WO2000071730A1 - Method for inserting a nucleic acid fragment in a vector, uses thereof and kits therefor - Google Patents

Abstract

The invention concerns a method for inserting a nucleic acid fragment in a vector, consisting in: (a) preparing a double-stranded nucleic acid molecule comprising the nucleic acid fragment to be inserted in a vector and on either side of said fragment a nucleic acid label; (b) performing the denaturation of said nucleic acid molecule and of a cleaved vector whereof the 5' and 3' ends of one of the strands are complementary to said nucleic acid labels; (c) incubating said nucleic acid molecule with the cleaved vector in conditions enabling said nucleic acid labels to be hybridised on the cleaved vector; (d) copying the cleaved vector and the nucleic acid fragment; (e) ligating the newly synthesised strands with the free ends; (f) optionally repeating steps (b) through (e).

Description

 METHOD OF INSERTING A FRAGMENT OF NUCLEIC ACID INTO A VECTOR, ITS APPLICATIONS AND THE KITS FOR ITS IMPLEMENTATION.

The present invention relates to the field of molecular biology and relates more specifically to the insertion of DNA fragments into a plasmid vector.

The insertion of DNA fragments is the basis of most DNA manipulations carried out in molecular biology laboratories. The conventional strategy consists in operating a ligation reaction between a DNA fragment cleaved at its ends by restriction enzymes on the one hand, and a plasmid cleaved by the same restriction enzymes, or at least restriction enzymes generating compatible sites. This conventional system involves the combined presence of numerous restriction sites on each plasmid, so as to be able to integrate DNA fragments of various ends.

More recently, a technique for cloning nucleic acid amplified by PCR has been described in international patent application PCT No. WO9206189. This technique known as "TA-cloning®" (Invitrogen catalog 97/98 page 54) allows the insertion of DNA fragments amplified by PCR in a plasmid cleaved by a restriction enzyme releasing at each of its ends a Thymidine 3 '- outgoing. This technique is based on the fact that the PCR products contain, at their ends, a 3 '-outgoing Adenine. The complementarity between the vector-T and the PCR-A product allows efficient ligation of one into the other by means of a ligase or a topoisomerase. This technique offers the advantage of being universal, since any PCR product can be inserted into the T-vector, but it poses several Droblems: the production and conservation of T-vectors is not easy,

- the use of thermostable polymerases containing an exonuclease activity, which is necessary to obtain good fidelity during amplification, is generally not possible, this activity eliminating the 3 ′ -adenins from the PCR product ,

- the insertion of DNA fragments is not done in an oriented fashion, and multiple insertions, successive or simultaneous, are difficult.

A technique for cloning PCR products has also been described in international patent application PCT No. VJ09222649. This technique known as "CLONEAMP®" (Life Technologies catalog 97/98 page 152) proposes to amplify a DNA fragment by PCR with primers associated with sequences containing dUMP, then to treat the products of amplification with the UDG so as to obtain 3 'ends compatible with a corresponding vector. A cloning technique using ur hybridization. single-stranded insert in a single-stranded vector, having homologous labels so that both can hybridize, has been described in the PCT international patent application published under No. WO 91 07505. The technique described in this document provides steps for solid phase separation of the strands of the vector and of the insert so as to obtain single-strand products. This step requires the use of oligonucleotides coupled to biotin. This form of implementation complicates the technique and limits its advantage compared to other techniques for cloning a DNA fragment into a vector.

The present invention specifically relates to a new method of inserting an acid fraσment nucleic acid in a vector, which is universal and independent of compatibility between restriction sites. The method of the invention also makes it possible to construct oriented complementary DNA libraries, and offers the possibility of easily isolating 5 ′ parts of DNA sequences of which the 3 ′ part has known.

The method of inserting a nucleic acid fragment into a vector according to the invention is characterized in that it comprises the following steps: a) a double-stranded nucleic acid molecule comprising the acid fragment is prepared nucleic acid to be inserted into a vector, and 5 'of said fragment a first labeled nucleic acid comprising a nucleic sequence complementary to the 5' end of one of the strands of a cleaved vector, and 3 'of said fragment a second nucleic acid label comprising a nucleic sequence complementary to the 3 'end of the same strand of said cleaved vector, b) denaturing on the one hand said nucleic acid molecule and on the other hand a cleaved vector whose 5' and 3 'ends of one of the strands are complementary to said first and second labeled nucleic acids, c) said nucleic acid molecule is incubated with the cleaved vector, the 5 'and 3' ends of which are complementary to the first and sec und labeled nucleic acids, under conditions allowing the hybridization of said first and second labeled nucleic acids on the cleaved vector, d) copying:

on the one hand, the vector cleaved in the 5 ′ to 3 ′ direction from the 3 ′ end of the second label nucleic acid hybridized to the 3 ′ end of the cleaved vector, and on the other hand, the nucleic acid fragment in the 5 ′ to 3 ′ direction from the 3 ′ end of the cleaved vector, e) optionally and preferably, ligation: on the one hand, the 3 end 'of the neosynthesized strand with the vector as a template, with the 5' end of the first hybrid label nucleic acid at the 5 'end of the cleaved vector, and - on the other hand, the 3' end of the neosynthesized strand with the template the fragment to be inserted, with the 5 'end of the cleaved vector, f) optionally repeating steps (b) to (e), g) optionally, transforming a cell host to select the vectors having integrated the acid fragment nucleic acid. This step of transforming a host, for example into competent bacteria, if it is only possible, is however highly preferable.

A schematic representation of the steps of the method of the invention is given in Figure 4 in the appendix.

The nucleic acid molecule comprising the nucleic acid fragment to be integrated into a vector is prepared by amplification of said fragment by a polymerase chain reaction (PCR) from two priming oligonucleotides. As shown in Figure 1 in the appendix, each of these oligonucleotides is composed of two parts:

- a 3 ′ part homologous to a part of the fragment to be inserted. a 5 ′ part, constituting the labeled nucleic acid, comprising a nucleic sequence homologous to one of the ends of a cleaved vector. Advantageously, each labeled nucleic acid has the following characteristics:

The nucleic acid sequence homologous with the end of the cleaved vector is characterized in that it is complementary to a sequence of the vector bordered by a 3 'Thymidine (T). In other words, the sequence of the cleaved end of the vector which is complementary to said nucleic acid sequence homologous to the labeled nucleic acid is bordered without containing it by a 3 'Thymidine (T). The position of this Thymidine (T), by its homology with an Adenine (A), added during step (c) of the process of the invention, makes it possible to avoid possible problems of mismatches.

The 3 ′ end of the nucleic acid sequence homologous to the end of the cleaved vector is characterized in that: either it ends at the last nucleotide of said cleaved vector, and then the labeled nucleic acid consists only of said homologous sequence at the end of the cleaved vector, either it is at least one nucleotide longer than the sequence which is homologous to it, and then the labeled nucleic acid consists of said homologous sequence at the end of the cleaved vector, extended by 3 'of at least one nucleotide.

Consequently, the method of the invention is characterized in that in step (a) a nucleic acid molecule is prepared comprising: - the nucleic acid fragment to be inserted into a vector, on either side of said fragment, a labeled nucleic acid molecule consisting of a nucleic sequence, of the order of 10 to 150 nucleotides and preferably of the order of 15 to 100 nucleotides, homologous at one end of a cleavage vector, said homologous nucleic sequence possibly being separated from the nucleic acid fragment to be integrated by at least one nucleotide and preferably between i and of the order of 130 nucleotides.

Preferably, the nucleic acid sequence homologous with the end of the cleavage vector included in the labeled nucleic acid is characterized in that it is complementary to a sequence of the vector bordered by a 3 'Thymidine (T).

Advantageously, each nucleic acid sequence homologous with one end of the cleaved vector, included in the labeled nucleic acid is sufficiently nomologic to hybridize at said end of the cleaved vector at a temperature between 30 and 90 ° C, preferably between 30 and 70 ° C and most preferably at a temperature of 50 ° C.

An example of a labeled nucleic acid according to the invention, hybridized to the end of a cleaved vector, is represented in FIG. 2 in the appendix. This figure shows in bold:

- the T base which borders 1 end of the cleavage vector complementary to the nomologic nucleic sequence of the labeled nucleic acid, and - the G base which separates the nucleic acid fragment to be inserted from the nomologic nucleic sequence of the cleaved vector.

All the vectors, in particular plasmids, advantageously containing a marker for resistance to an antibiotic can be used in the method of the invention. The vector chosen must be cleaved at one or two restriction sites, so as to be linearized. In the case where the vector is cleaved at two restriction sites, different identical ta, the small fragment released during this cleavage must be eliminated by purification of the linear vector by any means known to those skilled in the art, such as, for example, purification on agarose gel, on a Sephacryl column.

The steps of copying (d) and possible ligation (e) of the method of the invention, also jointly designated hereinafter "combined reaction" or "combined chain reaction", consists in subjecting, after the denaturation of the step (b) and the hybridization of step (c) the nucleic acid molecule prepared in step (a) and the vector cleaves, to the action of a thermostable polymerase and of a ligase, then , advantageously, to repeat steps (b) to (e) according to a series of temperature cycles. Steps (d) and (e) can be carried out either successively or substantially simultaneously after the denaturation and then hybridization steps.

The combined reaction allows both: total replication of the cleaved vector from a primer consisting of the 3 'end of the nucleic acid molecule prepared in step (a), then simple ligation oπn of l 'end 3' newly generated on the 5 'end, phosphorylated, of the nucleic acid molecule which served as a primer. - Replication of the nucleic acid fragment from a primer constituted by the end of the cleaved vector, then single-strand ligation of the newly generated end on the 5 ′, phophorylated end, of the vector of the molecule. nucleic acid.

The ligation step can be omitted, since it is capable of being carried out naturally, after transformation, by the ligases of the bacteria. However, the absence of thermostable ligase in the reaction is associated with a lower cloning efficiency. The synthesis by a thermostable polymerase of a strand representing the full size of the vector can be difficult to carry out, also, according to a preferred form of implementation of the method of the invention, step (d) is carried out in the presence relay, that is to say phosphorylated oligonucleotides homologous from different parts of the vector distributed homogeneously on each of the two strands of the plasmid, at a distance between them of 500 to 2000 base pairs.

As indicated above, the nucleic acid molecule comprising the nucleic acid fragment to be integrated into a vector is prepared in step (a) by amplification of said fragment by a polymerization chain reaction (PCR) from two priming oligonucleotides , each composed of two parts:

- a 3 'part homologous to one end of the fragment to be inserted. a 5 ′ part, constituting the labeled nucleic acid, comprising a nucleic sequence homologous to one of the ends of a cleaved vector.

Advantageously, the 3 ′ part of each primer oligonucleotide comprises from 5 to 20 nucleotides whose sequence is sufficiently homologous to that of each of the ends of the DNA fragment to hybridize at said end at a temperature between 30 and 90 ° C. , and preferably about 50 ° C.

As indicated previously, each labeled nucleic acid entering into the constitution of the priming oligonucleotides comprises a nucleic sequence homologous to the end of the cleaved vector which is complementary to a sequence of the vector bordered by a Thymidine (T) in 3 '. Also as indicated previously, each labeled nucleic acid entering into the constitution of the priming oligonucleotides comprises a nucleic sequence homologous to the end of the cleaved vector, the 3 'end of which: either ends at the last nucleotide of said cleaved vector, and then the labeled nucleic acid consists only of said homologous sequence at the end of the cleaved vector, - either is at least one nucleotide longer than its homologous sequence, and then the labeled nucleic acid consists of said sequence homologous to the end of the cleaved vector, extended 3 ′ by at least one nucleotide. Thus, each labeled nucleic acid entering into the constitution of the priming oligonucleotides consists of a nucleic sequence, of the order of 10 to 150 nucleotides and preferably of the order of 15 to 100 nucleotides, homologous at one of the ends. of a cleaved vector, said homologous nucleic sequence possibly being separated from the 3 ′ homologous part at one end of the fragment to be inserted by at least one nucleotide and preferably between 1 and of the order of 130 nucleotide.

Advantageously, each labeled nucleic acid entering into the constitution of the priming oligonucleotides comprises a nucleic sequence homologous to the end of the cleaved vector which is sufficiently homologous to hybridize to said end of the cleaved vector at a temperature of between 30 and 90 ° C., preferably between 30 and 70 ° C and most preferably at a temperature of 50 ° C.

The invention therefore also relates to the oligonucleoids constituting primers for the preparation by PCR of a nucleic acid molecule containing a nucleic acid fragment to be inserted into a cleaved vector, capable of being used in the method described above. Such an oligonucleotide is composed of two parts: a 3 ′ part homologous at one end of the fragment to be inserted, a 5 ′ part constituting a labeled nucleic acid, comprising a nucleic sequence homologous at one of the ends of a cleaved vector. the 3 ′ part of such an oligonucleotide comprises from 5 to 20 nucleotides whose sequence is sufficiently homologous to that of one end of the nucleic acid fragment to hybridize at said end at a temperature between 30 and 90 ° C. , and preferably about 50 ° C.

The 5 ′ part constituting the tag nucleic acid of such an oligonucleotide comprises a nucleic sequence homologous to the end of a cleaved vector which is complementary to a sequence of the vector bordered by a Thymidine (T) at 3 ′.

The 5 ′ part constituting the labeled nucleic acid of such an oligonucleotide advantageously consists of a nucleic sequence, of the order of 10 to 150 nucleotides and preferably of the order of 15 to 100 nucleotides, homologous to the one of the ends of a cleaved vector, said homologous nucleic sequence possibly being separated from the 3 'homologous part at one end of the fragment to be inserted by at least one nucleotide and preferably between 1 and of the order of 130 nucleotide. The 5 'part constituting the labeled nucleic acid of such an oligonucleotide preferably comprises a nucleic sequence homologous to the end of the cleaved vector which is sufficiently homologous to hybridize to said end of the cleaved vector at a temperature between 30 and 90 ° C, preferably between 30 and 70 ° C and most preferably at a temperature of 50 ° C.

The invention also relates to a pair of primers useful for a PCR amplification reaction of a nucleic acid fragment to be inserted into a cleaved vector consisting of two preceding oligonucleotides, as well as the amplification product obtained by a reaction. PCR implemented with said pair of primers. Such an amplification product is more particularly that prepared in step (a) of the process of the invention, consisting of a double-stranded nucleic acid molecule comprising the nucleic acid fragment to be inserted into a vector, and 5 'of said fragment a first labeled nucleic acid comprising a nucleic sequence complementary to the 5' end of one of the strands of a cleaved vector, and 3 'of said fragment a second labeled nucleic acid comprising a nucleic sequence complementary to l 'end 3' of the same strand of said cleaved vector,

The method of the invention is remarkable in that it can be used to insert into a vector an unknown or heterogeneous nucleic acid fragment, for example in the case of the preparation of DNA libraries. Such a nucleic acid fragment cannot be easily amplified by PCR using flanking oligonucleotides. It is then possible in step (a) to prepare a nucleic acid molecule comprising at one of its ends an adapter constituted by the first and second labeled nucleic acids. An example of preparation of a cDNA molecule having adapters is shown in FIG. 3. These adapters can be ligated to the nucleic acid fragment to be inserted into the vector by any method known to those skilled in the art.

The method of the invention is also useful for finding the 5 'ends of an acid sequence nucleic acid. In this case, for the preparation of the nucleic acid molecule of step (a), two oligonucleotides are used, each composed of two parts:

a 3 ′ part degenerated so as to be homologous to several nucleic acid sequences, and

a 5 ′ part, previously designated label nucleic acid, comprising a nucleic sequence homologous to one of the ends of a cleaved vector.

In order to increase the yield of the insertion of the nucleic acid fragment into the vector, the method of the invention comprises the repetition of the steps: b) denaturation, c) hybridization, d) copy, e) optional ligation, defining a temperature cycle each corresponding to the reaction considered.

The method of the invention finds numerous applications such as of course the subcloning of DNA fragments obtained by PCR, or even for example the construction of cDNA libraries, the search for 5 'ends of partially cloned genes, and the construction of complex molecular combinations.

The invention also relates to a kit for implementing the method of the invention. Such a kit comprises the following products, offered separately or in the form of a mixture: a plasmid vector cleaved in its polylinker at the level of one or preferably of two incompatible sites, a series of relay oligonucleotides complementary to regions of the vector, a buffer compatible with the simultaneous activity of a thermostable polymerase and a thermostable ligase, a thermostable polymerase preferably having an additional exonuclease activity,

- a thermostable ligase,

- a sufficient amount of each of the four deoxyribo-nucleotides. possibly competent bacteria capable of being transformed.

The invention also relates to a kit for the manufacture of cDNA libraries implementing the method of insertion of a DNA fragment into a vector described above. This involves, for example, building a cDNA library integrated into a plasmid, for example pBM.l, from messenger RNA isolated from a cell line or from a tissue. Such a kit is composed of the following products, offered separately or in the form of a mixture: a plasmid vector, cleaved in its polylinker at one or preferably two incompatible sites, a series of complementary oligonucleotides vector regions,

- an oligonucleotide necessary for the synthesis of the first strand,

- two oligonucleotides homologous to each of the strands of one of the ends of the cleaved vector, capable of being added as adapters to the cDNA once it has been synthesized, - a buffer compatible with the simultaneous activity of a thermostable polymerase and a thermostable ligase, a thermostable polymerase preferably having additional exonuclease activity, - a thermostable ligase, - a sufficient amount of each of the four deoxyribo-nucleotides. possibly competent bacteria capable of being transformed.

The method of the invention is also useful for finding the 5 'ends of a nucleic acid sequence. A kit for the implementation of the method of

1 invention in this application is composed of the following products, offered separately or as a mixture: a plasmid vector, cleaved in its polylinker at one or preferably two incompatible sites, a series of relay oligonucleotides complementary to vector regions,

- two oligonucleotides each composed of two parts:

- a 3 'part degenerated so as to be homologous to several nucleic acid sequences, and - a 5' part, previously designated label nucleic acid, comprising a nucleic sequence homologous to one of the ends of a cleaved vector . a buffer compatible with the simultaneous activity of a thermostable polymerase and a thermostable ligase, a thermostable polymerase preferably having an additional exonuclease activity,

- a thermostable ligase,

- a sufficient amount of each of the four deoxyribo-nucleotides. possibly competent bacteria capable of being transformed.

Other advantages and characteristics of the invention will appear from the following examples which are relate the preparation of nucleic acid molecule comprising the nucleic acid fragment to be inserted, and to applications of the method of the invention:

- simple insertion of a PCR product, - preparation of a complex construction,

- subcloning of mutants obtained obtained by overlapping PCR,

- protein fusion,

- case of a PCR using degenerate oligonucleotides, insertion of a cDNA library into the plasmid pBM.l.

Example 1: Preparation of nucleic acid molecule comprising the nucleic acid fragment to be inserted.

The DNA segment of known sequence is inserted into the plasmid by PCR using primer oligonucleotides constituted in two parts as described above. The quality of this amplification can be verified by migrating part of the PCR product on an agarose gel, and by observing the presence of a DNA band at the expected size. To avoid any risk of interference of the residual oligonucleotides during the subsequent steps, it may be necessary to purify the PCR product by one of the conventional techniques of purification on gel or on a column of sephacryl, etc.

In another embodiment of the method of the invention, the DNA fragments to be inserted are cDNAs, produced from mRNA, of unknown sequences. In this case, oligonucleotide adapters can be added to them so that one of the adapters is homologous at one of the ends of the plasmid into which each of these fragments is inserted. of DNA, and the second adapter is homologous at the other ends of the plasmid into which each of these DNA fragments is inserted.

The second step consists in carrying out a combined reaction using as a fractionated matrix on the one hand the cleaved vector as described above, and on the other hand the PCR product. To this matrix are added the enzymes necessary for the desired activity, thermostable polymerase containing a thermostable exonuclease and ligase, their buffers and substrates, as well as a series of phosphorylated oligonucleotides complementary at several points of the plasmid, intended to serve as relays during the combined reaction, the effectiveness of this activity decreases sharply with the length of the strand to be synthesized.

An example of a reaction mixture is as follows: PCR product 5 μl Purified cleaved vector (100 ng / μl) 1 μl Wind Polymerase (5 U) 0.5 μl

Tth ligase (40 U) 0.5 μl

Buffer 10X Taq Pol 1.25 μl

Buffer 5X Tth Ligase 2.5 μl Relay mixture 12 x 5 pmoles / μl 2 μl dNTP 2.5 nmoles / μl 0.5 μl

Deionized water 11.75 μl

Total 25 μl

The mixture is then subjected to a series of temperature cycles, such as [(94 °, l '); (50 °, l '); (72 °, 5 ')]. At the end of this combined reaction, all or part of the reaction mixture is directly transformed into competent bacteria using one of the conventional transformation techniques. The bacteria are then spread on a solid medium containing the selective medium corresponding to the resistance gene of the plasmid used.

After an adequate incubation period, the bacterial colonies are individually examined either by mini-preparation of plasmid DNA or, advantageously, by PCR on colonies. The presence of an insert corresponding to the expected size is then detected by electrophoretic migration of the DNA on agarose gel.

Example 2: Simple insertion of a product

PCR.

This example corresponds to the simplest use of the method of the invention. In this example, a large amount of a plasmid vector is prepared so that it can be used in the context of the present technique, and can thus be used for a large number of insertion of independent DNA fragments. This example is perfectly suited to the production of DNA fragment cloning kits, in particular obtained after amplification by PCR.

1) Preparation of the vector. pBM.l is a plasmid vector with a size of 4 kb.

A microgram of this plasmid is simultaneously digested by the two restriction enzymes Xhol and EcoRI, whose unique sites are present at the polylinker of the plasmid, under standard conditions. This digestion product is then purified using a Sephacryl column, so as to eliminate from the mixture the small fragments resulting from the double digestion, potentially harmful. The plasmid preparation is then adjusted to 100 ng / μl.

2) Choice of oligonucleotides.

The sequences of the primers necessary for the amplification by PCR of the gene to be inserted into the plasmid and the sequences of the two ends of the plasmid pBM.l cleaved by Xhol and EcoRI being known, the sequence of the oligonucleotides to be used in the context of the present invention can be determined.

The 3 'part of these oligonucleotides is fixed by the sequence of the DNA fragment to be inserted. In the present example, it is the ampicillin resistance gene, present on the plasmid SK + (Stratagene Catalog 97/98 p312), between two oligonucleotides distant by approximately 700 bases from each other, and antiparalleles.

The 5 ′ part of these oligonucleotides is fixed by the sequence of the ends of the cleaved plasmid pBM.l, and is further defined by the fact that it extends beyond this end (by a nucleotide in the present example), and that the first nucleotide upstream of the site of its binding on its complementary sequence is preferably a thymidine. Two distinct 5 ′ parts are defined, one being complementary to the Xhol end of the vector, the other to its EcoRI end. Part 5 'Xhol was associated with part 3'

AmpA so as to obtain an oligonucleotide of approximately 40 bases, and named FF32. The 5 'EcoRI part was combined with the 3' AmpB part so as to obtain an oligonucleotide of approximately 40 bases, and named FF34. 3) PCR reaction.

A PCR reaction is carried out using the plasmid SK + and the oligonucleotides FF32 and FF34. Twenty-five temperature cycles [(94 °, l '); (50 °, l '); (72 °, l ')] are carried out in the presence of 5 units of Vent polymerase (New England Biolabs) and concentrations of ions and dNTP compatible with the activity of this enzyme.

The efficiency of this PCR reaction is controlled by agarose gel electrophoresis of part of the product of this reaction.

4) Combined chain reaction.

A combined chain reaction is carried out according to the following protocol:

PCR product 5 μl pBM.l cleaved and purified (100 ng / μl) 1 μl

Wind Polymerase (5 U) 0.5μl

Tth ligase (40 U) 0.5 μl 10X Taq Pol buffer 1.25 μl

Buffer 5X Tth Ligase 2, 5 μl

Relay mixture 12 x 5 pmoles / ul 2 μl dNTP 2.5 nmoles / μl 0.5 μl

Deionized water 11.75 μl

Total 25 μl

This reaction mixture is subjected to 12 temperature cycles f (94 °, l '); (50 °, l '); (72 °, 5 ')].

5) Transformation of the combined reaction product.

Ten microliters of this reaction mixture are taken and transformed into competent bacteria of the DH10B strain using a conventional heat shock transformation protocol.

The transformation mixture is then spread on a petri dish supporting a solid medium containing an optimal concentration of anamycin, and incubated at least 16 hours.

6) Individual examination of the colonies.

Twenty-four of the bacterial colonies present on the petri dish at the end of the incubation are removed and tested for the presence of an insert at the level of the polylinker by the technique of PCR on colonies, using flanking oligonucleotides. The detection of inserts at the polylinker is done by electrophoresis of PCR products on colonies.

The results of this electrophoresis make it possible to identify 11 colonies out of 24 containing an insert at the level of the polylinker.

The complete sequencing of the insert present at two of these bacterial colonies confirms that the insertion of the DNA fragment using the technique of the invention took place without adding or removing nucleotides at the junctions.

Example 3: Preparation of a complex construction.

This example refers to the previous one, but differs from it in that, in step 2), six oligonucleotides are determined so as to obtain a construct composed of three PCR fragments amplified so that their ends are homologous in pairs as shown in the schematic representation given in Figure 5 in the appendix). The third step of the protocol in this example consists of three independent PCRs carried out with three pairs of oligonucleotides and three different matrices, as shown in FIG. 5 in the appendix. Five microliters of each of these three PCR products are mixed, and the other reagents necessary to carry out the combined reaction as described in the previous example are added to them.

The protocol is then identical to that of the previous example. 30% of the colonies examined contain an insert composed of the succession of the three PCR fragments as explained in FIG. 5.

Example 4: Subcloning of mutants obtained obtained by overlapping PCR.

A mutagenesis technique used by a large number of laboratories involves the production of two PCR products, the ends of which are homologous and consisting of an oligonucleotide containing the desired mutation. In a second step, the two PCR products are traditionally used as a template in a second PCR reaction, overlapping at the homologous ends, and using oligonucleotides flanking the assembly. This large PCR product is then subcloned into a plasmid.

The method of the invention presents an improvement of this technique, making it possible to group the overlapping PCR and subcloning steps. This example is close to the previous two, and respects the order and composition of their stages.

However, in the second step, the choice of oligonucleotides involves, in addition to the two oligonucleotides described in Example 3, two oligonucleotides internal to the fragment to be mutated, containing the desired and complementary mutations, called MutA and MutB here (Figure 6).

In the third step, two PCR reactions are carried out using as template the DNA fragment into which to introduce a mutation, the first using the oligonucleotides FF32 and MutA, the second using the oligonucleotides FF34 and MutB.

In the fourth step, the two PCR products are mixed, and this mixture is completed so as to be the support for a combined reaction, according to the protocol described in the first and second examples.

The remainder of the protocol is identical, and allows direct obtaining of the mutated DNA fragment subcloned into the plasmid pBM.l.

Example 5: Protein fusion.

In this example, it is sought to merge without disruption of the coding phase, two domains of two different proteins. The construction of DNA must therefore be carried out to the nearest nucleotide, which is difficult to carry out by techniques other than that of the present invention.

The gene coding for protein X is cloned into the vector pBM.l, the assembly subsequently being called pBM.l-X. The gene coding for protein Y is not inserted into the plasmid pBM.l.

1) Preparation of the vector. The vector pBM.lX is firstly cleaved at the single restriction site closest to the junction site, the restriction enzyme used being hereinafter called enzl. Secondly, this vector is cleaved at the polylinker site such that the fragment of the protein X gene to be eliminated is bounded by the two cleavage sites. The restriction enzyme used for this second cleavage is called enz2.

The vector is then isolated from the part of gene A to be eliminated by gel purification, using one or other of the multiple techniques for purifying DNA from the agarose band.

2) Choice of oligonucleotides.

The two oligonucleotides used in this example each consist of two parts. Their 3 ′ part allows the amplification of the desired DNA fragment using the protein Y gene as a template. Their 5 ′ ends are homologous to the ends of the vector pBM.1-X Enzl Enz2. The diagram of the enz2 junction is comparable to the junctions described in the second example.

The enzl junction is slightly different, in that the junction between the gene of protein X and that of protein Y, as previously determined, can be located at a relatively greater nucleotide distance than in the case of Example 2 This distance is bridged by the oligonucleotide used. Thus, the second oligonucleotide may contain up to one hundred nucleotides, necessary to effect the junction between X and Y on the one hand, and the position of the enzl site on the other hand.

In the third step, the PCR is carried out using the two oligonucleotides as described above, one being potentially much longer than the other. Apart from this particularity, this step takes place substantially similar to the third step of the second example.

The fourth, fifth and sixth steps take place in a manner comparable to the corresponding steps in Example 2. Example 6: Case of a PCR using degenerate oligonucleotides.

In this example, two oligonucleotides, each composed of two parts, are used, in a manner analogous to that described in example 2. The major difference resides in the fact that the 3 ′ part of one of these two oligonucleotides is degenerate, that is to say that it contains a series of 5 to 40, and preferably approximately 20 nucleotides randomly constituted at some of its positions, or at each, of a mixture of the four nucleotides.

This example is particularly suited to the case where an experimenter knows the sequence of only part of a gene, and where he seeks to isolate the part which is not yet known to him. This example is more particularly suited to the case where an experimenter has already isolated and sequenced the 3 'part of a gene, for example after screening a cDNA library, then seeking to isolate the 5' part of this same gene.

1) Preparation of the vector.

The first step is identical to that of Example 2.

2) Choice of oligonucleotides.

Two oligonucleotides each consisting of two parts must be determined. Their 5 'parts are identical to those described in Example 2. Their 3' parts are, for one, homologous to part of the known sequence. For the other, this sequence consists of a series of degenerate nucleotides.

Each of these two parts 3 'is associated either with one or the other of the parts 3'. For example, the degenerate oligonucleo ide is associated with The oligonucleotide Xhol, while the oligonucleotide homologous to the known part of the sequence is associated with the oligonucleotide EcoRI.

'3) Amplification by PCR.

A PCR is carried out using as a template a mixture of DNA containing the DNA fragment sought, and preferably complementary or genomic DNA purified from a tissue or a cell line in which the gene corresponding to the sequence sought is expressed.

The product of this PCR can be observed by electrophoresis on agarose gel. Observation of distinct bands indicates that the sequence sought has probably been amplified. The fourth, fifth and sixth steps are identical to those described in Example 2.

At the end of this series of steps, the plasmid DNA of certain bacterial colonies, selected on the criterion of the presence of a DNA insertion at the level of the polylinker of the vector, can be sequenced. This sequence may correspond to the sequence sought.

Example 7: Insertion of a cDNA Ban into the Plasmid pBM.l.

In this example, it is not a question of inserting a single DNA fragment into a cleaved and purified vector as in the previous examples, but of inserting a DNA library therein, that is to say a heterogeneous mixture. DNA fragments.

1) Preparation of the vector.

The first step is identical to that described in the second example. 2) Preparation of cDNA homologous at its ends to the cleaved and purified plasmid.

After extracting mRNA from a tissue or a cell line, a first strand of cDNA is synthesized according to one of the conventional procedures, using as an primer an oligonucleotide composed of two parts. The 3 'part of this oligonucleotide is composed of several thymidines (between 5 and 50, and preferably about 20). This 3 ′ part is intended to hybridize on the poly-A tail of the mRNAs. The 5 'part of this oligonucleotide is homologous to the EcoRI end of the plasmid pBM.l, and extends beyond a nucleotide. In addition, at 5 'of part 5' is added a rare restriction site (such as SwaI). The second strand of the cDNA is then synthesized according to one or other of the techniques known to those skilled in the art.

Adapters, made up of double-stranded oligonucleotides, are linked to this cDNA according to a conventional ligation procedure. These adapters are ligated either on the 5 ′ side and on the 3 ′ side of the cDNA.

The cDNAs thus obtained are then digested with the enzyme SwaI, so as to specifically excise the adapter bound in 3 ', undesirable. A combined chain reaction is then carried out as in step 4 of Example 2.

The product of this reaction is then transformed into competent bacteria, preferably using the electroporation technique, known to give optimum transformation efficiency.

A sample of this transformation mixture can be taken, deposited on a solid medium containing Kanamycme, and studied for example by PCR on colonies, as described in Example 2, so as to estimate the titer and the frequency in this mixture of bacteria containing a cDNA insert.

This cDNA library is then intended to be screened by any technique known to those skilled in the art, and in particular by the use of a labeled probe or of coupled antibodies.

Claims

1) Method of inserting a nucleic acid fragment into a vector, characterized in that it comprises the following steps: a) a double-stranded nucleic acid molecule comprising the nucleic acid fragment is prepared. inserting into a vector, and 5 'of said fragment a first labeled nucleic acid comprising a nucleic sequence complementary to the 5' end of one of the strands of a cleaved vector, and 3 'of said fragment a second labeled nucleic acid comprising a nucleic sequence complementary to the 3 'end of the same strand of said cleaved vector, b) denaturing said nucleic acid molecule and a cleaved vector in which the 5' and 3 'ends of one of the strands are complementary to said first and second labeled nucleic acids, c) incubating said nucleic acid molecule with the cleaved vector whose 5 'and 3' ends are complementary to the first and second labeled nucleic acids, in d the conditions allowing the hybridization of said first and second labeled nucleic acids on the cleaved vector, d) copying: - on the one hand, the vector cleaved in the 5 ′ to 3 ′ direction from the 3 ′ end of the second label nucleic acid hybridized to the 3 ′ end of the cleaved vector, and - on the other hand, the fragment of nucleic acid in the 5 ′ to 3 ′ direction from the 3 ′ end of the cleaved vector, e) optionally ligated: on the one hand, the 3 ′ end of the neosynthesized strand with the vector as matrix, with the 5 'end of the first hybrid label nucleic acid to the 5' end of the cleaved vector, and on the other hand, the 3 'end of the neosynthesized strand with the fragment to be inserted as a matrix, 5 with the 5' end of the cleaved vector, f) optionally repeating steps (b) to (e), g) optionally, transforming a cell host to select the vectors having integrated the 10. nucleic acid fragment. 2) Method of inserting a nucleic acid fragment into a vector according to claim 1, characterized in that in step (a) a 15 nucleic acid molecule comprising: - the nucleic acid fragment to be inserted into a vector, on either side of said fragment, a label nucleic acid molecule consisting of a 20 nucleic sequence, of the order of 10 to 150 nucleotides and preferably of the order of 15 to 100 nucleotides, homologous to one of the ends of a cleaved vector, said homologous nucleic sequence possibly being separated from the fragment of nucleic acid to be integrated by at least one 25 nucleotide and preferably between 1 and of the order of 130 nucleotides. 3) Method of inserting a nucleic acid fragment into a vector according to claim 2, 30 characterized in that the nucleic acid sequence homologous to the end of the cleaved vector included in the labeled nucleic acid is complementary to a sequence of the vector bordered by a 3 'Thymidine (T). 4) Method of inserting a nucleic acid fragment into a vector according to one of claims 1 to 3, characterized in that each nucleic acid sequence homologous with one end of the cleaved vector, included in the labeled nucleic acid , is sufficiently homologous to hybridize at said end of the cleaved vector at a temperature between 30 and 90 ° C, preferably between 30 and 70 ° C and most preferably at a temperature of 50 ° C. 5) Method of inserting a nucleic acid fragment into a vector according to any one of claims 1 to 4, characterized in that step (a) the nucleic acid molecule is prepared by amplification of the nucleic acid fragment to be inserted into the vector by a polymerase chain reaction (PCR) from two priming oligonucleotides each comprising a 5 ′ part constituting the labeled nucleic acid, including the 3 ′ end of the nucleic sequence homologous to the end of the cleaved vector: either ends at the last nucleotide of said cleaved vector, and then the labeled nucleic acid consists only of said sequence homologous to the end of the cleaved vector, - or is longer by at least one nucleotide, and then the labeled nucleic acid consists of said homologous sequence at the end of the cleaved vector, extended 3 ′ by at least one nucleotide. 6) Method of inserting a nucleic acid fragment into a vector according to any one of claims 1 to 5, characterized in that the vector is a plasmid vector advantageously containing a antibiotic resistance marker, and which is cleaved at one or two restriction sites. 7) Method of inserting a nucleic acid fragment into a vector according to any one of claims 1 to 6, characterized in that the copying (d) and ligation (e) steps consist in subjecting, after the denaturation of step (b) and the hybridization of step (c) the nucleic acid molecule prepared in step (a) and the cleaved vector, to the action of a polymerase and a thermostable ligase. 8) Method of inserting a nucleic acid fragment into a vector according to any one of claims 1 to 7, characterized in that the copying (d) and ligation (e) steps are carried out either successively or substantially simultaneously. 9) Method of inserting a nucleic acid fragment into a vector according to any one of claims 1 to 8, characterized in that step (d) is carried out in the presence of homologous phosphorylated relay oligonucleotides of different parts of the vector and distributed homogeneously on each of the two strands of the plasmid, at a distance between them of 500 to 2000 base pairs. 10) Method of inserting a nucleic acid fragment into a vector according to any one of claims 1 to 9, characterized in that in step (a) the nucleic acid molecule is prepared by amplification of the nucleic acid fragment to be inserted into the vector by a polymerase chain reaction (PCR) from two priming oligonucleotides, each of these oligonucleotides being composed of two parts: - A 3 ′ part homologous to one end of the fragment to be inserted, a 5 ′ part, constituting the labeled nucleic acid, comprising a nucleic sequence homologous to one of the ends of a cleaved vector. 11) Method of inserting a nucleic acid fragment into a vector according to claim 10, characterized in that the 3 'part of each primer oligonucleotide comprises from 5 to 20 nucleotides whose sequence is sufficiently homologous to that of each ends of the nucleic acid fragment to hybridize to said end at a temperature between 30 and 90 ° C, and preferably about 50 ° C. 12) Method of inserting a nucleic acid fragment into a vector according to one of claims 10 or 11, characterized in that the 5 ′ part of each primer oligonucleotide constituting the labeled nucleic acid comprises a homologous nucleic sequence of the end of the cleaved vector which is complementary to a sequence of the vector bordered by a Thymidine (T) in 3 '. 13) Method of inserting a nucleic acid fragment into a vector according to one of claims 10 to 12, characterized in that the 5 ′ part of each primer oligonucleotide constituting the labeled nucleic acid consists of a nucleic sequence, of the order of 10 to 150 nucleotides and preferably of the order of 15 to 100 nucleotides, homologous to one of the ends of a cleaved vector, said homologous nucleic sequence possibly being separated from the part in 3 homologous to one end of the fragment to be inserted with at least one nucleotide and preferably between 1 and of the order of 130 nucleotides. 14) Method of inserting a nucleic acid fragment into a vector according to one of claims 10 to 13, characterized in that the 5 ′ part of each primer oligonucleotide constituting the nucleic acid 5 label comprises a nucleic sequence homologous to the end of the cleaved vector which is sufficiently homologous to hybridize to said end of the cleaved vector at a temperature of between 30 and 90 ° C, preferably between 30 and 70 ° C and most preferably at a temperature of 10 50 ° C. 15) A kit for implementing a method according to any one of claims 1 to 14, characterized in that it comprises the following products, 15. Offered separately or as a mixture: a plasmid vector cleaved in its polylinker at one or preferably two incompatible sites, a series of relay oligonucleotides complementary to regions of the vector, a buffer compatible with the simultaneous activity of a thermostable polymerase and a thermostable ligase, a thermostable polymerase preferably having additional exonuclease activity, - a thermostable ligase, - a sufficient amount of each of the four deoxyribo-nucleotides. optionally, competent bacteria capable of being transformed. 16) Use of a method according to any one of claims 1 to 14, for the manufacture of cDNA libraries. 17) A kit for the implementation of a method according to any one of claims 1 to 14 applied to the manufacture of cDNA library, characterized in that it comprises the following products, offered separately or in the form of mixture: a plasmid vector, cleaved in its polylinker at one or preferably two incompatible sites, a series of relay oligonucleotides complementary to regions of the vector, - an oligonucleotide necessary for the synthesis of the first strand, two oligonucleotides homologous to each of the strands of one of the ends of the cleaved vector, capable of being added as adapters to the cDNA once it has been synthesized, a buffer compatible with the simultaneous activity of a thermostable polymerase and a thermostable ligase, - a thermostable polymerase preferably having additional exonuclease activity, - a thermostable ligase, - a sufficient amount of each of the four deoxyribo-nucleotides. - possibly, competent bacteria capable of being transformed. 18) Use of a method according to any one of claims 1 to 14, to insert an unknown or heterogeneous nucleic acid fragment into a vector. 19) Use of a method according to any one of claims 1 to 14, for the search for the 5 'ends of a nucleic acid sequence. 20) Use according to claim 19, characterized in that the two priming oligonucleotides used for the preparation of the nucleic acid molecule of step (a) are each composed of two parts: - a 3 'part degenerated so as to be homologous to several nucleic acid sequences, and a 5' part, constituting the labeled nucleic acid, comprising a nucleic sequence homologous to one of the ends of a cleaved vector . 21) A kit for implementing a method according to any one of claims 1 to 14 applied to the search for the 5 ′ ends of a nucleic acid sequence, characterized in that it comprises the following products , offered separately or as a mixture: a plasmid vector, cleaved in its polylinker at one or preferably two incompatible sites, - a series of relay oligonucleotides complementary to regions of the vector, - two oligonucleotides each composed of two parts: - a 3 'part degenerated so as to be homologous to several nucleic acid sequences, and a 5' part, constituting the labeled nucleic acid, comprising a nucleic sequence homologous to one of the ends of a cleaved vector . a buffer compatible with the simultaneous activity of a thermostable polymerase and a thermostable ligase, - a thermostable polymerase preferably having an additional exonuclease activity, - a thermostable ligase, - a sufficient amount of each of the four deoxyribo-nucleotides. possibly competent bacteria capable of being transformed. 22) An oligonucleotide constituting a primer for the preparation by PCR of a nucleic acid molecule containing a nucleic acid fragment to be inserted into a vector, capable of being used in a method according to any one of claims 1 to 14, characterized in that each said oligonucleotide is composed of two parts: - a 3 'portion homologous to one end of the fragment to be inserted, - - a 5' portion, constituting a labeled nucleic acid, comprising a nucleic sequence homologous to one of the ends of a cleaved vector. 23) An oligonucleotide according to claim 22, characterized in that the 3 'part comprises from 5 to 20 nucleotides whose sequence is sufficiently homologous to that of one end of the nucleic acid fragment to hybridize at said end at a temperature between 30 and 90 ° C, and preferably about 50 ° C. 24) An oligonucleotide according to one of claims 22 or 23, characterized in that the 5 ′ part constituting the labeled nucleic acid comprises a nucleic sequence homologous to the end of a cleaved vector which is complementary to a sequence of the vector bordered by a 3 'Thymidine (T). 25) An oligonucleotide according to one of claims 22 to 24, characterized in that the 5 ′ part constituting the labeled nucleic acid consists of a nucleic sequence, of the order of 10 to 150 nucleotides and preferably of the order of 15 to 100 nucleotides, homologous to one of the ends of a cleaved vector, said homologous nucleic sequence being optionally separated from the part in 3 ′ homologous to one end of the fragment to be inserted with at least one nucleotide and preferably between 1 and of the order of 130 nucleotide. 26) An oligonucleotide according to one of claims 22 to 25, characterized in that the 5 ′ part constituting the labeled nucleic acid comprises a nucleic sequence homologous to the end of the cleaved vector which is sufficiently homologous to hybridize to said end of the cleaved vector at a temperature between 30 and 90 ° C, preferably between 30 and 70 ° C and most preferably at a temperature of 50 ° C. 27) A pair of primer useful for a PCR amplification reaction of a nucleic acid fragment to be inserted into a cleaved vector, characterized in that it consists of two oligonucleotides according to any one of claims 22 to 26. 28) An amplification product obtained by a PCR reaction implemented with a pair of primers according to claim 27.