WO2008065505A2

WO2008065505A2 - Simultaneous mixed site-specific mutagenesis method

Info

Publication number: WO2008065505A2
Application number: PCT/IB2007/003629
Authority: WO
Inventors: Ciro Isidoro; Carlo Follo
Original assignee: Universita degli Studi del Piemonte Orientale -Amedeo Avogadro
Current assignee: Universita degli Studi del Piemonte Orientale -Amedeo Avogadro
Priority date: 2006-11-29
Filing date: 2007-11-19
Publication date: 2008-06-05
Anticipated expiration: 2009-05-29
Also published as: WO2008065505A3; ITTO20060848A1

Abstract

Method of introducing mutations into a circular DNA template molecule comprising the steps of : i) providing two mutagenic primers; ii) annealing the mutagenic primers to the DNA template molecule; iii) synthesising two mutated DNA strands by polymeric chain reaction; iv) annealing the two mutated DNA strands obtaining a linearized mutated DNA molecule; and v) amplifying the linearized mutated DNA molecule by polymeric chain reaction; wherein the mutagenic primers present the following features : - each mutagenic primer comprises at its 3' terminal a portion having a nucleotide sequence complementary to a portion of the DNA template molecule to be mutated; the mutagenic primers anneal to the DNA template molecule back-to-back in opposite directions; and the mutagenic primers anneal to different DNA template molecule strands.

Description

Simultaneous mixed site-specific mutagenesis method

*★*

Field of the invention

The present invention relates to a site-specific mutagenesis method, and more particularly to a site- specific mutagenesis method that allows to introduce in a single-step all three types of nucleotide sequence mutation (deletion, insertion and substitution) .

Background of the invention

Site-specific mutagenesis is a powerful way to modify target portions of the DNA sequence that helps to identify regions of interactions with other molecules or, in case of sequences coding for peptides, to explore the structure-function relationship of the protein with regard to its targeting, folding, enzyme activity and post-translational modifications or interactions with other molecules such as transporters, inhibitors or substrates. Typical modifications that can be obtained by site-specific mutagenesis include generation of cysteine-less proteins, alanine-scanning mutagenesis, codon optimisation for heterologous expression, deletion or insertion of peptide regions (for review see 1, 2) .

A variety of methods have been proposed for site- specific mutagenesis (3, 4, 5, 6, I)₁ several of them being available as commercial kits. Among these methods, the PCR-based ones that do not require subcloning of the sequence of interest into new plasmids are certainly the most appealing because of their simplicity and rapidity of execution (8, 9, 10, 11, 12) . Other examples of PCR-based site-specific mutagenesis methods are disclosed i.a. in EP-A-O 466 083, EP-B-O 497 798, EP-B-O 620 858, EP-A-O 987 326, US-A-2003/0224492, US-A-5 284 760, US-A-5 955 363, WO- A-03/025118, WO-A-03/040376, WO-A-2004/054492, US-B-6 391 548, US-A-2003/0032037, US-A-2004/0253729 and WO-A- 97/20950.

The site-specific mutagenesis method disclosed by Chiu et al. (12) requires the use of four primers - two tailed primers and two short primers - which are able to anneal back-to-back to the DNA sequence to be mutated. The two tailed primers carry a mutagenic sequence in the tails at the 5' terminal, wherein the mutagenic sequences on the two tailed primers are complementary to each other. This procedure generates four PCR products, two of which are not productive from the mutagenesis point of view (one carries the mutated sequence on both termini and the other does not contain any sequence mutation) . The PCR amplification is carried out by employing two types of DNA polymerase (Tag and Platinum Pfx) . DNA template must necessarily be first cloned in DAM+ E. coli and need to be removed by Dpnl restriction enzyme digestion. A crucial step inthis method, that strongly affects the efficiency, is the formation of several heteroduplexes, only two of which undergo spontaneous circularization by overhanging. It is, in fact, a specific feature of the primers employed to have complementary 5' overhangs that can form stable DNA circles. This reaction requires two different buffers, the Dpnl enzyme and is carried out in two steps. Moreover, the use of two tailed primers having the mutagenic sequences complementary to each other reduces the length of the sequence to be mutagenized and the efficiency of the method. Finally, it is to note that the PCR program has to be adapted depending on the type of mutagenesis desired. This method has been tested by the authors only for insertion or deletion of six codons and for a substitution of one nucleotide (12) .

In general, PCR-methods for site-directed mutagenesis yield a high percentage of non-mutated or incorrectly mutated clones. Other limitations offered by current methods are represented by the length of the sequence that can be modified, the number and the types of modifications that can be introduced at one time, the rate of mutagenized plasmids produced.

Description of the invention

It is therefore object of the present invention to provide a site-specific mutagenesis method that allows the introduction in a single-step of all three types of nucleotide sequence mutations, i.e. deletion, insertion and substitution, substantially without any limitation on the length of the nucleotide sequence to be mutagenized. According to a first preferred embodiment of the present invention, the Simple Method for Simultaneous Mixed Site-specific Mutagenesis (called herein 3SM method) comprises the use of two primers, one of which is 5' -phosphorylated, able to anneal back-to-back to the target sequence inserted in a plasmid. By inverse PCR the entire circular plasmid is amplified and the mutation can be inserted anywhere in the plasmid. The PCR product incorporates the desired mutagenesis and upon ligation the plasmid is ready for cloning in bacteria. Original template can be cloned in DAM+ E. coli, so that wild-type plasmid is degraded by Dpnl digestion. This is however not mandatory. The mutagenic sequence (for substitution "and/or insertion) is included in the tail of the primers, which do not contain any sequence that can undergo to spontaneous circularization by overhanging. In a preferred embodiment of the present invention, the method allows to amplify a circular plasmid up to 12 Kbp introducing mixed mutations in a wide coding region. Object of the present invention is also the kit used to carry out the method of the invention.

The objects of the present invention are achieved thanks to the solutions claimed in the ensuing claims, which form integral part of the technical teaching herein provided.

Brief description of the drawings

Figure 1. Schematic representation of the mutagenesis procedure. The box indicate the step a) and b) that were performed in one tube. Step: a) PCR reaction; b) Dpnl digestion; c) T4 DNA ligase reaction; d) competent cells transformation . Figure 2. Schematic representation of primer' s features to obtain substitution (a) or insertion (b) or deletion (c) or or a mix of substitution/deletion (d and e) . The desired region to mutate with an aminoacid deletion or substitution is in dark grey. The primers tails is in white and grey.

Figure 3. A) Agarose gel electrophoresis of mutagenesis PCR product (the PCR total volume was subdivided in two lanes) after Dpnl digestion. Expected band was 7000 bp. B) Agarose gel electrophoresis of screening PCR product. Expected band was 1350 bp. Lane 1-10 and 12-21: control PCR performed on colonies. Lanes 11 and 22: negative control PCR performed on colonies resulting from E. coli TOP 10 transformation with re-ligation product of the wild type vector digested with EcoRI restriction enzyme. C) electropherogram from sequencing of clone 1. The new mutagenic sequence is in black box.

Figure 4. A) Agarose gel electrophoresis of mutagenesis PCR product after Dpnl digestion. Expected band was 5700 bp. B) Agarose gel electrophoresis of screening PCR product. Expected band was 1070 bp (1350 bp for wild type) . Lane 2- 11: control PCR performed on colonies. Lanes 1 and 12: negative control PCR performed on colonies resulting from E. coli TOP 10 transformation with re-ligation product of the wild type vector digested with EcoRI restriction enzyme. C) electropherogram from sequencing of clone 1. The new mutagenic sequence is in black box.

Detailed description of the invention

The. invention will now be described in detail by way of non-limiting examples. Site-specific mutagenesis at one or multiple sites has recently become an invaluable strategy in functional proteomic studies and genetic engineering. Modification of structural and functional characteristics of proteins by DNA mutagenesis is mostly useful in biomedical and industrial fields.

The 3SM method object of the present invention has the advantage of introducing all types of mutation (insertion, deletion, substitution) simultaneously, at any site within any circular DNA plasmid and with a relative high mutant to wild-type ratio. The method is based on inverse PCR and employs two mutagenic primers, one of which is 5' -phosphorylated, that are designed to anneal in opposite direction to the gene/sequence of interest. The first cycle of PCR generates linear plasmid molecules from both template strands. Subsequent rounds of PCR further amplify these linear plasmid molecules which are then ligated and used for transformation of competent E. coli (see Figure 1) . For PCR amplification the present inventors propose to use Platinum Pfx with its buffer, which guarantees an elevated primer specificity, a broader range of optimal magnesium concentrations, broad annealing temperatures, and improved thermostability. Original DNA template is removed by Dpnl restriction enzyme digestion. PCR mutagenesis and Dpnl-mediated removal of template are run in one tube. Dpnl-mediated digestion step can be skipped with no impact on mutagenesis efficiency. Thus, it is not mandatory to amplify DNA template in DAM+ bacteria. In this case, the linearized PCR product bearing the mutation is purified by affinity using published method and commercial kits. ,As the PCR proceeds the efficiency of amplification is likely to increase since annealing of mutagenic primers to modified DNA strands involves the whole sequence. This should be true, in particular, for substitution and/or insertion types of mutagenesis. In the case of deletion type of mutagenesis, primers anneal to both template and PCR product with the same efficiency, still the latter has the advantage of being shorter and therefore requires a minor time of amplification. 10 cycles of amplification rise an amount of DNA sufficient for the following steps. At the end of PCR amplification, and eventually after Dpnl digestion, the mutated DNA must be re-ligated. For this reaction it is usually sufficient to take 1:10 or 1:20 of PCR product (depending on the number of amplification cycles) . Prior to this step the PCR product can be subjected to agarose gel purification. In this case, mutated DNA of expected size is excised from the gel and a fraction of it is used for ligation. Gel purification can be avoided, though efficiency of subsequent steps

(ligation and bacteria transformation) is negatively affected. In the experience of the present inventors, when PCR product was not gel-purified efficiency of transformation dropped to about 50 %. Also in this case the number of positive colonies is anyhow sufficiently high to guarantee the success of the procedure. Same efficiency was achieved also when the PCR product was directly purified (preceded or not by gel separation) skipping the Dpnl digestion step.

The present inventors tested the flexibility and power of 3SM method to introduce extensive mixed mutagenesis of a rather long stretch of aminoacids . In all applications colonies bearing the mutated DNA were obtained at a relative high rate. In particular, efficiency is higher than 80 % when PCR product is gel- purified prior to ligation and transformation steps. The present inventors used in performing experimentation competent, not supercompetent , E. coli for transformation. It is likely that efficiency would increase by using supercompetent bacteria.

In general, the efficiency of mutation is also influenced by the extent of the region to be modified and varies depending on the type of mutation, being maximal for deletion, regardless of the length of the region involved. It is worth to noting that the method allows to delete a region as long as desired, virtually with no limits.

An interesting application of DNA mutagenesis in functional proteomic studies is the alanine scanning. This technique is most useful to study the influence of aminoacids lateral chains in protein function and consists in changing one or more codon codifying for an aminoacid in the wild type sequence to a codon codifying for alanine. 3SM method allows to perform the alanine substitution of a large number of aminoacids in one step, even if the aminoacids to be substituted in the sequence of interest are discontinuous.

3SM method is rapid (mutant colonies can be generated in 1 day) and leads to a substantial number of transformants bearing the mutated plasmid (the desired mutation is introduced at a frequency greater than 80 % using competent E. coli). The present inventors observed a relative low efficiency only for extensive substitution of a long sequence, such as in the case of alanine scanning of as many as 19 aminoacids. In this case, the mutated DNA was found in about 50% of transformant colonies. It should be considered, however, that this situation is rather complex and particular, since mutagenic primers are unusually long and contain an elevated number of similar codons for alanine, close to each other. In all cases, sequencing confirmed the data of PCR screening on positive colonies. At present, the unique limit is the length (around 8.000 bp) of the plasmid that Platinum Pfx DNA polymerase can amplify. However, it is predictable that the procedure 3SM can work with plasmids of up to 12 kbp or longer, provided that a good and efficient DNA polymerase is employed.

MATERIALS AND METHODS

Mutagenesis and. control-screening primers Mutagenic primers and control-screening primers for the respective mutagenesis are reported in Table 1. PCR primers were designed with the aid of the Gene Runner software (Hastings Software Inc.) . Primers were from MWG-BIOTECH AG (Ebersberg, Germany) . Note that one of the mutagenic primers is phosphorylated at the 5' end. Table 1. Type of gene

Mutagenesis primers Screening primers modification

F-MYChCD 5'-AGC GAG GAG GAC

CTG TCC TTC TAC CTG AGC AGG F-MYC contr. 5' -GCA

GAC CCA GAT GC -3 AAA ACT CAT CAG CGA

Generation of (SEQ ID NO. :1) GGA GG-3' (SEQ ID NO. :9) epitope by substitutions R-MYChCD P-5'-GAT GAG TTT TTG

CTC CAT CAG GTT GTC GAA GAC R-BGH 5' -TAG AAG GCA

GGG CAG CAC G -3' CAG TCG AGG-3'

(SEQ ID NO. :2) (SEQ ID NO. :10)

F-mpr 5 -GGC GAC ATC GTC GTG

GAG AGG CAG GTC TTT GGG GAG F-mpr contr. 5' -AGC

GCC A-3' TGC ACC ATC GGC GAC

Substitution of (SEQ ID NO. :3) ATC GTC-3'

19 ammoacids by (SEQ ID NO. : 11) deletions/ R-mpr P-5'-GAT 3GT GCA GCT insertions GTC CTG GCT CAG GTA CCC GGA R-BGH 5' -TAG AAG GCA

GAG GCT- 3' CAG TCG AGG-3'

(SEQ ID NO . : 4 ) (SEQ ID NO. :12)

F-mprAlaT P-5' - GCT GCG GCA

GCG GCA GGC GGT GCG GCA GTG

GAG AGG CAG GTC TTT GGG GAG F-contr .AIaT 5' -AGC

GCC A-3' AGC AGC CGC TGC GG-3'

Alanine scanning (SEQ ID NO . : 5 ) (SEQ ID NO. :13) in 19 ammoacids region R-mprAlaT 5'-GGC TGC TGC TGC R-BGH 5'-TAG AAG GCA

GCA AGC TGC CGA AGC AGC GTC CAG TCG AGG-3'

CTG GCT CAG GTA CCC GGA GAG (SEQ ID NO. :14)

GCT-3'

(SEQ ID NO. :6)

F-BECNl 5'-GCC CGA ATT

F-delta ECD P-5'- -TAT CTG GAG CGG GAT GGA AGG GTC

TCT CTG ACA GAC AAA TC-3' TAA GA-3'

(SEQ ID NO. :7) (SEQ ID NO. :15)

Deletion of 93 ammoacids

R-delta ECD 5'-CAG CTC CAG R-Becnl GFP 5'- -CTC CTT

CTG CTG TCG TTT A-3' GGC CTC CAT CTG TAA

(SEQ ID NO. :8) CTG TTC ACT GTC ATC _ ^ '

! (SEQ ID NO. : 16)

3SM PCR-amplification

The 2038 bp length cDNA of human cathepsin D (CD, SEQ ID NO.: 17 - GeneBank: M11233) (Faust, et al 1985) subcloned into pcDNA 3.1 Zeo (-) (Invitrogen Corp.) and the cDNA of human Beclin 1 (BECN, only coding sequence, SEQ ID N0.:18 - GeneBank: AF139131) subcloned into pEGFP-Nl vector (Clontech, Takara Bio Inc., Otsu, Japan) were used as templates. 50 ng of the template plasmid purified from DAM+ E. CoIi bacteria were used for PCR. Optimized conditions for DNA Polymerase Platinum Pfx (Invitrogen Corp., Carlsbad, CA, USA) directed amplification of template in the presence of long primers with high Tm were assessed. The high Tm of mutagenic primers was adjusted to the 68⁰C annealing temperature using PCR Enhancer Solution (3x final concentration for mutagenesis n. 1-3; 0,5x final concentration for mutagenesis n. 4) . The PCR conditions were: initial denaturing step at 94°C for 5', followed by a standard two-steps cycling protocol (denaturation at 94⁰C for 15''; extension at 68⁰C for 1'/Kb). 2.5 U Platinum Pfx (or other recombinant DNA Polymerase from Thermococcus KOD with proofreading 3 'to 5' exonuclease activity available from any producers: for example KOD HiFi DNA Polymerase from Novagen) and 2x final Platinum Pfx amplification buffer concentration were used. Sufficient amount of PCR product for further use was obtained with 10 to 20 denaturation-extension cycles. At the end, digestion of template plasmid was performed at 37°C for 1,5 h with 10 U of the restriction enzyme Dpnl (Stratagene, La Jolla, CA, USA) , directly added into the PCR tube. As a control, 50% of the PCR product was purified by agarose gel electrophoresis and the expected band of DNA excised from the gel (QIAEX II Gel Extraction kit, Qiagen, Hilden, Germany) . This step can be omitted. 1/10 of the gel-purified (or not-purified) product was used for the ligase reaction (T4 DNA ligase, Invitrogen Corp., or Pfu DNA Ligase, Stratagene) and subsequently used to transform E. coli TOPlO competent cells. Conditions for ligase reaction were: 0.5 U ligase, 26°C for 1 h. PCR screening of colonies

Colonies of E. coli TOP 10 transformed with the ligated 3SM PCR product were screened by PCR using recombinant Taq DNA Polymerase (Invitrogen Corp.) following manufacturer's protocol. PCR products were analyzed by agarose gel electrophoresis. Colonies of E. coli TOP 10 transformed with re-ligation product of the wild type vector digested with EcoRI restriction enzyme were also included as controls. Primers for screening of colonies are reported in Table 1.

Sequencing of the DNA

The mutant cDNA was subjected to automatic sequencing (ABI PRISM 7200, Applied Biosystems, Foster City, CA, USA) . Primers for sequencing are indicated in Table 2.

Table 2.

Schematic overview of 3SM method

The whole procedure is carried out in one tube as depicted in Figure 1. Alternative strategies to obtain the mutated PCR product for ligation are schematized in panel B of Figure 1. DNA template (whether or not previously amplified in DAM+ bacteria) is amplified by reverse PCR (10-20 or more cycles) in the presence of mutagenic primers. The employed primers do not contain any nucleotide sequence which can determine spontaneous circularization by overhanging. If required, at the end of the PCR amplification the wild-type plasmid (DNA template) is specifically digested by adding directly to the tube the restriction enzyme Dpnl from Stratagene or other type II methyl-directed restriction enzyme (subtype M) (e.g. Bisl from SibEnzyme Ltd.; Mall from SibEnzyme Ltd) . (11, 13) . This enzyme cleaves the GATC sequence only if adenine is methylated (at position 6) , as it occurs in DNA plasmids produced in DAM+ bacteria. If Dpnl digestion step is skipped, the linearized PCR product bearing the mutation is purified by affinity using published method and commercial kits (e.g.: ChargeSwitch PCR Clean-Up Kit, from Invitrogen) . If desired, the PCR product is further purified by agarose gel electrophoresis and DNA extraction from the band of expected size '(if predicted on the basis of the mutagenesis introduced) . This gel-purification step is dispensable. Re-circularization of the linear PCR product (whether gel-purified or not) is achieved by ligase reaction. The final product is a plasmid carrying the mutation and with a nick in one strand that is ready to use for transformation of standard competent E. coli TOPlO bacteria.

Design of 3SM primers

Two primers that are not complementary to each other are employed. General features, of mutagenic primers are the following: 1) the two mutagenic primers are designed to perform the DNA polymerase synthesis in the opposite directions on the two strands, starting from the region of interest; 2) one of the mutagenic primers (usually the shorter one) is phosphorylated at its 5' end; 3) the 3' -terminal portion of the mutagenic primer that hybridizes to the DNA strand of the template is preferably 25-30 nucleotides long and rich (about 60%) of GC so that annealing temperature can be adjusted to 68°C as required for the two-step protocol PCR with Platinum Pfx; 4) the two primers do not contain any nucleotide sequence which can determine spontaneous circularization by overhanging. The design of mutagenic primers is strictly dependent on the type(s) of desired mutagenesis. The overall strategy is illustrated in Figure 2. To introduce aminoacid(s) substitutions or insertion the primers extend at their 5' -terminus with a tail of polynucleotides that concurs to form the new sequence. For substitution type of mutagenesis (Figure 2A) each mutagenic primer bears at its 5' -terminus a portion of the mutant coding sequence. In other word, given the new desired sequence, this is split into two parts that form each the extension of the mutagenic primer. There are no restrictions in choosing the point of split in the mutagenesis sequence (even within a codon) and the two tails in mutagenic primers can be of different length. For insertion type of mutagenesis (Figure 2B), the 5' tails of the two primers are made up of nucleotides to be inserted. In this case, the 5' nucleotides of the hybridizing portion of mutagenic primers complement to the nucleotides delimit the point of insertion in the respective DNA strand of the template. PCR extension will result in the insertion of the polynucleotides as dictated by the sequence of the primers tail (Figure 2B). For deletion type of mutagenesis (Figure 2C), the two primers do not bear any tail. In this case primers are made up of oligonucleotides that perfectly match a sequence in each opposing strand and the extent of the region to be deleted is defined only by the distance between their 5' -terminal ends. PCR extension will exclude this region in amplified product (Figure 2C). It is possible to introduce mixed mutageneses such as deletions and substitutions/insertion by using a combination of the primers designed as above (Figure 2D-E) .

EXAMPLES

EXAMPLE 1. Amlnoaclds substitution

The generation of an antigenic epitope in an exposed loop ^'of a protein by modifying adequately the cDNA sequence was performed. This strategy is particularly convenient since allows the generation of a peptide tag, useful for detection and isolation with specific antibody, with minor modification of the primary sequence and, possibly, without altering the tertiary structure. The 9E10^MYC epitope was generated within the sequence of the human enzyme Cathepsin D

(CD, SEQ ID NO.: 17). The coding region to be mutated was chosen based on the following criteria: 1. the epitope should be located within an exposed loop of the protein; 2. the mutagenesis should introduce minimum changes in the original sequence. The 9E1O^MYC epitope corresponds to the EQKLISEEDL peptide (SEQ ID NO.:32).

The present inventors identified a 10 aminoacids sequence (QQKLVDQNIF, SEQ ID NO.: 33) in a convenient region of the protein that could be changed as desired. Note that the codons of the conserved tri-peptide QKL have been changed (see the sequence of the mutagenic primers in Table 1) . Thus, the mutagenesis de facto corresponds to a substitution by simultaneous deletion/insertion. Each mutagenic primer bears at the 5' -end a tail coding for half (5 aminoacids) of the new sequence .

PCR was performed as described above. The PCR product was subjected to Dpnl digestion, split into two aliquots and analyzed by agarose gel electrophoresis (Figure 3A). DNA from one excised band (corresponding to half of the PCR product) was ligated and used for transformation. The electrophoresis analysis of the PCR screening of 20 colonies of transformed E. coli is shown in Figure 3B. Based on this test, the yield of colonies with the mutated plasmid approached 80 % (16 positive out of 20 screened) . All clones positive to PCR screening were also positive to sequencing. The chromo-electropherogram of the sequencing of one positive clone is shown in Figure 3C.

EXAMPLE 2. Aminoacids deletion and substitution by insertion

The 3SM method was carried out to mutagenize a wide region spanning 19 aminoacids in human cathepsin D. In the mutant protein this region is substituted with 8 aminoacids of the corresponding region in Clupea harengus cathepsin D(SEQ ID NO.:34 - GeneBank: AF312364). The sequence coding for the new 8 aminoacids was split into the two extensions forming the tails of the mutagenic primers (see Table 1, SEQ ID NO.: 3 and 4) . Thus, each primer bears the sequence for 4 aminoacids in the tail and hybridizes at the ends of the region of interest (coding for the 19 aminoacids). The principle for designing these primers is illustrated in Figure 2E. PCR extension will lead to deletion of the sequence coding for the 19 aminoacids and contemporary insertion of the sequence coding for the 8 aminoacids. After PCR amplification (20 cycles) and Dpnl digestion, 1/20 of the PCR product was taken for ligation. Half of the PCR product (Dpnl digested) was separated on agarose gel electrophoresis and DNA was recovered from the expected band for ligation. Ligated plasmids (not gel-purified and gel-purified) were used for transformation. Screening analysis of transformant colonies revealed that efficiency of transformation with not gel-purified plasmid was of 50 % (10 out of 20 colonies showed the presence of mutated DNA) , compared to 80 % of efficiency when transformation was performed with the plasmid ligated after gel-purification. The presence of mutated DNA in positive clones obtained with the two strategies was confirmed by sequencing (data not shown) .

EXAMPLE 3. Alanine scanning

3SM method was utilized to perform a systematic mutational analysis of a large sequence in the human cathepsin D protein (SEQ ID NO.:17). The aim was to study the functional role of the aminoacids lateral chains in the sequence T¹⁵⁵VSVPCQSASSASALGGVK¹⁷³ (SEQ ID NO. :35) . To this end, each aminoacid was individually mutated into an alanine, with the exception of S¹⁵⁷, C¹⁶⁰ and GG^170"171. The new final sequence is A¹⁵⁵ASAACA7yyy^AAAAGGAA¹⁷³ (SEQ ID NO.: 36). The primers utilized for this mutagenesis are shown in Table 1 (SEQ ID NO. :5 and 6) . The mutagenic nucleotide sequence differs completely from the wild type sequence even in the codons of the conserved aminoacids. In this particular application, mutated DNA was found in about 50 % of transformant colonies. Sequencing confirmed the data of PCR screening. Inventors have also applied 3SM to generate mutants in which couples of aminoacids of the sequence T¹⁵⁵VSVPCQSASSASALGGVK¹⁷³ (SEQ ID NO.:35) were changed into alanines (not shown) . In this application of 3SM the Dpnl digestion step of the PCR products was skipped. Also in this case positive colonies were obtained with approximately 80 % of efficiency.

EXAMPLE 5. Aminoacids deletion

A fourth example of application of 3SM is represented by a wide deletion encompassing the sequence coding for 93 aminoacids in the cDNA of the human oncosuppressor protein Beclinl. In this experiment, as many as 279 nucleotides were deleted in a 6000 bp long vector. Primers for this mutagenesis were designed as indicated in Figure 2C and are reported in Table 1 (SEQ ID NO. : 7 and 8) . PCR amplified DNA was subjected to Dpnl digestion and gel purification prior to ligation. Screening of E. coli transformed with the ligated plasmid revealed . the presence of mutated DNA in more than 80 % colonies. Sequencing of some of the positive clones confirmed this data. Representative data of this experiment are shown in Figure 4. Naturally, while the principle of the invention remains the same, the details of construction and the embodiments , may widely vary with respect to what has been described and illustrated purely by way of example, without departing from the scope of the present invention as defined in the appended claims.

Claims

References

1. Braman, J. (ed. ) (2002) In Vitro Protocols, 2nd edn. Humana Press, Totowa, NJ. 2. Ishii, T. M., Zerr, P., Xia, ^.M., Bond, CT. , Maylie, J. and Adelman, J. P. (1998) Site-directed mutagenesis. Meth. Enzymol . , 293, 53-71.

3. Kunkel, T. A. (1985) Rapid and efficient site- specific mutagenesis without phenotypic selection. Proc Natl Acad. Sci. USA, 82, 488-492.

4. Sarkar, G. and Sommer, S. S. (1990) The λmegaprimer' method of site-directed mutagenesis. BioTechniques, 8, 404-407.^'

5. Deena, M. KE., Docktor, CM. arid DiMaio, D. (1994) Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes. Nucleic Acids Res., 22, 1593-1599. 6.^' Wang, W. and Marcolm, B. A. (1999) Two-stage PCR protocol allowing introduction of multiple mutations, deletion and insertion using QuickChange™ site-directed mutagenesis. BioTechniques, 26, 680-682.

7. Zheng, L., Baumann, U. and Reymond, JL. (2004) An efficient one-step site-directed and site- saturation mutagenesis protocol. Nucleic Acids Res., 32, ell5

8. Hemsley, A., Arnheim, N., Toney, M. D., Cortopassi, G. and Galas, J. (1989) A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res., 17, 6545-6551. 9. Landt, 0., Grunert, H. P. and Hahn.U. (1990)

A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene, 96, 125-128.

10. Marini, F., Naeem, A. and Lapeyre, JN. (1993)

An efficient 1-tube PCR method for internal site- directed mutagenesis of large amplified molecules. Nucleic Acids Res., 21, 2277-2278.

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Claims

1. Method of introducing mutations into a circular DNA template molecule comprising the steps of: i) providing two mutagenic primers; ii) annealing the mutagenic primers to the DNA template molecule; iii) synthesizing two mutated DNA strands by polymeric chain reaction; iv) annealing the two mutated DNA strands obtaining a linearized mutated DNA molecule; and v) amplifying the linearized mutated DNA molecule by polymeric chain reaction; wherein the mutagenic primers present the following features : each mutagenic primer comprises at its 3' terminal a portion having a nucleotide sequence complementary to a portion of the DNA template molecule to be mutated;

- the mutagenic primers anneal to the DNA template molecule back-to-back in opposite directions; and the mutagenic primers anneal to different DNA template molecule strands.

2. Method according claim 1, characterized in that at least one mutagenic primer presents a mutagenic nucleotide sequence in a tail at its 5' terminal.

3. Method according to claim 1, characterized in that each mutagenic primer presents a mutagenic nucleotide sequence in a tail at its 5' terminal and the two mutagenic nucleotide sequences in the two mutagenic primers are uncomplementary to each other.

4. Method according to any one of claims 1 to 3, characterized in that the mutagenic primers do not contain any nucleotide sequence which can undergo to circularization by overhang.

5. Method according claim 1, characterized in that step iii) is carried out by inverse polymerase chain reaction.

6. Method according to any one of the preceding claims, characterized in that the method further comprises the step of digesting the circular DNA template molecule.

7. Method according to claim 6, characterized in that the step of digesting is performed by Dpnl restriction enzyme or type II methyl-directed restriction enzyme.

8. Method according to any one of the preceding claims, characterized in that the method further comprises the step of purifying the amplified mutated DNA molecule.

9. Method according to claim 8, characterized in that the step of purifying is performed by agarose gel electrophoresis .

10. Method according to any one of the preceding claims, characterized in that the method further comprises the step of ligating the amplified mutated DNA molecule to obtain a circular mutated DNA molecule.

11. Method according to claim 10, characterized in that the step of ligating is performed by a thermostable DNA ligase, preferably T4 DNA Ligase or Pfu DNA Ligase.

12. Method according to any one of the preceding claims, characterized in that the complementary portion of the mutagenic primers to the DNA molecule to be mutated is 10-50 nucleotides long.

13. Method according to any one of the preceding claims, characterized in that the complementary portion of the mutagenic primers to the DNA molecule to be mutated is 20-40, preferably 25-30 nucleotides long.

14. Method according to any one of the preceding claims, characterized in that the complementary portion of the mutagenic primers to the DNA molecule to be mutated is rich in GC nucleotide couple.

15. Method according to the preceding claim, characterized in that the complementary portion of the mutagenic primers contains at least 60% of GC nucleotide couples.

16. Method according to any one of the preceding claims, characterized in that one mutagenic primer is phosphorilated at the 5' terminal.

17. Kit for use in the method of- any one of claims 1 to 16 comprising a DNA polymerase and instructions for carrying out the method.

18. Kit according to claim 17 further comprising individual nucleotide triphosphates or mixtures of nucleotide triphosphates.

19. Kit according to claim 17, characterized in that the DNA polymerase is selected from DNA polymerase Platinum Pfx or recombinant DNA Polymerase from

Thermococcus KOD with proofreading 3' to 5' exonuclease activity.