FR2883885A1 - In vitro production of recombinant polynucleotides comprises preparing a mixture of plasmid molecules and a phosphorylated hybrid oligonucleotide primer, and subjecting the mixture to elongation cycle - Google Patents

Abstract

In vitro production of recombinant polynucleotides which leaves at least 2 different polynucleotides comprises: (a) preparing a mixture of plasmids including each of the different polynucleotides, and at least one oligonucleotide which self-hybridises to a conserved region in the circular polynucleotide molecules; and (b) subjecting the mixture to several cycles of elongation where the duration of elongation for at least one of the cycles is adapted so that the elongation is stopped at the level of the polynucleotides of interest. Independent claims are included for: (1) a recombinant polynucleotide bank; and (2) a protein bank obtained from an expression bank.

Description

The present invention relates to the field of molecular biology and

  more particularly that of the evolution in vitro.

  The evolution of proteins in vitro is an activity in full expansion, whose objective is to generate mutants with modified characteristics, or having acquired new activities compared to the natural protein. In vitro evolution is inspired by natural evolution to improve in vitro molecules of industrial, medical or biotechnological interest. Two essential steps are identified: the creation of a genetic diversity (by mutagenesis and / or recombination) on the one hand, and on the other hand the mass selection or the individual screening of this diversity, in search of mutants having acquired the characteristics of interest. In vitro evolution allows the improvement of proteins, mainly enzymes, antibodies and therapeutic proteins. The improvement of enzymes is of major economic interest: indeed, many industrial enzymes are used in various processes - such as the synthesis of antibiotics or vitamins, the brewery, the treatment of textiles - or are incorporated into products as diverse as laundry or animal feed. Natural enzymes are most often imperfect for this purpose, and it is necessary to obtain improved enzymes, in particular on the criteria of their activity or their stability at high temperature, in order to be able to use them in these industries. The improvement of the enzymes makes it possible to reduce the costs of the corresponding processes, or makes it possible to implement new ones. The use of enzymes in substitution of conventional chemical methods often polluting carries environmental benefits (we speak of green chemistry). The current enzyme market is estimated at billions of euros a year. The evolution in vitro can also make it possible to improve the stability or the activity of the therapeutic proteins (peptides, hormones, cytokines, interferons, vaccines, antibodies, ...), or serve to control the specificity of antibodies used in diagnosis or in analytical chemistry. In vitro evolution also makes it possible to directly improve functional nucleic acids, for example ribozymes, deoxyribozymes, or nucleic acids useful in the context of DNA computers (Breaker RR Nature 2004, 432: 838-45). Schubert S et al., Curr Drug Targets 2004, 5: 667-81, Parker J. EMBO Rep. 2003, 4: 7-10). The evolution in vitro thus finds a large number of applications in key areas such as health or the chemical industry. It is therefore understandable why the techniques intended to facilitate this gene and protein enhancement activity have been the subject of important research efforts.

  To create the genetic diversity on which selection is based, natural evolution involves two basic processes: mutation, and recombination. Mutations are introduced at a rate of order of magnitude of one mutation per megabase and per generation. They may be due to spontaneous errors in the polymerase causing DNA replication, or changes in nucleotides as a result of ultraviolet radiation. Recombination is an active phenomenon, mediated by DNA-binding proteins.

  Similarly, in the field of in vitro evolution, mutagenesis and recombination both exist. Mutagenesis can be done randomly, rationally, or in an intermediate mode. Rational mutagenesis is based on the structural and functional characteristics of the protein, when available. Predictions are made from this information, and the corresponding mutants are generated individually by site-directed mutagenesis. In the intermediate mode between the random and the rational, many mutants having an increased chance of chance of conveying an improvement are generated, without the probability associated with each being sufficiently important for us to really speak of prediction. In all cases, these mutants are intended to be selected in bulk or individually screened, in order to identify improved clones.

  In vitro recombination of genes makes it possible in the first place to recombine the improved mutants in a mutagenesis-based approach. Mutants combining several of the enhancer mutations are likely to have an even higher level of the desired trait. In vitro recombination also makes it possible to mix together various natural genes of totally or only partially known sequences, in order to associate several characteristics of interest of these genes and / or to generate new characteristics. In this case, a preparatory step consists in isolating homologous genes (the level of homology required is variable from one technique to another), either by cloning from the strains cultured in vitro, or by using an approach allowing to get rid of this in vitro culture. This latter approach, referred to as metagenome (Streit WR et al., Curr Opin Biotechnol 2004, 15: 285-90), seems to be the most effective because it allows access to a large number of genes by producing a large number of genes. limited effort.

  In all cases, it should be noted that the in vitro evolution experiments involve several turns (typically 3 to 30 generations of improved molecules). Also, it seems desirable that each elementary step of mutation, selection or, in this case, recombination, is of minimal cost, complexity and duration.

  The subject of the invention relates to an in vitro recombination technique: the technological background of this activity is more precisely explained in the following paragraphs.

  The first in vitro recombination technique was described in 1993 and is now known by the trade name DNA-shuffling (Stemmer WPC, Proc Natl Acad Sci USA 1993, 91: 10747 51, Stemmer WPC Nature 1994). 370: 389-91). Since then, DNA recombination has been successfully applied in contexts as diverse as the generation of a thermostable and organic solvent tolerant enzyme (Hao J et al., Protein Eng Des Sel., 2004; 17: 689-97). ; a penicillin G acylase variant with broad substrate specificity (Hsu JS Appl Environ Microbiol 2004, 70: 6257-63); an enzyme increasing plant resistance to glyphosate herbicide; a DNA polymerase capable of incorporating non-standard nucleotides (Ghadessy FJ et al Nat Biotechnol 2004; 22: 755-9); improved vaccines (Locher CP et al., Expert Opin Biol Ther., 2004; 4: 589-97); enzymes with controlled enantioselectivity (Reetz MT, Proc Natl Acad Sci USA 2004, 101: 5716-22); modified tropism virus (Soong NW et al Nat Genet 2000, 25: 436-9). This list is far from being exhaustive and the advent of in vitro recombination has allowed a qualitative leap in what could be done in the field of in vitro evolution, with the important industrial issues described above.

  Two broad classes of in vitro recombination methods can be distinguished.

  The first class, which includes most of the methods described to date, involves a fragmentation / assembly process. From the sequences to be recombined, fragments are created in a first step, which are assembled in a second step into new complete sequences. Thus, in the case of DNA shuffling mentioned above, the parent DNA is cleaved in an uncontrolled DNAse then the fragments serve as a primer for a PCR reaction performed without other primers (US 5,605,793, US 5,830,721, US 6,506,603 ). The fragmentation / assembly type recombination techniques are described below, without this description being considered exhaustive. A first alternative is to treat matrices incorporating dUTPs with exonuclease V (Miazaki K. Nucleic Acids Res 2002; 30: e139). The amount of product obtained is very small and the product is reamplified by a second PCR which contains appropriate primers.

  DNA can also be randomly fragmented by sonication or simple mechanical disruption. Random chimeragenesis of transient templates (RACHITT) is another fragmentation / assembly technique that can recombine DNA molecules in vitro (Coco WM et al., Nat Biotechnol 2001, 19: 354-359 and Coco WM. Mol Biol 2003: 231: 111-127). One strand of a particular parental DNA containing dUTP serves as a template on which the other parental DNAs are paired. The latter are then digested with an exonuclease and the resulting holes are filled with a DNA polymerase. Compared with DNAShuffling, RACHITT makes it possible to increase the number of recombinations per molecule. In all cases, fragmentation is not completely random: the DNAses, for example, preferentially cut certain positions, which leads to creating bias. Three other in vitro recombination / fragmentation techniques are conceptually very close to DNA-shuffling: L-shuffling (WO0009679), particularly adapted to the case of long genes, FIND (W003097834), which isolates single-stranded DNAs to increase the recombination rate and the synthetic reassembly (US 6,537,776). ITCHY (incremental truncation for the creation of hybrid enzymes) is another fragmentation / assembly recombination technique that consists of ligating libraries of generated fragments after the truncation of two template sequences, where each template is digested from opposite strands (Ostermeier M et al., Nat Biotechnol 1999; 17: 1205-9 and Lutz S. Nucleic Acids Res 2001; 29: e19). Fragments of a template digested with exonuclease III and S nuclease from the 5 'portion of the gene are thus assembled with the fragments of another template digested from the 3' portion of the gene. To rapidly generate a variety of fragment sizes, ITCHY varies the digestion time or introduces nuclease-resistant phosphothioate bonds into the matrices. It is possible to add a DNA-Shuffling step after an ITCHY step in order to obtain molecules having several recombinations. The ITCHY technique has the advantage of not requiring homology between the sequences to be recombined. However, this technique requires the use of a suitable plasmid, is limited to the recombination of two genes and is of a relatively complex implementation, including various enzymatic treatments and several DNA purifications. Two other fragmentation / assembly methods have recently been described, which make it possible to make site-specific recombination between genes of sequences that can be very similar. The first uses specific primers to precisely generate gene fragments which are then assembled by overlapping extension (O'Maille PE et al., J. Mol Biol 2002; 321: 677-91). The second introduced by mutagenesis of the restriction enzyme sites in the matrices to be recombined then fragments these matrices with the corresponding endonucleases (Higara K et al., J. Mol Biol 2003; 330: 287-96). In all cases of site-specific recombination, the generation of the fragments to be recombined involves a high number of PCR reactions and purification steps. Their implementation remains complex and they are reserved for cases where the structure of the target protein is well known. In contrast, when recombining at random (ie without site specificity) very different sequences, we obtain most often a majority of non-functional proteins because folding back badly.

  The fragmentation / assembly methods are therefore relatively complex to implement, because of the multiplicity of assembly steps, and do not constitute a tool that could easily be used by any molecular biology laboratory for evolution experiments. in vitro routine. These methods are, moreover, not applicable to very short (<200 bp) or very long (> 3 kbp) genes.

  The second class of in vitro recombination methods rely on the interrupted amplification of homologous genes. Staggered Extension Process (StEP) is a method that does not involve fragmentation / assembly of sequences, but consists in amplifying by PCR a set of templates using at least two primers flanking the gene of interest, with cycles containing a very short elongation phase (of the order of one second). Thus, at each cycle, the product detached from a matrix can reappear with a different matrix, thus giving rise to a recombination event (US 6,153,410, US 6,177,263, Zhao et al., Nat Biotechnol 1998: 16: 258-61 and Aguinaldo AM et al., Methods Mol Biol 2003: 231: 105-110). StEP has on all the fragmentation / assembly techniques described above the major advantage of its relative simplicity of implementation. Although in theory this step is not essential, the published works using StEP involve an additional step of amplification of the products obtained as a result of short cycle amplification. The DNA obtained is finally purified and then cloned by double-stranded ligation in a linearized plasmid vector.

  StEP is implemented substantially simplified compared to fragmentation / reassembly techniques, although in practice it is not always easy to adjust the amplification conditions so as to promote the production of recombinant molecules. However, the products obtained after recombination with StEP are scarce and must generally be amplified by PCR.

  In the two classes of processes (fragmentation / assembly, or STEP), an exponential amplification of the DNA exists: it can take place during the assembly PCR, during the StEP reaction, or during the PCR that follows StEP. The exponential character of this amplification can lead to introduce into the bank an amplification bias. The existence of bias in favor of certain recombinant molecules will be amplified in an approximately geometric progression, and these molecules will ultimately be strongly overrepresented in the final library. This effect is all the more troublesome as the gene of interest is long and the number of cycles is large.

  Also, in all cases, a final double-stranded ligation step in a linearized plasmid (preferably adapted to expression) is necessary. This double-stranded ligation step, besides the fact that it increases the cost, the difficulty and the duration of the manipulation, leads to limit the diversity of the bank generated. In addition, the DNA to be ligated must be purified beforehand, generally on an agarose gel, which further increases the cost and the level of human intervention.

  In the field of site-directed mutagenesis, the first techniques described relied on an overlapping PCR step to introduce the desired point mutations, followed by purification of the amplicon and double-strand ligation of the insert into a plasmid vector. (Kadowaki H et al., Gene 1989, 76: 161-6). These techniques have since been largely replaced by techniques based on circular replication (Hemsley et al., Nucleic Acids Res.1989; 17: 6545: 51), allowing the introduction of mutations directly onto the initial plasmid and thus without ligation. Today, the QuickChange (US2004253729) and Massive Mutagenesis (W00216606) techniques, in particular, make it possible to generate mutations in a rapid and simple manner in any plasmid, without the need to perform double-stranded ligation. In addition, there is the development of techniques for fast DNA subcloning without using ligase such as TOPO-cloning (W002061034 and WO0216594), or Gateway system (US5,888,732).

  In summary, the state of the art analysis of DNA recombination shows that there are a variety of effective techniques. These techniques all involve several stages, often relatively complex. This complexity makes their use relatively limited in comparison with other approaches for creating genetic diversity, such as random mutagenesis for example. In particular, the PCR, DNA purification and double-ligation ligation steps inherent in existing in vitro recombination techniques contribute significantly to the cost, duration and complexity of the recombination protocols. These steps also affect the quality of the diversity generated, especially in terms of the size of the diversity and because of the amplification bias during the PCR. There is therefore a strong interest for a simple and rapid in vitro recombination technique, which takes place in a minimum number of steps, and involves neither exponential amplification nor double-stranded ligation.

  Finally, the present invention further relates to a library of polynucleotides of recombinant interest produced by the method according to the present invention as well as a protein library derived from the expression of this library. It also relates to the DNA resulting from the amplification or cleavage of this library, the RNA resulting from the in vivo or in vitro transcription of this library.

Summary description

  The subject of the present invention is an in vitro genetic recombination method, that is to say the production, from a mixture of several parent matrix polynucleotides, of a progeny of many new polynucleotides whose sequences each combine two parental sequences or more. Starting from a set of polynucleotides sharing a certain degree of homology, the method according to the invention is characterized by obtaining in just one simple and rapid step a mixture containing a large proportion of recombinant molecules in one or more sites. . The technique of the invention is characterized in particular by the fact that no double-strand ligation step, which constitutes a limiting factor for the techniques according to the prior art, is necessary. The technique according to the invention is also characterized by the absence of exponential amplification of genetic material, generally necessary in the techniques of the prior art, and source of the introduction of bias in the generated diversity. The invention also relates to the polynucleotides obtained and the peptides, polypeptides or proteins that they make it possible to obtain. The technique according to the invention is a recombination technique for genes of homologous sequences characterized by the fact that the recombination takes place directly between two circular DNA molecules. Indeed, in the technique according to the invention, the genes to be recombined are present in the form of closed circular DNA molecules, preferably double-stranded, and at no time these circular molecules are linearized beforehand or during the step recombination, as in the techniques of the prior art. This technical element constitutes a major difference compared to the techniques according to the prior art, where a double-strand ligation step (preceded by a purification of the insert), which is always a source of many difficulties, is necessary to recreate double-strand circular molecules.

  In addition, the technique according to the invention is characterized in that a single oligonucleotide is necessary to introduce recombination. In the prior art techniques based on short-cycle polymerization (StEP), recombination is generated using two homologous oligonucleotides of opposite strands, so as to trigger a PCR reaction allowing the exponential amplification of the DNA strands. targeted. Fragmentation / assembly techniques also involve exponential amplification. This exponential amplification reaction is associated with bias in the representativity of the mutations. In contrast, in the method according to the invention, a single oligonucleotide primer, preferably complementary to a part of the circular DNA molecules not being subject to variability, is necessary. The DNA amplification that can result from the polymerization from this oligonucleotide is not exponential in nature, but instead is linear. The amplification bias therefore disappears since it is always the parental plasmid molecules that serve as a matrix. However, it should be noted that the method according to the present invention does not exclude the use of several primer oligonucleotides. In a particular embodiment, the primer oligonucleotides all hybridize on the same strand of the circular DNA molecules. This makes it possible to increase the frequency of the recombinations. In an alternative embodiment, the primer oligonucleotides hybridize on both strands of the circular DNA molecules. This makes it possible to increase the percentage of recombinant molecules relative to the parent plasmids, with the counterpart of introducing a possible amplification bias.

  Thus, the present invention relates to a method for producing in vitro recombinant polynucleotides of interest from at least 2 polynucleotides of different interest, characterized in that it comprises: a) preparing a mixture comprising circular polynucleotide molecules comprising each of said polynucleotides of different interest, and at least one primer oligonucleotide hybridizing to a conserved region in said circular polynucleotide molecules; b) subjecting the mixture to several elongation cycles each comprising an elongation phase, and the elongation time for at least one of the cycles is adapted so that the elongation is interrupted at said polynucleotides of interest; said method resulting in the production of newly synthesized circular polynucleotide molecules comprising recombinant polynucleotides of interest.

  Preferably, the method further comprises a step c) of selecting the newly synthesized circular polynucleotide molecules. In a particular embodiment, the selection c) is carried out by digestion of the circular polynucleotide molecules resulting from step b) by one or more specific enzymes of the methylated DNA, preferably selected from the following group: DpnI, NanII, NmuDI and NmuEI. In an alternative embodiment, the initial circular polynucleotide molecules of step a) were amplified in ung- strains and the selection c) is performed by transforming ung + strains by circular polynucleotide molecules resulting from step b). . In an additional embodiment, the primer oligonucleotide has one or more mismatched bases that deletes, in the newly synthesized circular polynucleotide molecules, a restriction site of an endonuclease present in the initial circular polynucleotide molecules of the step a), and the selection c) is performed by digesting the initial circular polynucleotide molecules with said restriction enzyme.

  In a preferred embodiment, the number of polynucleotides of different interest is between 2 and 1011, preferably between 2 and 106.

  Preferably, the elongation steps have durations between 0 s and 100 s, and preferably between 0 s and 5 s. In a preferred embodiment, the elongation is carried out by a thermostable polymerase, preferably selected from the group of DNA polymerases Taq, Pfu, Vent, Pfx, KD or a mixture thereof. In a particular embodiment, a thermostable DNA ligase (preferably: Taq Ligase, Tth Ligase or Amp Ligase) is added to said mixture.

  Preferably, the polynucleotides of interest have a sequence identity greater than 50%, and preferably greater than 85%. In a particular embodiment, the polynucleotides of interest are derived from the homologous cloning of a gene in several different organisms. In an alternative embodiment, the polynucleotides of interest are produced by direct cloning - by so-called DNA metagenome techniques from an environmental source. In another embodiment, the polynucleotides of interest are produced by a mutagenesis technique. The mutagenesis technique used may be random mutagenesis, site-directed mutagenesis, or Massive Mutagenesis.

  Preferably, the circular polynucleotide molecules are double-stranded, preferably they are a plasmid. In a preferred embodiment, the circular polynucleotide molecules essentially differ only by the polynucleotides of interest.

  More particularly, the primer oligonucleotide has a size of between 6 and 100 bases, and preferably between 15 and 25 bases. In a preferred embodiment, the method uses a single primer oligonucleotide. In an alternative embodiment, the method employs two or more primer oligonucleotides, preferably between two and five primer oligonucleotides. Preferably, the primer oligonucleotide hybridizes to the circular polynucleotide molecules outside the polynucleotides of interest. Preferably, the primer oligonucleotide is phosphorylated.

  In a particular embodiment, the circular polynucleotide molecules resulting from step c) are resubmitted in step b).

  In a preferred embodiment, the resulting recombinant polynucleotides of interest are then subjected to a low-throughput, high-throughput or ultra-high-throughput screening step. Preferably, the screening step is a functional selection step. The functional selection method can be selected from the following techniques: selection on the surface of a ribosome ribosome display, selection on the surface of a phage phage display, selection on the surface of a cell cell-surface display, selection to the surface of a plasmid display plasmid, mRNA-peptide fusion, in vitro compartmentalization, compartmentalized self-replication, and in vivo selection.

detailed description

  The method according to the invention is a process of in vitro recombination of homologous sequence genes characterized by its ease of manipulation. In particular, the present invention relates to a method of in vitro recombination of parent plasmids characterized in that the recombination takes place directly between the circular molecules of the parent plasmids and therefore without any molecular cloning and in that at least one complementary primer oligonucleotide or partially complementary to a region of one of the strands of the parent plasmids is used to initiate a cyclic linear amplification reaction by a polymerase, preferably thermostable, with very short elongation steps producing plasmid son molecules that combine one or more sequences of one or more parent plasmids. The method preferably comprises the following steps: a) In a tube,

  a mixture containing: Circular DNA molecules, preferably double-stranded, of different natures, preferably present in equimolar concentrations; At least one oligonucleotide of sufficient size for hybridization, and therefore preferably between 6 and 100 bases, and more preferably still between 15 and 30 bases, and homologous to a region common to the DNA molecules, or a region being sufficiently homologous on the templates to allow hybridization of the oligonucleotide; and a polymerase, preferably thermostable, and all the reagents (in particular substrates, buffers and cofactors) allowing the polymerization of DNA.

  b) The mixture prepared in a) is subjected to several elongation cycles, preferably comprising: a high temperature phase so as to dissociate the DNA strands with each other; - a lower temperature phase so that the oligonucleotide (s) primer (s) hybridizes (s) on its binding site at different plasmids; and an elongation phase, the elongation time for at least one of the cycles is adapted so that the elongation is interrupted at the level of the part to be recombined. This elongation time can be calculated from the rate of procession of the polymerase.

  c) Preferably, the reaction obtained in b) is subjected to a step of selecting newly synthesized strands. This selection is essentially done by eliminating the parent strands.

  The circular DNA molecules described in step a) preferably consist of a part of a vector (preferably identical for all the molecules, and containing an antibiotic resistance gene and the elements sufficient for its replication ) and on the other hand an insert consisting essentially of the polynucleotide of interest. The vector is preferably a plasmid. The polynucleotide of interest is preferably constituted by a coding phase. It may also be a polynucleotide exhibiting a biological activity such as a promoter, an antisense, a ribozyme, etc. This polynucleotide of interest is different from one DNA molecule to another. The vector may further contain a promoter allowing the expression of the insert in prokaryotic or eukaryotic organisms or in vitro expression. If the polynucleotide of interest is a promoter, it will preferably be operably associated with a coding sequence, for example a reporter gene coding sequence. Preferably, the insertion of the (different) inserts will have been made at the same site in the vector (single sites present at the level of the cloning multisite), or at neighboring sites.

  In step a), the polynucleotides of interest have close sequences, preferably with a degree of similarity or identity greater than 50%, 60% or 70%, and even more preferably greater than 80%, 85% , 90%, 95% or 99%. In a first embodiment, the polynucleotides of interest present in step a) are genes of the same family, cloned by homology cloning from different organisms or by isolation of the genes from cultures of organisms. in vitro, either by a technique of direct isolation of homologous genes from DNA isolated from environmental samples (approach still called metagenome). In this embodiment, the objective is to recombine these genes to obtain variants with improved or cumulative properties, or new characteristics.

  In a second embodiment, the polynucleotides of interest are mutants of the same parental gene, differing from it, and between them, only by a limited number of modifications. In this second embodiment, the objective is to recombine these previously selected variants, in order to obtain one or more secondary variants resulting from the recombination of these primary variants, these secondary variants having an even higher level of improvement. These polynucleotides of interest are present at a total concentration in the mixture of step a) preferably between 1 and 100 ng / l.

  In a preferred embodiment, the circular DNA molecules used in step a) are double stranded. In an alternative embodiment, the circular DNA molecules used in step a) are single-stranded.

  In another particular embodiment, the circular DNA molecules used in step a) are composed in part of non-conventional bases, such as, for example, deoxy-uridine, making it possible to implement in step c) certain efficient selection modes of newly synthesized molecules.

  In step a), the oligonucleotide or primers used are preferably complementary to a region of the starting circular DNA molecules which is located on the vector, at a variable distance from the polynucleotide of interest. These primer oligonucleotides are present at a concentration in the mixture of step a) preferably between 0.01 M and 10 M.

  In step b), the heat-stable polymerase used is, for example, a polymerase or a mixture of polymerases selected from: Taq, Pfu, Vent, Pfx, and KOD. Preferably, a polymerase having good read fidelity is used, the generation of random mutations not being sought here. In a preferred embodiment, a thermostable ligase (eg, Taq Ligase, Tth Ligase, or Amp Ligase), buffer, and optional co-factors are added to the mixture described in step a). In the latter case, the oligonucleotide or oligonucleotide (s) used for initiating the polymerization will have or will have been preferentially phosphorylated using a polynucleotide kinase under appropriate conditions. Alternatively to this enzymatic pathway, phosphorylation of the oligonucleotide or oligonucleotides can be made by chemical coupling.

  In step b), the melting temperature of the two DNA strands can conventionally be 94 ° C., this temperature being known to dehybridize all the strands of double-stranded DNA. The melting time is not discriminating, and is preferably between one second and two minutes.

  In this same step b), the hybridization temperature of the oligonucleotide (s) on the plasmid strands can conventionally be chosen to be around 50 ° C. Indeed, most oligonucleotides of conventional size (20 mer) hybridize efficiently on their matrix at such a temperature. In the first cycle, the only hybridization sought is that of the oligonucleotide (s) serving as a primer on its homologous sequence or sequence of the parental plasmid strands. During this first cycle, the hybridization time is not discriminating, and is preferably between one second and one minute. In the subsequent cycles, the hybridization of the strands obtained during the previous cycles, of a length of several tens to several thousand bases, is sought in priority, and it may be advantageous to use a temperature and a hybridization time which favors the hybridization of these strands with respect to the hybridization of the starting oligonucleotide (s). Indeed, when these single-stranded extension products, obtained during previous cycles by extension on a parental matrix x, hybridize on a parental matrix y, a new extension begins on this matrix y; this hybridization is more or less homologous, on the 3 'side of the neosynthesized strand, and may therefore require particular hybridization conditions, and in particular a suitable hybridization temperature, and a longer duration of hybridization, of several minutes, or even of several tens of minutes. Optionally, several hybridization temperatures may be used, so as to allow strands with different degrees of homology to hybridize, each at their optimum temperature.

  The elongation temperature used during this step b) is conventionally between 68 and 72 ° C., the temperature range of maximum efficiency of the thermostable polymerases. In general, the temperature to be used here is the optimum temperature of activity of the thermostable polymerase used. The duration of this elongation step is very discriminating. Indeed, it is desired that the elongation of the DNA strand is such that it is interrupted upon arrival at the non-homologous zone, where it is desired to recombine parental strands. Since the elongation is not totally synchronous, the interruption will not take place exactly at the same level from one molecule to another, and real diversity will therefore be generated in the recombined strands. To do this, it is possible to measure the distance between the hybridization zone of the primer oligonucleotide and the region where it is desired to interrupt the synthesis, and to calculate the time required to cross the distance between the oligonucleotide and the targeted zone, the rate of progression of the polymerase being known (this speed depends on the exact nature of the enzyme used, and is generally indicated by the supplier). For example, if 1500 bases separate the oligonucleotide (at the 3 'end of which will be initiated the polymerization of the neoformed strand), and the region where it is desired that the polymerase is interrupted, and if the polymerase used has a speed of 750 bases per minute, two minutes of elongation will be necessary. In general, the polymerization does not really begin when the temperature reaches the plateau (at 68 or 72 C for example), but gradually during the temperature rise phase. It is therefore necessary to take this element into account in order to calculate the duration necessary for this phase of elongation. Most often, it is preferable to identify the optimal duration empirically: then, in a preliminary phase, several tests are carried out in order to identify the optimal duration allowing to obtain a high level of recombined molecules, and / or a number of maximum recombination events per molecule.

  The number of elongation cycles is generally between 2 and 200, preferably between 5 and 50. These cycles may all be identical or the lengths and temperatures of the hybridization and elongation phases may be different depending on the cycles.

  In a particular embodiment, the elongation cycles performed in step b) can be followed by a series of additional cycles, associated with a longer elongation time, the function of which is to complete the circular replication around of the plasmid serving as a template, and optionally to allow the ligation of the 3 'end of the neoformed strand on the phosphorylated 5' end of the oligonucleotide (s) having served as a primer.

  The oligonucleotide (s) used for priming may or may be homologous to a region situated at a variable distance from the zone not being homologous between the parental strands, more particularly the region comprising the polynucleotide of interest.

  In a first embodiment, the oligonucleotide (s) used for priming is located as far as possible from the zone where it is desired to interrupt the synthesis. Thus, as the distance to be traveled is the longest possible, it is expected that the variations in the precise stall zone, from one molecule to another, will also be maximal, the variability being conserved as a percentage of the distance traveled, but increased if one measures in number of bases. In a second embodiment, on the other hand, the oligonucleotide (s) serving as priming is located as close as possible to the non-homologous part of the parental strands, that is to say of the region comprising the polynucleotide of interest. The elongation time is minimal, for example one second, or even zero. Thus, there is no more phase of elongation itself, but only a rise in temperature between the hybridization temperature and the denaturation temperature. The polymerization takes place during the rise in temperature, and the slope (in C / s) of this rise is then decisive. The choice of volumes subjected to thermocycling, the tube or plate used, the thermocycler and the program are therefore critical and may need to be adjusted empirically. In a preferred embodiment, the method uses a single primer oligonucleotide. In an alternative embodiment, the method uses a plurality of primer oligonucleotides. In a particular embodiment, the primer oligonucleotides all hybridize on the same strand. Preferably, they will hybridize at different sites distributed on the vector at variable distances of the polynucleotide of interest. Thus, the diversity of the recombination sites can be increased. In another particular embodiment, the primer oligonucleotides hybridize on both strands of the vector. In a preferred embodiment, at the end of the recombination step, a series of elongation cycles is carried out, comprising a phase at a temperature permitting the activity of the polymerase and optionally of the ligase, of a duration sufficient to allow the plasmid to be turned round and possibly the subsequent binding to the 5 'end, preferably phosphorylated, of the oligonucleotide used for priming.

  In step c), any means known to those skilled in the art for selecting the neo-synthesized strands are used. Indeed, the parental strands used in the mixture are still, at the end of the reaction carried out in b), a large fraction and most often the majority of DNA strands present in the mixture. In a first embodiment, an approach based on the differences in methylation between the DNA synthesized in bacteria (methylated) and the DNA synthesized in vitro (unmethylated) is used. It is a question of using the enzyme DpnI, specific for sites present on the methylated DNA, but not on the unmethylated DNA (Lacks et al., Methodes in Enzymology, 1980; 65: 138). The enzymes NanII, NmuDI and NmuEI can also be used to fulfill the same purpose. In a reaction by circular extension of a hybridized oligonucleotide on a plasmid, these enzymes digest the parental strands (produced in vivo by bacteria), but not the strands synthesized during the reaction, unmethylated. In a second embodiment, one of the oligonucleotides used for priming is homologous to a region containing a unique restriction site, and differs at one or more nucleotides, preferably located at its central portion. . The neosynthesized strand will therefore not carry this restriction site. A digestion step using a suitable restriction enzyme, that is to say specific for said restriction site, will have the effect of making linear the parental matrices, but not the neosynthesized strand, which alone can be efficiently transformed into competent bacteria. In a third embodiment, the parent matrices were generated from cultures obtained in dutung- (dUTPase deficient) bacteria, introducing into the synthetic DNA a number of deoxy-uridine instead of the deoxy-thymidines. During the reaction of step b), the neosynthesized DNA strands will, on the contrary, consist only of the four conventional bases, including deoxy thymidine. Transforming the reaction mixture into a ung + bacterial strain will have the effect of eliminating the parental strands (most of the strains conventionally used in the laboratory are ung +, these bacteria possess an enzyme which selectively destroys DNA strands carrying deoxyuridines) . This experimental approach is analogous to that described by Kunkel in the context of site-directed mutagenesis (Kunkel, TA Proc Natl Acad Sci USA 1985, 488-92). These three selection systems, which in no way constitute an exhaustive list, can be used in combination in order to obtain an optimal level of selection.

  In a preferred embodiment, the reaction mixture obtained in c) is intended to be transformed into competent bacteria, using any method for effectively transforming DNA into bacteria, and preferably a method of transformation. by thermal shock or a method of transformation by electric shock (electroporation). In the case of electroporation, it is necessary to get rid of the majority of the salts present in the mixture, for example by membrane dialysis or by using suitable columns (for example the MinElute PCR Purification Kit from Qiagen). The transformed bacteria can then be spread on a solid culture medium, containing a selection agent, so as to obtain individualized bacterial colonies. Each of these colonies most often contains a single plasmid, some of which correspond to one of the parental plasmids (products not sought here), and for others to plasmids containing fragments belonging to several different parental plasmids, one or more recombination events having then taken place.

  *** Embodiment 1: Circular in vitro recombination of gene variants obtained by mutagenesis; selection by Dpnl.

  In a first embodiment, the method according to the invention is characterized by the in vitro recombination of variants of a gene obtained by mutagenesis and by the succession of the following steps: a) A gene of interest G1 cloned is available in a suitable vector: a plasmid containing in particular, besides G1, the regions necessary for its replication, possibly for the expression of G1, and one or more selection markers, for example an antibiotic resistance gene.

  b) Any known random mutagenesis method (preferably: mutagenic PCR) is synthesized by a B1 library of variants of the G1 gene cloned into the initial plasmid. Alternatively, by site-specific mutagenesis (preferably using a commercial site directed mutagenesis kit, for example the Stratagene QuickChange TM kit or the Promega GeneEditorTM kit), a set of directed mutants of the G1 gene is synthesized. Optionally, several sets of mutants directed in parallel are made and then mixed to create the bank B1. Alternatively, and preferably, Massive Mutagenesis is synthesized by a combinatorial library B1 of many directed mutants of the G1 gene (typically 105 to 1010 different mutants).

  c) In a single tube, a mixture is produced containing: the B1 library obtained in step b); an oligonucleotide of sufficient size for hybridization, and therefore preferably between 6 and 100 bases, and more preferably still between 15 and 30 bases, and homologous to a region common to all the genes of the B1 library, preferably a region of the vector, which is unique, and not the insert, which has different variants; a thermostable polymerase, or a mixture of thermostable polymerases, and all the substrates, buffers and cofactors allowing the polymerization of DNA; optionally a thermostable ligase.

  d) The reaction prepared in c) is subjected to a number of temperature cycles (preferably 5 to 100 cycles) comprising: - a high temperature phase so as to dissociate the DNA strands with each other (typically between 80 C and 110 C, preferably 95 C); a lower temperature phase so that the oligonucleotide hybridizes on its binding site at the level of the plasmids of the B1 library (typically between 20 ° C. and 60 ° C., preferably at 50 ° C.); an elongation phase, typically at a temperature of between 60 ° C. and 80 ° C. (preferably between 65 ° C. and 75 ° C.), with a duration of preferably between 0 seconds (in which case the polymerization takes place only during the rise and the descent in temperature) and 100 seconds (when it is desired to obtain relatively long recombined regions).

  e) The reaction obtained in d) is subjected to a step of selecting the newly synthesized strands. For this purpose, the plasmids produced in step d) are digested (preferably for a period of between 10 minutes and 10 hours and at a temperature of between 25 and 45 ° C.) with a restriction enzyme specific for the methylated DNAs. preferably one or more enzymes belonging to the group of enzymes DpnI, Nanl, NmuDI and NmuEI. Even more preferentially, the reaction is digested for half an hour at 37 ° C. with the enzyme DpnI. A B2 library enriched with recombinant plasmids is obtained.

  f) Optional step. It is removed by membrane dialysis or any other suitable technique (for example phenol / chloroform extraction followed by ethanol or isopropanol precipitation or the use of a commercial purification kit). essential salts present in B2 bank.

  g) The B2 library obtained in step e) (or f) is converted by a suitable technique into cells (for example, cells of bacteria, yeasts, plants, insects, mammals or humans) . Preferably, the plasmid DNA obtained in step f) is transformed by electroporation of E. coli cells. coli.

  h) The transformed cells are grown on a selection medium corresponding to the selection marker present on the initial plasmid.

  i) The plasmid DNA is prepared from the cells that have grown. A B2 'bank is thus obtained.

  In a particular embodiment, several rounds of circular in vitro recombination are carried out (preferably 1 to 5 rounds): iterates steps c) to i) but in step c) the B1 library is not used but bank B2 'obtained at the end of step i) of the previous round.

  *** Embodiment 2: Circular in vitro recombination of gene variants obtained by mutagenesis; selection by deleting a restriction site in the amplified plasmids. *** In a second embodiment, the method according to the invention is characterized by the in vitro recombination of gene variants obtained by mutagenesis and by the following succession of steps: a) a gene is available; interest cloned in a suitable vector: a plasmid containing in particular, besides the gene of interest G1, the regions necessary for its replication, possibly for the expression of the G1 gene, and one or more selection markers, for example a gene resistance to an antibiotic.

  b) Any known random mutagenesis method (preferably mutagenic PCR) is synthesized by a method B1 of variants of the G1 gene. Alternatively, by site-specific mutagenesis (preferably using a commercial site-directed mutagenesis kit, for example Stratagene's QuickChange MultiSite ™ kit or Promega's GeneEditor ™ kit), a set of directed mutants of the G1 gene is synthesized. Optionally, several sets of mutants directed in parallel are made and then mixed to create the bank B1. Alternatively, and preferably, Massive Mutagenesis is synthesized by a combinatorial library B1 of many directed mutants of the G1 gene (typically 105 to 1010 different mutants).

  c) In a single tube, a mixture is produced containing: the B1 library obtained in step b); an oligonucleotide of sufficient size for hybridization, and therefore preferably between 6 and 100 bases, and even more preferably between 15 and 30 bases, and partially homologous to a region common to all of the genes of bank B1. This oligonucleotide, preferably homologous to a region of the vector, which is unique, and not to the insert, which has different variants, is not completely homologous and has a small number of mismatches (1 to 10, preferably 1 to 4) designed to remove in the plasmid a unique restriction site R1 (a site that had a single occurrence previously throughout the sequence of the original plasmid); a thermostable polymerase, or a mixture of thermostable polymerases, and all the substrates, buffers and cofactors allowing the polymerization of DNA; optionally a thermostable ligase.

  d) The reaction prepared in c) is subjected to a number of temperature cycles (preferably 5 to 100 cycles) comprising: - a high temperature phase so as to dissociate the DNA strands with each other (typically between 80 C and 110 C, preferably 95 C); a lower temperature phase so that the oligonucleotide hybridizes on its binding site at the level of the plasmids of the B1 library (typically between 20 ° C. and 60 ° C., preferably at 50 ° C.); an elongation phase, typically at a temperature of between 60 ° C. and 80 ° C. (preferably between 65 ° C. and 75 ° C.), with a duration of preferably between 0 seconds (in which case the polymerization takes place only during the rise and the descent in temperature) and 100 seconds (when it is desired to obtain relatively long recombined regions).

  e) The reaction obtained in d) is subjected to a step of selecting the newly synthesized strands. For this purpose, the plasmids produced in step d) are digested (preferably for a period of between 10 minutes and 10 hours and at a temperature of between 25 and 45 ° C.) with a restriction enzyme specific for the R1 site. We obtain a B2 bank. The plasmids which have just been synthesized in step d) do not carry the R1 site, are not digested and remain circular. In contrast, the initial plasmids carry the R1 site, are cleaved by the enzyme and become linear. These linearized plasmids have a very low transformation efficiency and will thus be substantially depleted in the B2 'library which will be obtained in step i).

  f) Optional step. It is removed by membrane dialysis or any other suitable technique (for example phenol / chloroform extraction followed by ethanol or isopropanol precipitation or the use of a commercial purification kit). essential salts present in B2 bank.

  g) The B2 library obtained in step e) (or f) is converted by a suitable technique into cells (for example, cells of bacteria, yeasts, plants, insects, mammals or humans) . Preferably, the plasmid DNA obtained in step f) is transformed by electroporation of E. coli cells. coli.

  h) The transformed cells are grown on a selection medium corresponding to the selection marker present on the initial plasmid.

  i) The plasmid DNA is prepared from the cells that have grown. A B2 'bank is thus obtained.

  In a particular embodiment, several rounds of circular in vitro recombination are carried out (preferably 1 to 5 rounds): iterates steps c) to i) but in step c) the B1 library is not used but the bank B2 'obtained at the end of step i) of the previous round.

  *** Embodiment 3: Circular in vitro recombination of gene variants obtained by mutagenesis; selection by use of ung- strains. *** In a third embodiment, the method according to the invention is characterized by the in vitro recombination of variants of a gene obtained by mutagenesis and by the succession of the following steps: a) a gene is available; interest cloned in a suitable vector: a plasmid containing in particular, besides the gene of interest G1, the regions necessary for its replication, possibly the expression of the G1 gene, and one or more selection markers, for example a gene of resistance to an antibiotic.

  b) Any known random mutagenesis method (preferably mutagenic PCR) is synthesized by a method B1 of variants of the G1 gene. Alternatively, by site-specific mutagenesis (preferably using a commercial site directed mutagenesis kit, for example the Stratagene QuickChange TM kit or the Promega GeneEditorTM kit), a set of directed mutants of the G1 gene is synthesized. Optionally, several sets of mutants directed in parallel are made and then mixed to create the bank B1. Alternatively, and preferably, Massive Mutagenesis is synthesized by a combinatorial library B1 of many directed mutants of the G1 gene (typically 105 to 1010 different mutants). This library is transformed into dut-ung bacteria (deficient in dUTPase), introducing into the DNA in synthesis a number of deoxy-uridine instead of deoxy thymidines. This experimental approach using particular bacteria is analogous to that described by Kunkel in the context of site-directed mutagenesis (Kunkel, TA Proc Natl Acad Sci USA 1985, 488-92). Amplified plasmid DNA is prepared by the machinery of ung- bacteria, and a B '1 library is recovered.

  c) In a single tube, a mixture is produced containing: the library B '1 obtained in step b); an oligonucleotide of sufficient size for hybridization, and therefore preferably between 6 and 100 bases, and more preferably still between 15 and 30 bases, and homologous to a region common to all the genes of the B'1 library, preferably a region of the vector, which is unique, and not the insert, which has different variants; a thermostable polymerase, or a mixture of thermostable polymerases, and all the substrates, buffers and cofactors allowing the polymerization of DNA; optionally a thermostable ligase.

  d) The reaction prepared in c) is subjected to a number of temperature cycles (preferably 5 to 100 cycles) comprising: - a high temperature phase so as to dissociate the DNA strands between them (typically between 80 C and 110 C, preferably 95 C); a phase at a lower temperature so that the oligonucleotide hybridizes on its binding site at the level of the plasmids of the library B '1 (typically between 20 ° C. and 60 ° C., preferably at 50 ° C.); an elongation phase, typically at a temperature of between 60.degree. C. and 80.degree. C. (preferably between 65.degree. C. and 75.degree. C.), of a duration preferably of between 0 seconds (in which case the polymerization takes place only during the rise and fall in temperature) and 100 seconds (when it is desired to obtain relatively long recombined regions). At the end of the amplification reaction, a B2 library containing recombinant plasmids is obtained.

  e) Optional step. It is removed by membrane dialysis or any other suitable technique (for example phenol / chloroform extraction followed by ethanol or isopropanol precipitation or the use of a commercial purification kit). essential salts present in B2 bank.

  f) The B2 library is converted by a suitable technique into E. coli cells. standard colt ung +; these bacteria have an enzyme that selectively destroys DNA strands carrying desoxy-uridines and this allows to counter-select the initial plasmids and enrich plasmids synthesized in step c).

  g) The transformed cells are grown on a selection medium corresponding to the selection marker present on the initial plasmid.

  h) Plasmid DNA is prepared from the cells that have grown. A B2 'bank is thus obtained.

  In a particular embodiment, several rounds of circular in vitro recombination (preferably: 1-5) are carried out: steps c) to h) are iterated but in step c) the B1 library is not used but a B3 library which corresponds to the B'2 library of step h) that will have been re-amplified by ung- bacteria.

  *** Embodiment 4: Circular in vitro recombination of genes obtained by metagenome techniques In a fourth embodiment, the method according to the invention is characterized by the in vitro recombination of variants of a gene. obtained by a so-called metagenome technique and by the succession of the following steps: a) One of the so-called available metagenome techniques is a B1 library of environmental genes. In a first embodiment, genes homologous to a gene of known interest are amplified by PCR and then cloned into a plasmid to obtain a B1 library. In a second embodiment, a set of environmental genes is cloned directly by shotgun cloning into a suitable plasmid, which is then selected by a functional test to obtain, after selection, a library B1.

  b) In a single tube, a mixture is produced containing: the B1 library obtained in step a); an oligonucleotide of sufficient size for hybridization, and therefore preferably between 6 and 100 bases, and more preferably still between 15 and 30 bases, and homologous to a region common to all the genes of the B1 library, preferably a region of the vector, which is unique and not the insert, which has different variants; a thermostable polymerase, or a mixture of thermostable polymerases, and all the substrates, buffers and cofactors allowing the polymerization of DNA; optionally a thermostable ligase.

  c) The reaction prepared in b) is subjected to a number of temperature cycles (preferably 5 to 100 cycles) comprising: - a high temperature phase so as to dissociate the DNA strands between them (typically between 80 C and 110 C, preferably 95 C); a phase at a lower temperature so that the oligonucleotide hybridizes on its binding site at the level of the plasmids of the B1 library (typically between 20 ° C. and 60 ° C., preferably at 50 ° C.); an elongation phase, typically at a temperature of between 60 ° C. and 80 ° C. (preferably between 65 ° C. and 75 ° C.), with a duration of preferably between 0 seconds (in which case the polymerization takes place only during the rise and the descent in temperature) and 100 seconds (when it is desired to obtain relatively long recombined regions).

  d) The reaction obtained in c) is subjected to a step of selecting the newly synthesized strands. For this purpose, the plasmids produced in step d) are digested (preferably for a period of between 10 minutes and 10 hours and at a temperature of between 25 and 45 ° C.) with a restriction enzyme specific for the methylated DNAs. preferably one or more enzymes belonging to the group of enzymes DpnI, Nanl, NmuDI and NmuEI. More preferably still, one digests for half an hour at 37 C with the enzyme DpnI. A B2 library enriched with recombinant plasmids is obtained.

  e) Optional step. It is removed by membrane dialysis or any other suitable technique (for example a phenol / chloroform extraction followed by ethanol or isopropanol precipitation or the use of a commercial purification kit). essential salts present in B2 bank.

  f) The B2 library obtained in step d) (or e) is converted by a suitable technique into cells (for example, cells of bacteria, yeasts, plants, insects, mammals or humans) . Preferably, the DNA obtained in step e) is transformed by electroporation of E. coli cells. coli.

  g) The transformed cells are grown on a selection medium corresponding to the selection marker present on the initial plasmid.

  h) Plasmid DNA is prepared from the cells that have grown. A bank B2 'is thus obtained.

  In a particular embodiment, several rounds of circular in vitro recombination (preferably: 1-5) are carried out: steps b) to h) are iterated but in step b) the B1 library resulting from metagenome but B2 'bank obtained at the end of step h) of the previous round.

  In a particular embodiment, the selection method used does not involve methylation, but the disappearance of a restriction site (see embodiment 2) or the use of particular bacterial strains (see embodiment 3).

  *** Embodiment 5: Circular in vitro recombination of homologous genes extracted from different organisms *** In a fifth embodiment, the method according to the invention is characterized by the in vitro recombination of homologous gene variants extracted from different organisms and by the succession of the following stages: a) A small number N1 (between 2 to 1000, and preferably between 3 to 50) of organisms known to be cultivated in the laboratory and whose genome contains for each one is chosen. a different version of the same G1 gene. These organisms are independently cultured, amplified by PCR using primers adapted to the G1 gene, and the N1 versions of the G1 gene present in the N1 organisms are independently cloned into N1 identical plasmids. For example, G1 can encode a thermostable protein and G1 is cloned from several organisms (such as Thermus aquaticus, Bacillus stearothermophilus, Pyrococcus species GB-D, Thermococcus, etc.) living in very warm marine environments. By mixing the N1 plasmids containing the different versions of G1, one obtains a bank B1. Preferably an equimolar mixture of the plasmids is made.

  b) In a single tube, a mixture is produced containing: the B1 library obtained in step a); an oligonucleotide of sufficient size for hybridization, and therefore preferably between 6 and 100 bases, and more preferably still between 15 and 30 bases, and homologous to a region common to all the genes of the B1 library, preferably a region of the vector, which is unique and not the insert, which has different variants; a thermostable polymerase, or a mixture of thermostable polymerases, and all the substrates, buffers and cofactors allowing the polymerization of DNA; optionally a thermostable ligase.

  c) The reaction prepared in c) is subjected to a number of temperature cycles (preferably 5 to 100 cycles) comprising: - a high temperature phase so as to dissociate the DNA strands between them (typically between 80 C and 110 C, preferably 95 C); a lower temperature phase so that the oligonucleotide hybridizes on its binding site at the level of the plasmids of the B1 library (typically between 20 ° C. and 60 ° C., preferably at 50 ° C.); an elongation phase, typically at a temperature of between 60.degree. C. and 80.degree. C. (preferably between 65.degree. C. and 75.degree. C.), of a duration preferably of between 0 seconds (in which case the polymerization takes place only during the rise and fall in temperature) and 100 seconds (when it is desired to obtain relatively long recombined regions).

  d) The reaction obtained in c) is subjected to a step of selecting the newly synthesized strands. For this purpose, the plasmids produced in step d) are digested (preferably for a period of between 10 minutes and 10 hours and at a temperature of between 25 and 45 ° C.) with a restriction enzyme specific for the methylated DNAs. preferably one or more enzymes belonging to the group of enzymes DpnI, Nanl, NmuDI and NmuEI. More preferably still, one digests for half an hour at 37 C with the enzyme DpnI. A B2 library containing recombinant plasmids is obtained.

  e) Optional step. It is removed by membrane dialysis or any other suitable technique (for example phenol / chloroform extraction followed by ethanol or isopropanol precipitation or the use of a commercial purification kit). essential salts present in B2 bank.

  f) The B2 library obtained in step d) (or e) is converted by a suitable technique into cells (for example, cells of bacteria, yeasts, plants, insects, mammals or humans) . Preferably, the DNA obtained in step e) is transformed by electroporation of E. coli cells. coli.

  g) The transformed cells are grown on a selection medium corresponding to the selection marker present on the initial plasmid.

  h) Plasmid DNA is prepared from the cells that have grown. A B2 'bank is thus obtained.

  In a particular embodiment, several rounds of circular in vitro recombination (preferably: 1-5) are carried out: steps b) to h) are iterated but in step b) the BI bank resulting from cloning of homologous genes of different organisms but B2 'bank obtained at the end of the previous round.

  In a particular embodiment, the selection method used does not involve methylation, but the disappearance of a restriction site (see embodiment 2) or the use of particular bacterial strains (see embodiment 3).

  *** Embodiment 6: Circular in vitro recombination and functional selection ** In a sixth particular embodiment, as before, we start from genes obtained either by mutagenesis or by metagenome techniques or else by cloning homologous genes in different then the method is obviously adapted for those skilled in the art so that at the end of the last step of the embodiments described above, the recombinant plasmid library can be subjected to a method of functional selection in vivo or in vitro. Preferably, the selection method used is one of the following methods: in vivo selection, phage display, cell-surface display, compartmentalized self-replication, in vitro compartmentation, ribosome display, mRNA-peptide fusion, mRNA display, plasmid display . Plasmids obtained following the application of this functional selection method (and possible cloning) can be used for individual expression to isolate a small number of significantly improved variants or used directly for a new circular in vitro recombination round. Preferably, a functional selection method is used which takes place directly on the circular plasmids so that several rounds of circular in vitro recombination / functional selection can be linked quickly and economically.

  *** Embodiment 7: Circular in vitro recombination using several oligonucleotides * In a seventh embodiment, the method according to the invention is characterized by the use, within the mixture which undergoes the circular amplification reaction. recombinant, of several oligonucleotides pairing at different locations along the sequence of the plasmids to be recombined. This makes it possible to increase the frequency of the recombination events (ie to decrease the average distance between two recombination points).

  In a particular embodiment, the process according to the invention is characterized by the use, within the mixture which undergoes the recombinant circular amplification reaction, of one or more oligonucleotides corresponding to one of the strands of the plasmids to recombine while one or more other oligonucleotides pair with the other strand. This makes it possible to increase the proportion of recombinant plasmids relative to parental plasmids in the product obtained after amplification.

Examples

  Example 1 Recombination of two mutated matrices

  A first study model, intended to measure the recombination efficiency between two completely identical sequence matrices except for two positions, has been put in place. Both parental matrices were composed of the human IL15 gene inserted into the plasmid pORF (Invivogen).

  Parental matrix A contained a mutation at position 457, canceling a BsrGI restriction site, unique over the entire plasmid and insert.

  Parental matrix B contained a mutation at position 208, canceling a MsII restriction site, unique over the entire plasmid and insert.

  The oligonucleotide used for initiation of the polymerization, IL15 (A), was homologous to a 25 base region after the IL15 initiation ATG. Its sequence, from 5 'to 3', was GTATTTCCATCCAGTGCTAC (SEQ ID No. 1).

  In a preliminary step, 1.5 nmol of oligonucleotide IL15 (A) was enzymatically phosphorylated in a reaction volume of 20 L, using a sufficient amount of phosphonucleotide kinase and its buffer, and a final concentration in the reaction of 1 mM ATP. The reaction mixture was incubated for one hour at 37 ° C. The enzyme was then inactivated by incubating the mixture at 65 ° C for 20 minutes.

  parental matrix A (75 ng / l) 1 l parental matrix B (75 ng / l) 1 l IL15 (A) phosphorylated oligonucleotide (diluted to 0.75 M) 1 l dNTP (25 mM) 1 l ATP (10 mM) 1 μM MgSO 4 (100 mM) 1 μ buffer 10 × pfu pol 2.5 μl T th ligase 0.8 μM NAD (100 mM) 0.2 μl The following reaction mixture was then carried out: dTT (1 M) 0.2 0.8 ml of polymerase 0.5 l Distilled water 14 l Total 25 l A second reaction mixture, similar but not including an oligonucleotide (negative control), was carried out in parallel.

  Both reaction mixtures were then subjected to a series of five temperature cycles such as [94 C, 1 sec; 50 C, 1 sec; 68 C, 1 sec], followed by twelve temperature cycles such as [94 C, 1 sec; 50 C, 1 sec; 68 C, 20 min].

   1 of each reaction mixture were then taken and placed in a new tube. 4 μl of buffer 4 (NEB), 0.5 μl of Dpn I enzyme at 20,000u / ml (NEB), and 10.5 l of distilled water were added thereto.

  After thirty minutes of incubation and twenty minutes of inactivation at the temperature (65 ° C.), 4 μl of each reaction mixture were taken and placed on a dialysis membrane (Millipore).

  After fifteen minutes, 4 μl of each dialyzed reaction is used to transform electrocompetent DH10B bacteria using standard conditions. After an incubation period of one hour in a SOC medium containing no selective agent, the transformed bacteria were spread on a petri dish containing ampicillin.

  The next day, 1380 colonies were counted on the petri dish corresponding to the complete reaction mixture, while only 6 colonies were counted on the box corresponding to the negative control. Ninety-six colonies were individually subcultured and incubated with shaking in 100 l of ampicillin-containing liquid medium.

  PCR reactions were then carried out on these cultures, using two oligonucleotides of opposite direction, flanking the coding region, and respectively designated IL15 + (CTTGGAGCCTACCTAGACTCA (SEQ ID No. 2), homologous to a region located approximately 270 bp before the codon ATG); and IL15- "(TGTCTCGGTGTTGACCAAAGG (SEQ ID NO: 3), homologous to a region about 100 bp after the STOP codon.) The amplified fragment using these oligonucleotides had a size of 1079 bp.

  The amplified DNA fragments were individually subjected to digestion by the BsrGI enzyme on the one hand and by the Ms1I enzyme on the other hand. The digests were migrated on an agarose gel. In this way, the presence of each of the two restriction sites could be measured for each of the 96 clones, and each clone could be classified into one of four possible categories: BsrG IR Msl Is; BsrG Is Msl IR; BsrG IR Msl IR; BsrG Is Ms1 Is (the exponent R here means that the amplified fragment was resistant to digestion with the restriction enzyme, the exponent S means that said fragment was sensitive thereto). The first two of these categories corresponded to the parental matrices. The last two categories corresponded to clones that had at least one recombination event.

  The two parent populations BsrG IR Msl Is and BsrG Is Ms1 IR accounted for 44% and 32% of the population (41 and 30 colonies out of 96 respectively). The recombinant BsrG IR Ms1 IR and BsrG Is Ms1 Is populations represent respectively 20% and 3% of the total population (respectively 19 and 3 clones out of 96). Their precise nature was verified by sequencing a statistical sample of these recombinant clones.

  A significant frequency of recombinant clones (23%) was therefore observed in this experimental example, which thus demonstrates the feasibility of the technology according to the invention.

  Example 2 Evolution of an Enzyme by In Vitro Evolution and Recombination

  We start from a plasmid containing a gene coding for a protein whose activity we wish to increase. A library of plasmids containing variants of this gene of interest, each carrying a variety of point mutations, is rapidly constructed by Massive Mutagenesis (WO0216606). These genes are expressed and screened to isolate a fraction of positive plasmids containing genes encoding proteins with improved activity. The precise sequence of these genes is not known, but we know that their sequences are quite similar, and identical to the original gene apart from various point mutations. This P1 mixture of positive plasmids is diluted to a concentration in the region of 100 ng / g.

  A primer oligonucleotide A1 complementary to the region located elsewhere than on the gene of interest is preferably chosen, preferably at the level of the plasmid vector. This oligonucleotide is synthesized and then 1.5 nmol of the oligonucleotide Al is phosphorylated in a reaction volume of 20 l, using a sufficient amount of phosphonucleotide kinase and its buffer, and a final concentration in the reaction of 1 mM ATP. . The reaction mixture is then incubated for one hour at 37 ° C. The kinase enzyme is then inactivated by incubating the mixture at a temperature of 65 ° C. for 20 minutes.

  Mixture of parental matrices P1 (100 ng / l) 2 l phosphorylated Al oligonucleotide (diluted to 0.75 M) 1 l dNTP (25 mM) 1 l ATP (10 mM) 1 l MgSO4 (100 mM) 1 gl Buffer 10x pfu pol 2.5 l Tth ligase 0.8 l NAD (100 mM) 0.2 1 dTT (1 M) 0.2 l Pfu polymerase 0.8 l Taq polymerase 0.5 g Distilled water 14 l Total 25 l The reaction mixture is then subjected to a series of five temperature cycles such as [94 C, 1 sec; 50 C, 1 sec; 68 C, 1 sec], followed by twelve temperature cycles such as [94 C, 1 sec; 50 C, 1 sec; 68 C, 20 min].

  After these temperature cycles, 25 μl of the reaction mixture are taken and placed in a new tube. 4 μl of buffer 4 (NEB), 0.5 μl of Dpn I enzyme at 20,000 μ / ml (NEB), and 10.5 μl of distilled water are added thereto. After thirty minutes of incubation and twenty minutes of thermal inactivation (65 C), 4 l of the reaction mixture are removed and arranged on a dialysis membrane (Millipore). After 15 minutes, the following reaction mixture is then carried out: 15 μl of each dialyzed reaction is used to transform electrocompetent DH10B bacteria using standard conditions. After an incubation period of one hour in a SOC medium containing no selective agent, the transformed bacteria are spread on a petri dish containing ampicillin and incubated at 37 C. The next day, we recover the petri dish and 940 colonies are taken and individually incubated in 96-well plates for three hours under agitation in 100 μl of liquid medium containing ampicillin to allow protein expression. Care is taken to place two controls in wells of known localization: a culture medium alone and a culture medium containing a clone carrying the plasmid of initial interest, which will therefore produce an enzyme having the basic activity. After expression, the enzymatic activity of the protein of interest present in each well is individually tested and compared with that of each of the two controls. Clones whose enzymatic activity is measured as significantly greater than that of the initial clone are recovered.

  Example 3 Recombination of natural genes sharing a certain degree of homology.

  A set of homologous genes from different organisms is cloned into the same plasmid containing an ampicillin resistance marker gene. This mixture P 1 is diluted to a concentration of 100 ng / l. The concentration of parental matrices P 1 is in total DNA amount per gl (for example, in the case of an equimolar mixture of 4 genes, 100/4 = 25 ng / l of each of the genes is introduced).

  A primer oligonucleotide A1 complementary to the region located elsewhere than on the genes of interest, preferably at the level of the plasmid vector, is chosen. This oligonucleotide is synthesized and then 1.5 nmol of the oligonucleotide Al is phosphorylated in a reaction volume of 20 l, using a sufficient amount of phosphonucleotide kinase and its buffer, and a final concentration in the reaction of 1 mM ATP. . The reaction mixture is then incubated for one hour at 37 ° C.

  The kinase enzyme is then inactivated by incubating the mixture at 65 C for 20 minutes.

  The following reaction mixture is then carried out: Mixture of parental matrices P1 (100 ng / gl) 2 l phosphorylated Al oligonucleotide (diluted to 0.75 M) 1 gl dNTP (25 mM) ATP (10 mM) MgSO4 (100 mM) Buffer 10x pfu pol Tth ligase NAD (100 mM) dTT (1 M) Pfu polymerase Taq polymerase Distilled water 1 l 1 l 1 l 2,5 l 0,8 l 0,2 gl 0,2 pl 0,8 l 0,5 l 14 g Total 25 l The reaction mixture is then subjected to a series of five temperature cycles such as [94 C 1 sec; 50 C 1 sec; 68 C 1 sec], followed by twelve temperature cycles such as [94 C 1 sec; 50 C 1 sec; 68 C 20 min].

  After these temperature cycles, 25 l of the reaction mixture are taken and placed in a new tube. 4 μl of buffer 4 (NEB), 0.5 μl of DpnI enzyme at 20,000 μ / ml (NEB), and 10.5 μl of distilled water are added thereto. After thirty minutes of incubation and twenty minutes of thermal inactivation (65 C), 4 l of the reaction mixture are removed and arranged on a dialysis membrane (Millipore). After fifteen minutes, 4 μl of each dialyzed reaction is used to transform electrocompetent DH10B bacteria using standard conditions. After an incubation period of one hour in a SOC medium containing no selective agent, the transformed bacteria are spread on a petri dish containing ampicillin and incubated at 37 ° C.

  The next day, the petri dish is recovered and 48 colonies are collected for quality control purposes and are individually incubated for three hours under stirring in 100 l of liquid medium containing ampicillin. The plasmid DNA of these 48 clones is prepared and the region of interest is sequenced using appropriate oligonucleotides. The analysis of the sequences obtained must show that the library constructed by in vitro recombination of variants of a gene from different organisms contains genes with several recombinations between the initial variants.

  Several hundreds or thousands of the recombinant genes are then blinded (i.e. without their sequence being previously known) using an activity test. Genes with a modification of the studied property are then sequenced and characterized more completely.

Legends of the figures.

  Figure 1. Principle of circular in vitro recombination.

  A set of parent plasmids (pl, p2) are identical for their vector part (origin of replication, antibiotic resistance gene, ... solid line) and carry as insert different versions (having a certain degree of similarity sequence) of a gene of interest (hatching). To simplify the diagram, we chose to illustrate a case where there are only 2 parent plasmids; in reality, there may be a large number of different parent plasmids. This set of plasmids is amplified with very short elongation cycles using a single primer oligonucleotide. The recombined products obtained (progeny: p3, p4, p5, p6) all have the common portion of the starting plasmids (solid lines) and their inserts (hatching) combine the inserts of the starting plasmids. Thus, for example, the gene of interest of p3 has a sequence which is the juxtaposition of the beginning of the sequence of the pl insert and the end of the sequence from p2. For plasmid p4, there is also a unique recombination between p1 and p2 but the order is reversed. Plasmids p5 and p6 illustrate the types of products that can be obtained when there are two recombination events, with sequences of the type (beginning of the insert of the central part of the insert of p2 end of the insert of pl) and (beginning of the insert of p2 central part of the insert pl pl end of the insert of p2) reciprocally. It is possible according to this principle to obtain a very large number of different products depending on the position of the recombinations and their number.

  Figure 2. Principle of circular recombination by short cycle elongation.

  (b) In this example, four parent plasmids p1, p2, p3 and p4 differ from a single primer oligonucleotide complementary to or partially complementary to one strand of these four plasmids.

  (c) Thermostable polymerase, optionally thermostable ligase and deoxyribonucleotide triphosphate are added to a suitable buffer and the mixture is thermocycled. In the first cycle, the primer oligonucleotide hybridizes with p1 and a portion of p1 is copied, generating a plasmid fragment complementary to a strand of p1. In the second cycle, after detaching, this portion of plasmid hybridizes with p2 and after polymerization a complementary recombinant molecule of a fragment of a strand of p1 and a fragment of a strand of p2 is obtained. In the third cycle, after being detached, this molecule hybridizes with p3 and a recombinant molecule complementary to a fragment of a strand of p1, a fragment of a strand of p2 and a fragment is obtained. one strand of p3. At the end of a fourth cycle of denaturation, hybridization, elongation, a complete plasmid is obtained, which recombines pl, p2, p3 and p4.

  Figure 3. Circular recombination of point mutants.

  (a) Point-directed mutants of a single plasmid were made using oligonucleotides 1, 2, 3 and 4.

  (b) A collection of 4 simple mutants is obtained.

  (c) The method according to the invention makes it possible to recombine these simple mutants to obtain double, triple and quadruple mutants. The product mixture also contains wild-type plasmids as well as simple mutants.

Claims (27)

Claims.   In vitro production process for polynucleotides of interest of at least two different polynucleotides of different interest, characterized in that it comprises: a) preparing a mixture comprising circular polynucleotide molecules each comprising one of said polynucleotides, different interest, and at least one primer oligonucleotide hybridizing to a conserved region in said circular polynucleotide molecules; b) subjecting the mixture to several elongation cycles each comprising an elongation phase, and the elongation time for at least one of the cycles is adapted so that the elongation is interrupted at said polynucleotides of interest; said method resulting in the production of newly synthesized circular polynucleotide molecules comprising recombinant polynucleotides of interest.   2. Method according to claim 1, characterized in that the method further comprises a step c) of selecting the newly synthesized circular polynucleotide molecules.   3- Method according to claim 1 or 2, characterized in that the number of polynucleotides of different interest is between 2 and 1011, preferably between 2 and 106.   4. Process according to any one of claims 1 to 3, characterized in that the elongation steps have durations between 0 s and 100 s, and preferably between 0 s and 5 s.   5. Method according to any one of claims 1 to 4, characterized in that said polynucleotides of interest have a sequence identity greater than 50%, and preferably greater than 85%.   6. Process according to any one of claims 1 to 5, characterized in that said circular polynucleotide molecules are double-stranded, preferably a plasmid.   7- Method according to claim 6, characterized in that said circular polynucleotide molecules differs essentially only by the polynucleotides of interest.   8- Method according to any one of the preceding claims, characterized in that said primer oligonucleotide has a size of between 6 and 100 bases, and preferably between 15 and 25 bases.   9- Method according to any one of the preceding claims, characterized in that the method uses a single oligonucleotide primer.   10. Process according to any one of claims 1 to 8, characterized in that the process uses two or more primer oligonucleotides, preferably between two and five primer oligonucleotides.   11- Method according to any one of the preceding claims, characterized in that the primer oligonucleotide hybridizes on said circular polynucleotide molecules outside the polynucleotides of interest.   12- Method according to any one of the preceding claims, characterized in that the elongation is carried out by a thermostable polymerase, preferably selected from the group of DNA polymerases Taq, Pfu, Vent, Pfx, KD or a mixture thereof. this.   13. A process as claimed in any one of the preceding claims, characterized in that a thermostable DNA ligase (preferably: Taq Ligase, Tth Ligase or Amp Ligase) is added to said mixture.   14- Method according to any one of the preceding claims, characterized in that the primer oligonucleotide is phosphorylated.   15- Method according to any one of the preceding claims, characterized in that the selection c) is carried out by digestion of the circular polynucleotide molecules resulting from step b) by one or more specific enzymes of the methylated DNA, preferably selected from the following group: DpnI, NanII, NmuDI, NmuEI.   16- Method according to any one of the preceding claims, characterized in that the initial circular polynucleotide molecules of step a) have been amplified in ung- strains and that the selection c) is carried out by transforming ung + strains by circular polynucleotide molecules resulting from step b).   17. The method as claimed in claim 1, wherein said primer oligonucleotide has one or more mismatched bases which remove, in the newly synthesized circular polynucleotide molecules, a restriction site of an endonuclease present in the initial circular polynucleotide molecules of step a), and in that selection c) is performed by digesting the initial circular polynucleotide molecules with said restriction enzyme.   18- Method according to any one of claims 1 to 17, characterized in that said polynucleotides of interest are derived from the homologous cloning of a gene in several different organisms.   19. A method according to any one of claims 1 to 17, characterized in that said polynucleotides of interest are produced by direct cloning - by so-called metagenome techniques - of DNA from an environmental source.   20- Method according to any one of claims 1 to 17, characterized in that said polynucleotides of interest are produced by a mutagenesis technique.   21- Method according to claim 20, characterized in that the mutagenesis technique used is random mutagenesis, site-directed mutagenesis or Massive Mutagenesis 20.   22. A method according to any one of claims 2 to 17, characterized in that the circular polynucleotide molecules resulting from step c) are subjected again to step b) of claim 1.   23. Method according to any one of the preceding claims, characterized in that said resultant recombined polynucleotides of interest are then subjected to a low-throughput, high-throughput or ultra-high-throughput screening step.   24. A method according to any one of the preceding claims, characterized in that said resulting recombined polynucleotides of interest are subjected to a functional selection step.   25. A method according to claim 24, characterized in that the functional selection method is selected from the following techniques: selection on the surface of a ribosome ribosome display, selection on the surface of a phage phage display, selection on the surface. of a cell-surface display cell, selection on the surface of a plasmid display plasmid, mRNA-peptide fusion, in vitro compartmentalization, compartmentalized self-replication, and selection in vivo.   A library of polynucleotide sequences of recombinant interest produced by the method of any one of the preceding claims.   27- Protein bank derived from the expression of a library according to claim 26.