WO2006050062A2 - Polymerase-based protocols for the introduction of deletions and insertions - Google Patents

Abstract

The present invention relates to improved methods for the creation of insertions, deletions, and point mutations without subcloning 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.

Description

POLYMERASE-BASED PROTOCOLS FOR THE INTRODUCTION OF DELETIONS AND INSERTIONS

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/623,015, filed on October 28, 2004. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

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. 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. In the first stage, 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. After production of a suitable number (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. 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.

One drawback of the INSULT system described above is that it appears to be less reliable in larger gene-plasmid systems, a property; it shares with other mutagenesis procedures, including all current commercially available kits.

SUMMARY OF THE INVENTION

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;

(c) hybridizing at least one mutagenized oligonucleotide primer to the single- stranded polynucleotide template, thereby obtaining a first heteroduplex; (d) extending the first heteroduplex with a polymerase, thereby obtaining an extended product;

(e) reacting the extended product with a ligase, thereby obtaining ligated product;

(f) denaturing the ligated product, thereby obtaining a closed single stranded mutated polynucleotide;

(g) optionally repeating steps (c)-(f) (e.g., via thermal cycling);

(h) 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. In one embodiment, 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.

In another embodiment the invention provides for a kit for use in the methods described herein comprising: (a) a vector comprising a cloning site;

(b) a generic oligonucleotide primer;

(c) a polymerase;

(d) a ligase;

(e) optionally, one or more helper primers (f) instructions for carrying out the method.

In an alternative embodiment, 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:

(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 and a single-stranded polynucleotide complement; (c) hybridizing at least one mutagenized oligonucleotide primer and at least one helper primer to the single-stranded polynucleotide template, (d) extending the mutagenized oligonucleotide primer and the helper primer with a polymerase, thereby obtaining an extended product; (e) reacting the extended product with a ligase, thereby obtaining ligated product;

(f) denaturing the ligated product, thereby obtaining a closed single stranded mutated polynucleotide;

(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. In one embodiment, 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. BRIEF DESCRIPTION OF THE DRAWINGS

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. 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.

DETAILED DESCRIPTION OF THE INVENTION

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. 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 Figure 1.

Like other mutagenesis methods, 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. In one embodiment of the invention, 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. Merely shortening the 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. In larger systems (~10kB) the extension time could not be shortened enough to obtain good results, even with only ten cycles in the initial stage.

In conjunction with shortening the extension/ligation time, 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.

In another embodiment 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.

In the basic INSULT method a single mutant 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. In one improvement on the basic INSULT method, one or more helper primers are also annealed to the same strand of the denatured template as the mutagenic primer. As used herein a first stage "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. either mutagenic primer or, during the second stage, the generic primer), but preferably does not overlap any portion of the mutation and is not complementary to any portion of the mutagenizing primer. To be effective, helper primers should be well spaced on the plasmid to reduce the number of base pairs added.

A polymerase, such as T4 or, preferably, thermophilic polymerase and thermophilic ligase (such as, Turbo pfu polymerase and Taq 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. Use of a single mutant primer produces linear amplification of the mutant strands. When T4 polymerase is employed, 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. 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. In another improvement of the INSULT system, parental strand suppression is achieved without the use of enzymatic activity. In this embodiment, 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.

In yet another improvement in accordance with the invention, 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. double strand is preferentially employed after the first cycle of stage one. 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 about 10) of single stranded mutant copies, a "generic" primer is introduced. A generic primer is also referred to herein as a "universal" primer. In one improvement embodiment of the invention, helper primers as discussed above may also be added along with the generic primer. In another improvement embodiment of the INSULT method, also referred to herein as the "double shot" method, 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. 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) that 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 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. Initial experiments testing the basic INSULT strategy (see WO 04/072245 incorporated herein by reference) were designed to produce point mutants in the small and tractable αA-crystallin pACYC184T7 system. The single mutation primers are shown in Table 1; the generic primers used in these experiments are simply the reverse primers that were originally used to copy the gene for introduction into the plasmid, and overlap the gene/plasmid junction. Transfonnation into BL21 cells with 1 uL of the reaction mixture produced a total of about forty colonies on six plates, two plates 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.

Table 1:

Clone name Primer Sequence Sequencing

Results

Alpha A Crystallin- R116G F: CGC GAG TTC CAC GGC CGC TAC CGC CTG CCT TCC (SEQ ID NO. 1) 2/3

R: CC CCT CAA GAC CCG TTT AGA GGC CCC (SEQ ID NO. 2)

Alpha A Crystallin- F:GGA GAT ATA CAT ATG GGC ATC GCC ATT CAG CAC CCC TGG (SEQ D2G ID NO. 3) 2/3

R: CC CCT CAA GAC CCG TTT AGA GGC CCC (SEQ ID NO.2)

Alpha A Crystallin- F: GAG GTC CGA TCC GAC CGG AGC AAG TTT GTC ATC TTC CTG G D69S (SEQ ID NO. 4) 3/3

R: CC CCT CAA GAC CCG TTT AGA GGC CCC (SEQ ID NO. 2)

Alpha A Crystallin- F: GCC CAG CTC TGC GCT GTG GAA GΘG dCT CGA GCA CCA CCA CCA ALWKG CC (SEQ ID NO. 5) 1/1

R: CC CCT CAA GAC CCG TTT AGA GGC CCC (SEQ ID NO. 2)

Chimera correction F: CAG CTC TGC GCC GfTC GTC CCT CGA GC (SEQ ID NO. 6) 2/2

R: CC CCT CAA GAC CCG TTT AGA GGC CCC (SEQ ID NO. 2) BoId= substitution; Highlights 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. Production of insertion and deletion mutants was also investigated in the small heat shock gene/ pACYC184T7 system; results from these trials are also summarized in Table 1, along with the primers used for these experiments. Insertions and deletions were obtained on the first attempt with mutation frequencies comparable to those obtained for the point mutants. An agarose gel (not shown) of the products of an INSULT mutagenesis procedure using small heat shock genes shows that the primers have been converted into two dominant species: the larger of these is the desired product, the closed circular homoduplex mutant DNA, and the other species is an artifact caused by incomplete closure by the ligase. This artifact does not interfere with mutagenesis and does not significantly transform cells because it is blunt ended and linear.

This success encouraged us to use the INSULT method on eNOS-pCWori+, and also on the smaller eNOS-pCRT7. The eNOS-pCWori+ gene/plasmid system of -9.5 kB represents a significant challenge because of its large size, GC-rich regions, and recurring short repeats. In order to compare results directly using the same primer set, the generic primers used in these experiments recognized the end of the eNOS gene rather than the vectors. Contrary to our expectations, we did not observe eNOS mutants on agarose gels in either plasmid in these trials. We also did not observe any successfully transformed competent BL21 cells from these trials. We did, however, consistently obtain the desired mutants in eNOS pCRT7 when we used Stratagene XLlO-GoId Ultracompetent cells for transformation; these trials produced approximately 150 colonies per plate, with mutational frequencies comparable to the results obtained with ocA-crystallin pACYC184T7. We did observe a few antibiotic-resistant colonies from mutagenesis of eNOS-pCWori+, but the procedure was not reliable.

Because of the apparent sensitivity of the procedure to the size of the gene- plasmid system, we surmised that the longer extension/ligation times used were inactivating the heat resistant polymerase and ligase enzymes too early in the protocol. We therefore shortened the extension/ ligation times to 12 minutes in parallel experiments using eNOS in both pCRT7 and pCWori+. The shortened time proved highly beneficial for pCRT7; we were able to observe the mutant circular homoduplexes on agarose in every case (not shown) and obtained colonies from transformation of competent JMl 09 cells, removing the requirement for ultracompetent cells. The results for pCWori+ were not promising; the system appears to be too large for this extension/ligation time. The results were consistent with enzyme inactivation, leading us to the present improvement of INSULT for larger gene plasmids.

In accordance with the present invention, 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. In a parallel experiment 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. Agarose gels of the results for single, double, and triple mutants are shown in Figure 2. The single mutant was a short insertion introducing a stop codon, while the other two mutations were deletions of 132 and 18 bp (see Table 2 for examples of primers).

Table 2: Selected primers and mutation results for indel and double indel mutations in eNOS and nNOS pCWori+.

Sequencing

Clone name Generic Primer Mutagenic Primer(s) Results eNOS (-838-843) ATG GGC AAC HG AAG AGT GTG C CCG CAC CCA GCT GGG AGG AGG GCC GCC TGG GCT

SI deletion (SEQ ID NO.7) GCC TT CTC CAG (SEQ ID NO. 8) 2/3

GT CCC CAG GGC CCC CGC ACT GTC TGT GTT GCT GGA

CTC Cπ TCT CTT CCG COG CCA GGA GGA CAC CAG CGG eNOS (-602-641) ATG GGC AAC TTG AAG AGT GTG GTC AGA GCA GGA GAC GCT GTT GAA GCG GAT CTT GTA

Al deletion (SEQ ID NO. 7) ACT Cπ GTG CTG TTC CGG CCG CGG GGA

GCTGTTGTAGGGCCCC (SEQ ID NO. 9) J4

#1 : C CCG CAC CCA GCT GGG AGG AGG GCC GCC TGG GCT eNOS (-838-843, - GCC π CTC CAG (SEQ ID NO. 10)

1166-1205) Sl, C #2: C CTC CTG GGT GCG CAG CGT fM GCC GAA AAT GTC terminal double ATG GGC AAC HG AAG AGT GTG CTC G fSEQ ID NO. 11) 1/2 * deletion (SEQ ID NO. 7)

#1 : πc ATC cπ CCA AΠ ACT GAT GAC ACC CAG AGC AGT

Gπ CCT CTC CTC (SEQ ID 12)

#2: GGC CAG GGG TCC AGT ACT πC AAT AGT ACT πc AAA

Gπ GTC TCT GAG GTC GGG TCC GTC GCC CGT ACT GAA nNOS (-1172-1177, AGC GGA GTC AGA ATA GGA GGA GAC GCT Gπ GAA TCG

-824-86I) SI₁ AI GAC ACA GAT ACC ATG GAA GAG GAC Cπ GTA GCT CTT TCT CTC CTC CTG CAC AGA Gπ double deletion AAC (SEQ ID 14) GGG GTG CCT CAT (SEQ ID 13) 1/4 *

BoId= substitution; Highlight= insertion; Underline= deletion

*Positive clones correspond to double mutants; other clones are single mutants Lanes 1, 2 and 3 in Figure 2 are mutagenesis trials by standard prior art circular PCR methodology; full length circular mutant DNA was obtained in good yield for the single mutants, but artifact production dominated the double and triple mutant trials; no full length mutant DNA was detectable for the triple mutant. Lanes 5, 6 and 7 show the corresponding trials for INSULT. In all cases the production of full length DNA was superior to circular PCR, and even in the double and triple mutant procedures this is the dominant species with little or no artifact production. Numerous variants of INSULT with the improvements of the invention 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 DPNl 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 improved 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.

Examples 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. In the first stage, 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.

After production of a suitable number (10-20) of single stranded mutant copies, 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.

Polymerase and ligase reactions were carried out simultaneously in the same vessel. 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.

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, 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.

Transformation of the same cell line (Stratagene XLlO-GoId 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. Example 2: "Double Shot" and "Helper Primer" Improvements.

Analysis of the failure of the initial INSULT procedure to make mutants in longer gene/plasmid systems led to the introduction of the 'double shot' INSULT procedure, in which another aliquot of thermostable polymerase and thermostable ligase was added along with the generic primer prior. Double shot was clearly effective at producing single, double, and triple mutants as shown in Fig. 2; the primers in each case were converted into mutant duplexes as the primary product.

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.

Many possible alternatives exist that allow the production of mutants without additional enzyme. Reducing the extension time was effective for pCRT7, but longer gene/plasmid systems place limitations on this strategy. Reduction of the number of stage I cycles would permit the generation of mutants in very large gene/plasmid systems, but the tradeoff would be a relatively low yield of mutant DNA. This would decrease the ratio of mutant to parental DNA. An additional possibility is the use of generic 'helper primers' to start DNA synthesis at several locations on the plasmid, allowing the extension time to be short even for large plasmids.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

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.

Claims

CLAIMSWhat is claimed is:

1. A method for polynucleotide mutagenesis comprising the steps of:

(a) cloning a parental polynucleotide into a vector comprising a cloning site, thereby obtaining a cloned product;

(b) denaturing the cloned product, thereby obtaining a single-stranded polynucleotide template and a single-stranded parental complement; (c) hybridizing at least one mutagenized oligonucleotide primer to the single- stranded polynucleotide template, thereby obtaining a first heteroduplex; (d) extending the first heteroduplex with a polymerase, thereby obtaining an extended product; (e) reacting the extended product with a ligase, thereby obtaining ligated product;

(f) denaturing the ligated product, thereby obtaining a closed single stranded mutated polynucleotide;

(g) hybridizing the single stranded mutated polynucleotides with a genericoligonucleotide primer thereby obtaining second hybridized complexes; (h) adding additional polymerase and ligase to the reaction mixture in step(g); and

(i) extending the second hybridized complex and ligating the double stranded product thereof, thereby obtaining a circular double stranded mutated polynucleotide.

2. The method of Claim 1, wherein steps (c)-(f) are repeated at least about four times.

3. The method of Claim 2, wherein the polymerase is a thermophilic polymerase.

4. The method of Claim 1, wherein the ligase is Taq DNA ligase.

5. The method of Claim 1, wherein steps (c)-(f) are repeated at least about 10 times.

6. The method of Claim 1, further comprising the step of transforming the double-stranded mutated polynucleotide of step (i) into a bacterial host, thereby obtaining transformants.

7. The method of Claim 1, wherein step (i) is subjected to a single cycle of DNA synthesis.

8. The method of Claim 1, wherein step (i) is subjected to a less than about 10 cycles of DNA synthesis.

9. The method of Claim 1 characterized by the absence of an oligonucleotide primer that repairs or inactivates a selection sequence.

10. The method of Claim 1, further comprising the step of destroying the parental strand prior to amplification of the first heteroduplexes.

11. The method of Claim 1 , wherein the mutagenized oligonucleotide primer inserts one or more nucleotides into the parental polynucleotide.

12. The method of Claim 1, wherein the mutagenized oligonucleotide primer inserts three or more nucleotides into the parental polynucleotide.

13. The method of Claim 1, wherein the mutagenized oligonucleotide primer inserts two or more codons into the parental polynucleotide.

14. The method of Claim 1 , wherein the mutagenized oligonucleotide primer deletes one or more nucleotides in the parental polynucleotide.

15. The method of Claim 1 , wherein the mutagenized oligonucleotide primer deletes three or more nucleotides in the parental polynucleotide.

16. The method of Claim 1, wherein the mutagenized oligonucleotide primer deletes two or more codons in the parental polynucleotide.

17. The method of Claim 1 , wherein the mutagenized oligonucleotide primer substitutes one or more nucleotides into the parental polynucleotide.

18. The method of Claim 1 , wherein the mutagenized oligonucleotide primer substitutes three or more nucleotides in the parental polynucleotide.

19. The method of Claim 1, wherein the mutagenized oligonucleotide primer substitutes two or more codons into the parental polynucleotide.

20. The method of Claim 1, wherein the mutagenized oligonucleotide primer substitutes five or more codons into the parental polynucleotide.

21. The method of Claim 1 , wherein two or more distinct mutagenized oligonucleotide primers are added in step (c).

22. The method of Claim 1, wherein five or more distinct mutagenized oligonucleotide primers are added in step (c).

23. The method of Claim 22, wherein each distinct mutagenized oligonucleotide primer substitutes two or more codons into the parental polynucleotide.

24. The method of Claim 22, wherein each mutagenized oligonucleotide primer substitutes five or more codons into the parental polynucleotide.

25. The method of Claim 1, wherein the parental polynucleotide comprises a coding sequence.

26. The method of Claim 1, wherein the generic oligonucleotide primer is not the complement of the mutagenized oligonucleotide primer or overlapped with it.

27. The method of Claim 26, wherein the generic oligonucleotide primer does not hybridize to the parental polynucleotide.

28. The method of Claim 26, wherein the generic oligonucleotide primer hybridizes to a sequence of the vector.

29. The method of Claim 1 , wherein the vector further comprises a replication origin of a filamentous bacteriophage.

30. The method of Claim 29, wherein the replication origin is an fl replication origin.

31. The method of Claim 1, wherein in step (c), 5 or more mutagenized oligonucleotide primers are added to the single-stranded polynucleotide template.

32. The method of Claim 31 , wherein the 5 or more mutagenized oligonucleotides hybridize to substantially the same sequences on the single- stranded polynucleotide template.

33. The method of Claim 31 , wherein the 5 or more mutagenized oligonucleotides hybridize to different sequences on the single-stranded polynucleotide template.

34. The method of Claim 33, wherein the 5 or more mutagenized oligonucleotides hybridize to non-overlapping sequences on the single- stranded polynucleotide template.

35. The method of Claim 1, wherein in step (c), 10 or more mutagenized oligonucleotide primers are added to the single-stranded polynucleotide template.

36. The method of Claim 1, wherein in step (c), 20 or more mutagenized oligonucleotide primers are added to the single-stranded polynucleotide template.

37. The method of Claim 1 , wherein the mutagenized oligonucleotide primer further comprises a unique sequence which hybridizes to the second oligonucleotide primer.

38. The method of Claim 37, wherein the unique sequence is at least about 4 nucleotides.

39. The method of Claim 38 further comprising the step of adding a blocking oligonucleotide that hybridizes to the parental polynucleotide at or proximal to the sequences the mutagenized oligonucleotide primer hybridizes, thereby providing a negative selection for the parental polynucleotide.

40. A kit for use in the method of Claim 1 comprising:

(a) a vector comprising a cloning site;

(b) a generic oligonucleotide primer;

(c) a polymerase;

(d) a ligase; and (e) instructions for carrying out the method.

41. A method of using a kit comprising:

(a) a vector comprising a cloning site;

(b) a generic oligonucleotide primer;

(c) a polymerase; (d) a ligase; and

(e) instructions for carrying out the method, in a method comprising the steps of:

(a) cloning a parental polynucleotide into a vector comprising a cloning site, thereby obtaining a cloned product; (b) denaturing the cloned product, thereby obtaining a single-stranded polynucleotide template and a single-stranded parental DNA complement;

(c) hybridizing at least one mutagenized oligonucleotide primer to the single- stranded polynucleotide template, thereby obtaining a first heteroduplex;

(d) extending the first heteroduplex with a polymerase, thereby obtaining an extended product;

(e) reacting the extended product with a ligase, thereby obtaining ligated product; (f) denaturing the ligated product, thereby obtaining a closed single stranded mutated polynucleotide;

(g) hybridizing the single stranded mutated polynucleotides with a generic primer thereby obtaining second hybridized complexes; (h) adding additional polymerase and ligase to the reaction mixture in step(g); and

(i) copying the second hybridized complex and ligating the double stranded product thereof, thereby obtaining a circular double stranded mutated polynucleotide.

42. The method of Claim 6, wherein parental DNA is bound to methylated DNA binding protein domains prior to the transformation step.

43. The method of Claim 42, wherein the methylated DNA binding protein domains are immobilized on a reusable support.

44. The method of Claim 1 further comprising the step of removing single- stranded parental complement contained in the reaction mixture of step (e) prior to step (f).

45. A method of site directed polynucleotide mutagenesis comprising the steps of: (a) cloning a parental polynucleotide into a vector comprising a cloning site, thereby obtaining a cloned product;

(b) denaturing the cloned product, thereby obtaining a single-stranded polynucleotide template and a single-stranded parental complement;

(c) hybridizing at least one mutagenized oligonucleotide primer and at least one helper primer to the single-stranded polynucleotide template;

(d) extending the mutagenized oligonucleotide primer and the helper primer with a polymerase, thereby obtaining an extended product;

(e) reacting the extended product with a ligase, thereby obtaining a ligated product;

(f) denaturing the ligated product, thereby obtaining a closed single stranded mutated polynucleotide;

(g) hybridizing the single stranded mutated polynucleotides with a generic oligonucleotide primer; and (h) extending the generic oligonucleotide primer and the second helper primer and ligating the double stranded product thereof, thereby obtaining a circular double stranded mutated polynucleotide.

46. The method of Claim 45 further comprising the step of suppressing single- strand parental DNA by binding the parental DNA to a support comprising methylated DNA binding protein domains.

47. The method of Claim 46, wherein the methylated DNA binding protein domains are immobilized on a reusable support.

48. The method of Claim 45 further comprising the step of removing single- stranded parental complement remaining in the reaction mixture of step (e) prior to step (f).

49. The method of Claim 45, wherein steps (c)-(f) are repeated at least about four times.

50. The method of Claim 45, wherein steps (c)-(f) are repeated at least about 10 times.

51. The method of Claim 45 further comprising the step of transforming the double-stranded mutated polynucleotide of step (h) into a bacterial host, thereby obtaining transformants.

52. The method of Claim 45, wherein step (h) is subjected to a single cycle of DNA synthesis.

53. The method of Claim 45, wherein step (h) is subjected to a less than about 10 cycles of DNA synthesis.

54. The method of Claim 45 characterized by the absence of an oligonucleotide primer that repairs or inactivates a selection sequence.

55. The method of Claim 45 further comprising the step of destroying or removing the parental strand prior to amplification of the first heteroduplexes.

56. In a method of site-directed polynucleotide mutagenesis, of the type wherein the first stage produces multiple, single-stranded copies of circular mutation bearing plasmid DNA and the second stage generates double stranded circular DNA bearing the desired mutation, the improvement comprising: adding an additional aliquot of polymerase and ligase prior to the second stage.

57. In a method of site-directed polynucleotide mutagenesis, of the type wherein the first stage produces multiple, single-stranded copies of circular mutation bearing plasmid DNA and the second stage generates double stranded circular DNA bearing the desired mutation, the improvement comprising: adding helper primers to the first stage.

58. The method of Claim 57 further comprising: adding helper primers to the second stage.