Patent Application
20060134624
In recent years a number of methods have come into common use that allow the generation of site directed mutants without subcloning based on polymerase activity. This technology is mature enough to allow the sale of a number of mutagenesis kits that are capable of producing point mutants and in some case insertion and deletion mutants (‘indels’).
An important class of 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. Although 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.
However, a need exists to further improve the efficiency of these methods.
INSULT, a novel 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. The method includes cycles of linear amplification with a thermophilic polymerase, and nick repair after each cycle with a thermophilic ligase. After production of multiple single stranded copies of circular mutation bearing plasmid DNA, addition of a ‘generic’ primer followed by one or more polymerase reaction cycles generates double stranded circular DNA bearing the desired mutation.
The present inventions relate to a set of methods which allow the production of site directed mutants via a novel polymerase based strategy which combines the strengths of both of the older methods. 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 kits and methods for site-specific in vitro mutagenesis or combinatorial mutagenesis comprising:
In one embodiment, the products 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.
In another embodiment the invention provides for a kit for use in the methods described herein comprising:
The invention also provides for primers and libraries of primers (e.g., two or more primers) for use in the claimed methods and methods of using mutagenized primers in the described methods.
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
INSULT, a novel 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. The method includes cycles of linear amplification with a thermophilic polymerase, and nick repair after each cycle with a thermophilic ligase. After production of multiple single stranded copies of circular mutation bearing plasmid DNA, addition of a ‘generic’ primer followed by one or more polymerase reaction cycles generates double stranded circular DNA bearing the desired mutation.
The basic strategy used in INSULT is outlined in
In one embodiment, 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. Alternatively, the method is carried out in the absence of an oligonucleotide primer that repairs or inactivates a selection sequence.
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) can be used that bind to the same, different, or overlapping or non-overlapping sequences of the parental polynucleotide. The preparation of mutagenizing primers is generally known in the art.
After production of a suitable number (e.g., preferably between about 10-20) of single stranded mutant copies, 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. If many mutations to genes carried in a vector are contemplated, 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.
In one embodiment, the mutagenized oligonucleotide primer further comprises a unique sequence (e.g. at least about 4 nucleotides) which hybridizes to the second oligonucleotide, or generic, primer, thereby introducing a simultaneous selection step in the DNA synthesis step.
Further adding a blocking oligonucleotide that 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 pcr 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.
It is sometimes convenient to run the first stage overnight, and to finish the procedure with the short second stage (e.g. about 1-5 cycles) the next morning. This allow transformation and plating on selective media on the second day.
The process can be practiced conventiently with currently available vectors and thermophilic enzymes. Currently available kits, such as the Promega and Clontech mu genesis 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 f1 replication origin.
Initial experiments were designed to produce point mutants in the αA-crystalline pACYC184T7 system. The single mutation primers are shown in
Transformation into BL21 cells with 1 uL of the reaction mixture produced about forty colonies on six plates, two for each mutant. As shown in table 1, the mutation frequency for the initial experiments was approximately 80%, and all three mutants were obtained on the first trial.
Production of insertion and deletion mutants was investigated using the same system (aA-crystallin pACYC184T7) with primers as indicated in Table 1. Results from these trials are summarized in Table 1. Insertions and deletions were obtained on the first attempt.
The eNOS pCWori+ system of approximately 9.5 kB represents a significant challenge for mutagenesis because of the presence of GC rich regions and recurring short motifs. Primers designed to insert a stop codon in the eNOS gene failed to produce any mutant colonies in several attempts with Stratagene QCM procedure or with our improved version using separate single primer linear amplification throughout, probably because of runaway PCR artifact.
Transformation of the same cell line (Stratagene XL10-Gold Ultracompetent cells) with the products of the mutagenesis procedure described here under the same conditions produced approximately 150 colonies per plate. As indicated in Table I, all of the colonies sampled were mutants, indicating that the mutation frequency is at least comparable to that obtained with the pACYC184T7 system. Direct comparison of these results suggests that for this mutant the new procedure is at least 1000 times more effective.
Selection Primer System. Several other selection systems are in use, including repair of antibiotic resistance genes and removal of restriction sites, which are features of the Promega and Clontech mutagenesis kits. The Promega kits was used to demonstrate the ability of the new procedure to use the selection protocol. The proprietary plasmid and repair primer generated colonies with the appropriate antibiotic resistance in the first attempt when transformed into Promega's competent cell line.
These results are significant because the new protocol transforms both the Promega and Clontech selection methods from a 50% theoretical mutation frequency to a 100% theoretical mutation frequency.
The production of mutant genes and their products without subcloning has been an important technical advance. Limitations of existing techniques flow naturally from flaws in strategy. QCM is well known to produce primer dimer in some situations, limiting its application in indel production. In addition, all QCM like procedures have the potential to degrade the mutant DNA they produce as the procedure is carried out. Although the mutant strands are never templates for the production of new mutant DNA from the mutagenic primers, the forward and reverse strands can prime each other for extension unless blocked by ‘wrong way’ (3′ out) primer binding. Where extension occurs, each strand is blunt ended, preventing the formation of circular DNA, and the gene is disrupted by the addition of a second copy of the primer sequence. To make matters worse, the duplexes destroyed by this process are now templates for runaway per, limiting the number of cycles of amplification that can be carried out. In favorable cases a high frequency of mutation can still be obtained, but the procedure still produces single stranded DNA requiring ultracompetent cells for transformation.
Clontech and Promega type strategies are limited by production of only a single copy of mutant DNA per parental, and by the production of hybrid duplexes which limits the selection power of antibiotic or restriction enzyme resistance. Production of high levels of mutant DNA is relatively easy by using thermostable enzymes that allow multiple copying steps. Introduction of a completely uncomplimentary, generic reverse primer makes INSULT qualitatively different from previous procedures, because the mutant copies produced are closed circular AND homoduplex. This is only possible because the multiple copies produced in stage 1 are in closed circular form; linear copies produced without ligase activity cannot be templates for synthesis of a reverse strand without introduction of primers to sites adjacent to the mutagenic primer, and this produces blunt ended linear duplexes.
Numerous variants of INSULT are feasible. Running a single cycle second stage decreases the amount of mutant DNA with the compensating advantage of introducing fewer copy errors. There are several options available for parental suppression. These include DPN1 digestion of methylated template as introduced by Strategene. Clontech and Promega selection strategies use a second forward ‘selection’ primer to repair an antibiotic resistance site or suppress a restriction site, and many other schemes are possible e.g., introduction of a mutation preventing induction of an inhibitory gene. These schemes are of real but limited utility in existing protocols because duplex DNA is a hybrid with one mutant and one parental strand, limiting selection efficiency to 50% with one transformation. Because INSULT produces homoduplexes, these selection schemes have a theoretical efficiency of 100% when applied within the INSULT context. This is true even in the limiting case when both the first and second stage are reduced to a single cycle, which would allow the use of T4 or other thermosensitive polymerase and ligase combinations. We believe that the T4 system is less desirable because of the lack of amplification of mutant DNA, but in view of the potential for total parental suppression this could be compensated for by increasing the level of template DNA.
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 mn mutants from mn primers when run together in the first stage. A single generic primer would suffice for the second stage. Use of a combinatorial mutagenic primer (a primer set in which all or many possible combinations of bases in a short stretch are present) would produce 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.
One skilled in the art will appreciate the many advantages that the method of the invention provides. For example, 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. Likewise 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. In yet other embodiments, 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.
Methods
Polymerase and ligase reactions were carried out simultaneously in the same vessel. The reaction mixture consisted of 5 ul of 10× Reaction buffer, 10 ng of template DNA, 125 ng of phosphorylated mutagenesis primer, 5 ul 10 mM NAD+ (ligase cofactor), 1 ul 20 mM dNTP mix, 1 ul Pfu Turbo, 1 ul Taq DNA ligase, and dH20 addded to make the final reaction mixture 50 uL.
The 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.
After holding at 4C, 2 ul 100 ng/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 60C 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.
Transformation of the same cell line (Stratagene XL10-Gold Ultracompetent cells) with the products of the mutagenesis procedure described here under the same conditions produced approximately 150 colonies per plate. As indicated in Table I, all of the colonies sampled were mutants, indicating that the mutation frequency is at least comparable to that obtained with the pACYC184T7 system. Direct comparison of these results suggests that for this mutant the new procedure is at least 1000 times more effective. CT CGA GCA CCA CCA CCA CC
TC GTC CCT CGA GC (SEQ ID NO. 9)
Bold = substitution
Highlight = insertion
Forward (mutagenic) and reverse (generic) primers for initial trails with INSULT mutagenesis. Sequencing results indicate the number of correct mutant sequences and total trials.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 60/445,689, filed on Feb. 6, 2003, U.S. Provisional Application No. 60/445,703, filed on Feb. 6, 2003, U.S. Provisional Application No. 60/446,045, filed on Feb. 6, 2003, U.S. Provisional Application No. 60/445,704, filed on Feb. 6, 2003 and U.S. Provisional Application No. 60/474,063, filed on May 29, 2003, Docket No. RPI-812, entitled “Parental Suppression via Polymerase-based Protocols for the Introduction of Deletions and Insertions.” The entire teachings of the above applications are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US04/03497 | 2/6/2004 | WO | 1/17/2006 |
| Number | Date | Country | |
|---|---|---|---|
| 60445689 | Feb 2003 | US | |
| 60445703 | Feb 2003 | US | |
| 60446045 | Feb 2003 | US | |
| 60445704 | Feb 2003 | US | |
| 60474063 | May 2003 | US |
