WO2006050062A2 - Protocoles reposant sur la polymerase pour l'introduction de deletions et d'insertions - Google Patents

Protocoles reposant sur la polymerase pour l'introduction de deletions et d'insertions Download PDF

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WO2006050062A2
WO2006050062A2 PCT/US2005/038873 US2005038873W WO2006050062A2 WO 2006050062 A2 WO2006050062 A2 WO 2006050062A2 US 2005038873 W US2005038873 W US 2005038873W WO 2006050062 A2 WO2006050062 A2 WO 2006050062A2
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polynucleotide
stranded
parental
oligonucleotide primer
obtaining
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PCT/US2005/038873
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WO2006050062A3 (fr
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John C. Salerno
Susan Smith
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Rensselaer Polytechnic Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • polymerase-based mutagenesis methods use two complementary or partially complementary primers together with a thermostable polymerase to produce linearly amplified, double stranded linear DNA.
  • the amplification is linear because primers binding to linear products face the wrong way (3' out) to serve as primers for elongation.
  • these methods are powerful, they contain flaws that limit their application and require expensive and delicate 'ultracompetent' cells for transformation because the products are linear.
  • a second class of mutagenesis methods use a T4 polymerase and a T4 ligase to make a single mutant copy which forms part of a hybrid circular duplex with the parental template from which it was copied.
  • a second forward selection primer is included allowing partial suppression of parentals based on repair of an antibiotic resistance gene or suppression of a restriction site.
  • the production of circular duplex DNA is highly desirable, but the hybrid nature of the duplex DNA limits the selection to 50% unless additional rounds plasmid preparation and transformation are included. This is so cumbersome that it is generally easier to sequence extra colonies. In addition, the single cycle limits the production of mutant DNA.
  • the present inventor has previously invented INSULT, a method for the creation of insertions, deletions, and point mutations without subcloning, requires only one new primer per mutant, and produces circular plasmids, obviating the need for special 'ultracompetent' cells. This method is described in WO 04/072245 incorporated herein by reference.
  • the basic strategy of INSULT is outlined in Figure 1.
  • a single primer bearing a mutation is annealed to one strand of a denatured template consisting of double stranded closed circular plasmid carrying the gene (or other sequence) to be mutagenized.
  • a thermophilic polymerase and thermophilic ligase are added and the temperature cycled to produce single stranded closed circular copies of the target strand.
  • Use of a single primer produces linear amplification of the mutant strands.
  • a 'generic' primer is introduced. This primer should not overlap the mutation, and it is desirable that no part of it be complementary to the mutagenizing primer.
  • the second stage preferably consists of one cycle of denaturation, annealing, and polymerase activity to produce closed circular duplexes of the mutant and parentals, with the mutant DNA in great excess.
  • the second stage could optionally include several more cycles.
  • the present invention relates to improved methods based on the INSULT system that provide the production of site directed mutants in larger gene-plasmid systems.
  • the results are high yields of mutant DNA, closed circular double stranded products which obviate the need for specialized 'ultracompetent' cells, and protocols which require only one new primer per mutant.
  • the invention relates to improved methods for site-specific in vitro mutagenesis or combinatorial mutagenesis comprising: (a) cloning a parental polynucleotide (such as polynucleotide comprising a coding sequence or gene) into a vector comprising a cloning site, thereby obtaining a cloned product; (b) denaturing the cloned product, thereby obtaining a single-stranded polynucleotide template;
  • a parental polynucleotide such as polynucleotide comprising a coding sequence or gene
  • steps (g) optionally repeating steps (c)-(f) (e.g., via thermal cycling);
  • step Q) hybridizing the single stranded mutated polynucleotides with a generic oligonucleotide primer thereby obtaining second hybridized complexes; (i) adding additional polymerase and ligase to the reaction mixture in step(h); (j) extending the second hybridized complex and ligating the double stranded product thereof, thereby obtaining a circular double stranded mutated polynucleotide; and (k) optionally transforming the double-stranded mutated polynucleotide into a bacterial host, thereby obtaining transformants.
  • the products of step Q) can be subjected to optional PCR amplification, or DNA synthesis, such as about four or more cycles, to further increase the number of mutant products.
  • the invention provides for a kit for use in the methods described herein comprising: (a) a vector comprising a cloning site;
  • helper primers instructions for carrying out the method.
  • the invention provides improved methods based on the INSULT system that provide the production of site directed mutants in larger gene-plasmid systems using generic "helper primers" to start DNA synthesis at several locations on the plasmid, allowing the extension time to be short even for large plasmids.
  • This method of site-directed mutagenesis comprises the steps of:
  • step (g) optionally repeating steps (c)-(f) (e.g., via thermal cycling); (h) hybridizing the single stranded mutated polynucleotides with a second oligonucleotide primer and optionally, at least one second helper primer; (i) extending the second oligonucleotide primer and optionally, the second helper primer and ligating the double stranded product thereof, thereby obtaining a circular double stranded mutated polynucleotide; and Q) optionally transforming the double-stranded mutated polynucleotide into a bacterial host, thereby obtaining transformants.
  • the products of step (i) can be subjected to optional PCR amplification, or DNA synthesis, such as about four or more cycles, to further increase the number of mutant products.
  • Figure 1 is a schematic representing the basic strategy used in INSULT, showing formation of multiple copies of closed mutant single stranded DNA in the first stage and binding of the generic primer to start the second stage.
  • a single cycle of polymerase activity produces mutant closed circular hornoduplex DNA; optional additional cycles PCR amplify the mutant product and linearly amplify one strand of parental DNA.
  • Figure 2 is an agarose gel showing the comparison of single, double and triple mutant generation via circular PCR and INSULT in eTNTOS pCWori+.
  • Lanes 1- 3 Circular PCR raw products for point mutant, (Lane 1), point mutant plus 132 bp deletion (Lane 2), point mutant plus 132 bp and 18 bp deletions (Lane 3).
  • Lanes 4 and 8 DNA ladders.
  • Lanes 5-8 raw products from INSULT mutagenesis corresponding to lanes 1-3.
  • the present invention describes improvements to a system referred to herein as "INSULT," a recently discovered method for the creation of insertions, deletions, and point mutations without subcloning, requires only one new primer per mutant, and produces circular plasmids, obviating the need for special 'ultracompetenf cells.
  • the basic INSULT method includes annealing a single primer bearing a mutation to one strand of a denatured template consisting of double stranded closed circular plasmid carrying the gene (or other sequence) to be mutagenized. Linear amplification of the mutant strands with a thermophilic polymerase, and nick repair after each cycle with a thermophilic ligase is then conducted.
  • INSULT is much less reliable for larger gene/plasmid systems.
  • Investigation of the problem using several gene/plasmid systems of different lengths suggested that a major contributor to the problem was the longer extension/ligation times necessary for a larger gene or plasmid cause inactivation of the heat resistant polymerase and ligase enzymes too early in the protocol.
  • the initial extension and ligation times are shortened and the first stage is limited to about 10 cycles, as it is believed that the polymerase is inactivated by this time.
  • extension/ligation times and reducing the number of first stage cycles significantly improved the results with the gene/plasmid systems up to 7.5 kB, allowing good production of mutants and producing strong bands on agarose gels consisting primarily of mutant homoduplex DNA.
  • extension time could not be shortened enough to obtain good results, even with only ten cycles in the initial stage.
  • additional polymerase/ligase is added along with the generic primer and the subsequent extension/ligation times are also shortened to about 1-10 cycles.
  • the method of adding more polymerase/ligase with the generic primer is also referred to herein as "double shot.”
  • the double shot method of the invention allows for the production of much higher levels of mutant DNA in several gene/plasmid systems of various sizes ranging from, for example about 6 base pairs (bp) to about 8 bp and particularly the larger gene/plasmid systems ranging from, for example about 8 bp to about 15 bp. Insertions and deletions ranging from, for example about 1 to at least about 150 base pairs are feasible with the double shot method of the invention.
  • a method to extend the reliability of the INSULT system for producing high levels of mutant DNA to larger gene/plasmid system comprises annealing one or more generic "helper primers" along with the mutant primer to start synthesis at several locations on the plasmid, allowing the extension time to be short, even for large plasmids.
  • This method in combination with reducing cycle times to about 10 cycles allows for the successful production of mutant DNA using larger gene/plasmid systems as described above for the double shot method.
  • helper primer is a generic primer that is complementary to a portion of the same strand of the denatured template that the mutant primer is annealed to and possesses the same orientation as the primer that the helper primer assists (i.e.
  • helper primers should be well spaced on the plasmid to reduce the number of base pairs added.
  • thermophilic polymerase and thermophilic ligase are added and the temperature cycled about 10 times to produce single stranded closed circular copies of the target strand as described in the methods section.
  • thermophilic polymerase and thermophilic ligase such as, Turbo pfu polymerase and Taq ligase
  • thermophilic ligase such as, Turbo pfu polymerase and Taq ligase
  • Use of a single mutant primer produces linear amplification of the mutant strands.
  • T4 polymerase one preferably adds enzyme prior to or during each cycle to maximize activity.
  • Thermophilic ligases can often be used without subsequently refreshing the reaction medium.
  • the parental strand is destroyed in the reaction medium or selected against after transformation, for example, by using a selection primer, such as those provided with commercial kits, such as the Clontech Transformer Kit.
  • the method is carried out in the absence of an oligonucleotide primer that repairs or inactivates a selection sequence.
  • parental strand suppression is achieved without the use of enzymatic activity.
  • methylated DNA binding protein domains are used to bind parental strands before transformation. These domains are small (65-70 residues) and easily produced, and can be readily immobilized. Parental DNA can be eluted from immobilized proteins, enabling the recycling of the filters, beads, or other immobilizing agents used in the reaction. In a preferred embodiment, this is done between the first and second steps (e.g. before the addition of generic primer). Any selection based on methylation state is preferably done after all the first stage copies are made but before any second stage copies are made.
  • parental strand suppression is achieved by selectively removing the single-stranded parental DNA complement (the parental DNA that was not the template for the mutagenic primer) prior denaturing the first heteroduplex produced in the first cycle.
  • the parental DNA complement may be removed after the first cycle by any number of methods including selectively digesting the parental DNA complement strand with an endonuclease as is known in the art, binding the parental DNA complement to an immobilized support via DNA (or other nucleic acid) complementary (antisense) to the parental DNA complement or via proteins capable of selectively binding methylated DNA as discussed above.
  • the immobilizing support is preferably reusable after the parental DNA has been eluted. Any selection method based on single vs.
  • the mutagenized oligonucleotide primer is capable of hybridizing to the polynucleotide sequence to be mutated and introduce one or more mutations.
  • the primer can insert, delete or substitute/change one or more nucleotides (such as three or more nucleotides) or one or more codons (such as two, five or more codons), for example.
  • Multiple primers e.g., about 5, 10 or 20 or more
  • the preparation of mutagenizing primers is generally known in the art.
  • a "generic" primer is introduced.
  • a generic primer is also referred to herein as a "universal" primer.
  • helper primers as discussed above may also be added along with the generic primer.
  • an additional aliquot of both polymerase and ligase are added to the reaction mixture along with the generic primer.
  • the generic primer should not overlap the mutation, and it is desirable that no part of it be complementary to the mutagenizing primer.
  • the generic primer can be made to a position in the vector outside the cloning site. If many mutations are to be made to a gene in different vectors, reverse or forward primers used for copying the gene, or internal sequencing primers which don't overlap the mutation primer, are suitable as long as the generic primer and the mutation primer anneal to opposite strands of the template.
  • the mutagenized oligonucleotide primer further comprises a unique sequence (e.g. at least about 4 nucleotides) that hybridizes to the second oligonucleotide, or generic, primer, thereby introducing a simultaneous selection step in the DNA synthesis step.
  • a blocking oligonucleotide that selectively hybridizes to the parental polynucleotide at or proximal to the sequences the mutagenized oligonucleotide primer hybridizes can additionally provide a negative selection for the parental polynucleotide.
  • One cycle of denaturation, annealing, and polymerase activity produces closed circular duplexes of the mutant and parentals, with the mutant DNA in great excess. Additional cycles of PCR, preferably no more than about 10 cycles, amplify the mutant DNA and linearly amplify one strand of the parental DNA. This leads to a huge excess of duplex mutant DNA, but many cycles of per could cause the accumulation of copy errors in the pool of mutants even with a high fidelity polymerase.
  • the methods of the invention can be practiced conveniently with currently available vectors and thermophilic enzymes.
  • Currently available kits such as the Promega and Clontech mutagenesis kits, can be adapted for use in the procedure, but the enzymes used in these kits are not thermostable. This limits them to a single thermal cycle per enzyme addition, which is not optimal.
  • the vectors used can comprise an insertion site for introducing the parental polynucleotide.
  • the vector can also further comprise a replication of origin, such as that of a filamentous bacteriophage, for example.
  • the replication of origin is preferably an fl replication origin.
  • the improvement to the INSULT system devised to overcome the obstacle of size for eNOS in pCWori+ was the addition of a second aliquot of polymerase and ligase added with the generic primer, or "double shot.”
  • the initial stage was shortened to 10 cycles, because it was clear that at least the polymerase was inactivated by this time, and ten cycles were selected as the nominal length of the second stage.
  • a true circular PCR protocol was followed, in which both primers were added simultaneously to the tube; we used the same primers, buffers, enzymes, etc. and the same thermocycler settings, but there was only one stage because both primers were present from the beginning.
  • Table 2 Selected primers and mutation results for indel and double indel mutations in eNOS and nNOS pCWori+.
  • the inherent ligase component of INSULT provides great potential for parallel introduction of multiple mutations. Multiple mutagenic primers would be extended by the polymerase to produce sections of DNA aligned along the circular template; the nicks separating the ends would be repaired by the ligase, generating multiple mutations in a single procedure. Limitations on this capability are imposed primarily by the need to not have the primers overlap, and in many cases closely spaced mutations could be carried on a single primer. Typically, the mutagenizing primers for point mutations are between about 15 and 35 basepairs (often 18-30 basepairs) in length. Mutations to two codons separate by less than half the primer length can most easily be accommodated by changing both codons in a single mutation.
  • Mutagenizing primer design is generally known in the art. Combinatorial numbers of mutants and 'limited chimera' can in principle be constructed with a limited number of primers by applying the multiple mutation approach with mixtures of mutagenic primers. (The chimera produced are limited in scope by the size of the individual primers used). For example, n sets consisting of m mutagenic primers each, binding to n different sites within a gene, would generate m n mutants from m n primers when run together in the first stage. A single generic primer would suffice for the second stage.
  • combinatorial mutagenic primer a primer set in which all or many possible combinations of bases in a short stretch are present
  • a combinatorial mixture of mutants concentrated in a single site Since in all cases the mutants are produced without subcloning and transform directly into cell lines capable of expression, the system has great potential for selection-based applications.
  • a primary advantage of INSULT is the ability of the relatively high levels of circular duplex mutant DNA to transform expression competent cells directly. In most cases this represents a greater economy than the need for only one primer per mutation. More importantly, it removes the need for a second cycle of transformation to produce mutant proteins, which in most cases is the object of the exercise. This streamlining of the procedure greatly reduces the time and effort involved. In addition to saving human time, it moves the entire process into a form amenable to 96 well plates and robotics until the point of scale up from colony selection to protein production. In most cases expensive 'Ultracompetent' cells are unnecessary. On the other hand, the use of such cells in the INSULT process can produce very large numbers of mutants compared to other methods and allows the rapid production of mutants.
  • the improved site-directed mutagenesis methods of the invention are useful in protein and enzyme engineering technologies (to impart desirable properties on proteins, enzymes, polynucleotides, etc.) for the production of drugs, diagnostics, research proteins and enzymes, agrochemicals, plant proteins, industrial proteins and enzymes such as detergent enzymes, enzymes useful for neutralizing contaminants, and enzymes suitable for improved or novel biosynthesis of compounds in industry, biotechnology, and medicine.
  • the methods of the invention are useful in protein engineering technologies for the production of proteins useful in the food and life sciences industries such as primary and secondary metabolites useful in the production of antibiotics, proteins and enzymes for the food industry (bread, beer), and combinatorial arrays of proteins for use in generating multiple epitopes for vaccine production.
  • the invention can also be used to manufacture novel polynucleotides, including DNAs and RNAs, such as RNA inhibitors.
  • the inventions can be used to manufacture protein tags, such as N-terminal addressing, affinity tags, labeling sites, etc.
  • the invention can be used in cell biology discovery and understanding protein-protein interactions. Fusion proteins for purification, targeting, labeling can be manufactured using the methods of the invention. For example, vectors with a GFP gene adjacent to a cloning site would allow easy conversion of a vector for expression of a target gene, e.g. via a linker.
  • Example 1 Methods and Materials eNOS in pCWori+, nNOS in pCWori+, eNOS in pCRT7, and small heat shock proteins in pACYC184T7 or pET20 were used as templates in the mutagenesis experiments described herein.
  • the basic strategy of INSULT is outlined in Figure 1.
  • a single primer bearing a mutation is annealed to one strand of a denatured template consisting of double stranded closed circular plasmid carrying the gene (or other sequence) to be mutagenized.
  • a thermophilic polymerase and thermophilic ligase are added and the temperature cycled to produce single stranded closed circular copies of the target strand.
  • Use of a single primer produces linear amplification of the mutant strands.
  • a 'generic' primer is introduced and in the "double shot" improvement of the invention, an additional aliquot of polymerase and ligase area added to the reaction.
  • the primer should not overlap the mutation, and it is desirable that no part of it be complementary to the mutagenizing primer.
  • the second stage consists of one cycle of denaturation, annealing, and polymerase activity to produce closed circular duplexes of the mutant and parentals, with the mutant DNA in great excess.
  • the reaction mixture consisted of 5ul of 1Ox Reaction buffer, 10 ng of template DNA, 125ng of phosphorylated mutagenesis primer, 5ul 1OmM NAD+ (ligase cofactor), IuI 2OmM dNTP mix, IuI Pfu Turbo, IuI Taq DNA ligase, and dH20 added to make the final reaction mixture 50 uL.
  • thermocycler program consisted of two stages. In the first, the template was denatured at 94C for 2', followed by annealing at 60C for 50 sec and extension for 10 minutes at 68C; on completion of extension around the plasmid the ligase closed the nicked product. Subsequent cycles (1-5) were identical except that the 94C step was shortened to 50 sec.
  • 2ul lOOng/ul phosphorylated universal primer was added to the reaction mixture in preparation for step 2. After denaturation at 94C for 2 minutes, the primers were annealed for 50 sec at 6OC and extended at 68C, followed by nick repair. Up to four additional cycles followed as in the first stage. 50 uL of competent BL21DE3 cells were transformed with 1 uL reaction mixture, and the resulting transformed cells were plated on LB antimycin plates for selection of colonies. A representative fraction of antibiotic resistant colonies were selected and sequenced to confirm the production of mutants.
  • Standard circular PCR mutagenesis with the same primer set produced nearly as good results with a single point mutant, but in contrast to INSULT produced primarily artifacts in double and triple mutagenesis trials (see Fig 2).
  • the large amount of closed circular homoduplex mutant DNA produced by the INSULT procedure allowed us to transform competent JM 109 cells, dispensing with the need for delicate and expensive ultracompetent cells.

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Abstract

La présente invention concerne des méthodes améliorées destinées à la création d'insertions, de délétions et de mutations ponctuelles sans sous-clonage qui engendrent la production de mutants dirigés vers des sites dans des systèmes plus importants de plasmide/gène. Les résultats se présentent sous forme de rendements élevés d'ADN mutant, de produits à double brin circulaires, fermés qui évitent d'avoir recours à des cellules spécialisées 'ultracompétentes', et de protocoles qui ne requièrent qu'une nouvelle amorce par mutant.
PCT/US2005/038873 2004-10-28 2005-10-27 Protocoles reposant sur la polymerase pour l'introduction de deletions et d'insertions WO2006050062A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008067035A2 (fr) * 2006-10-05 2008-06-05 Nationwide Children's Hospital Mutagenèse illimitée et procédés de clonage

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001094634A2 (fr) * 2000-06-06 2001-12-13 Xtrana, Inc. Procede et dispositif de multiplexage des reactions d'amplification
WO2004072245A2 (fr) * 2003-02-06 2004-08-26 Rensselaer Polytechnic Institute Protocoles bases sur la polymerase pour l'introduction de deletions et d'insertions
US20040175733A1 (en) * 2002-12-04 2004-09-09 Andersen Mark R. Multiplex amplification of polynucleotides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001094634A2 (fr) * 2000-06-06 2001-12-13 Xtrana, Inc. Procede et dispositif de multiplexage des reactions d'amplification
US20040175733A1 (en) * 2002-12-04 2004-09-09 Andersen Mark R. Multiplex amplification of polynucleotides
WO2004072245A2 (fr) * 2003-02-06 2004-08-26 Rensselaer Polytechnic Institute Protocoles bases sur la polymerase pour l'introduction de deletions et d'insertions

Non-Patent Citations (1)

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Title
THOMAS D.C. ET AL.: 'Amplification of Padlock Probes for DNA Diagnostics by Cascade Rolling Circle Amplification or the Polymerase Chain Reaction' ARCH. PATHOL. LAB. MED. vol. 123, 1999, pages 1170 - 1176, XP008001739 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008067035A2 (fr) * 2006-10-05 2008-06-05 Nationwide Children's Hospital Mutagenèse illimitée et procédés de clonage
WO2008067035A3 (fr) * 2006-10-05 2009-05-22 Nationwide Childrens Hospital Mutagenèse illimitée et procédés de clonage

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