.5, in the presence of 10 mM MgCl.sub.2 at 37° C. for 15 min. The deprotection reaction has the result of destroying the bond existing between the nucleotide and the protective group, and also any other group optionally associated with the protective group. According to certain preferred embodiments of the invention, this deprotection stage can simultaneously release the fragment from an interaction at its 3′ end.

(38) The DNA fragments in the course of synthesis are incubated at 20° for 10 min in a reaction chamber comprising a glass sheet to which DNA fragments having the following sequence:

(39) TABLE-US-00002 Seq. No. 2 3′-CCCCCCCCCCCCCCGACGTC-5′
have been fixed beforehand.

(40) The techniques for generating a DNA chip have been employed for the fixing by the 3′ end of DNA fragments having the sequence (Seq No. 2). The DNA fragments are thus immobilized in this way.

(41) The washing stages are carried out with a 25 mM Tris-HCl pH 7.2 solution according to a flow rate of 5 μL per second for 30 seconds.

(42) The stages for release of the DNA fragments are carried out by passing a 25 mM Tris-HCl pH 7.2 solution at 95° C. at the flow rate of 5 μL per second for 60 seconds.

(43) The transfers of the free DNA fragments are carried out by means of a system of valves which makes it possible to convey the stream of 25 mM Tris-HCL pH 7.2, in which the fragments are dissolved, into the second rector 12.

(44) The DNA fragments which have undergone the stage of enzymatic elongation using the protected nucleotides are then incubated at ambient temperature for 15 min in the presence of magnetic beads 2 exhibiting, at their surface, a molecule or protein which makes possible the interaction with the final nucleotide added. Magnetic beads coated with GST protein are a nonlimiting example of the type of beads which can be used.

(45) Using a permanent magnet, the magnetic beads 2 carrying the DNA fragments are rendered static while a 25 mM Tris-HCl pH 7.2 stream at a flow rate of 10 μL per second is applied for washing purposes for 30 seconds.

(46) The stage of amplification of the DNA fragments is carried out by the Platinum® Taq DNA Polymerase enzyme (Invitrogen) according to the following protocol: 10× buffer supplied 1×, 0.2 mM dNTP for each nucleotide, 1.5 mM MgCl.sub.2, 0.2 μM complementary primer, 1.0 U Polymerase for a total volume of 50 μL.

(47) The amplification cycles used are given by the following table: incubation at 94° C. for 60 sec., application of 30 amplification cycles with the following stages: denaturation at 94° C. for 30 s, then pairing at 55° C. for 30 s, then extension at 72° C. for 60 s per thousand nucleotides to be amplified; a stage of final extension at 72° C. for 5 min is added.

(48) The primer is chosen as a function of the state of progression of the synthesis of the DNA fragments. It has a sequence of approximately 20 nucleotides. The amplification conditions described above are adjusted so as to promote a very specific amplification of the fragments capable of pairing exactly with the complementary primer chosen. The size of this complementary primer is furthermore also chosen as a function of the sequence, so as to promote the most specific amplification possible.

(49) According to the principle disclosed by the present invention, the performances of the different stages were measured and are presented in table 1 below:

(50) TABLE-US-00003 TABLE 1 Content of good Content of bad fragments fragments Enzymatic addition 80% 20%  Purification (Wide 90% 2% removal stages) Amplification 90% 1%

(51) The wide removal stages, for example, make it possible to obtain 90% of the fragments having a correct sequence (regarded as “good”) and only 2% of the fragments having an incorrect sequence (regarded as “bad”).

(52) The repetition of the cycles employing these different stages carefully combined makes possible the synthesis of long fragments, the final purity of which is given by table 2 below (amount of initial primer of 2 pmol):

(53) TABLE-US-00004 TABLE 2 Elongation Purification Amplification Purity Cycle Good Bad Good Bad Good Bad Good Bad 0 9.63E+11 2.40E+11 8.67E+11 4.82E+09 8.67E+11 4.82E+09 99.44% 0.552% 1 6.93E+11 1.78E+11 6.24E+11 3.57E+09 6.24E+11 3.57E+09 99.43% 0.568% 10 3.60E+10 9.27E+9  3.24E+10 1.86E+08 3.24E+10 1.86E+08 99.43% 0.568% 50 70 870   18 224   63 783   365  63 783   365  99.43% 0.569% 100 4 609 640   1 185 337    4 148 676   23 707    4 148 676   23 707    99.43% 0.568% 500 1816 467 1634 10 1634 10 99.392% 0.608% 1000 2852 734 2566 15 2566 15 99.419% 0.581% 5000 94 719   24 356   85 247   488  7.67E+13 4.88E+09 99.994% 0.006%

(54) Thus, despite the imperfection inherent in each stage taken separately and despite the presence of fragments failing at various stages of the synthesis, the final result exhibits a purity of more than 99% and can thus be used directly by the experimenter, this being the case independently of the length of the fragment under consideration. The results given by this table are presented as examples and may be substantially different if the parameters (presented in table 1) of content of good and bad fragments generated at each stage, taken as parameters, are different.

INDUSTRIAL APPLICATION

(55) The process which is a subject matter of the present invention makes possible the synthesis of nucleic acids without loss in yield, independently of their length. It makes possible a substantial improvement in the performances of synthesis of nucleic acids with respect to the existing techniques, in particular in terms of simplicity of implementation, of synthesis costs, of synthesis time, of purity of the products obtained and of synthesis capacity. This process can be used for the production of genes or of synthetic sequences of nucleic acids. It is intended in particular for the synthesis of nucleic acids, such as DNA or RNA, for the purposes of research, development or industrial implementation in the field of biotechnology or more generally in the broad field of biology.