Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube me
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《核酸研究医学期刊》
1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia and 2 Nucleics Pty Ltd, Suite 145, National Innovation Centre, Australian Technology Park, Eveleigh, Sydney, NSW 1430, Australia
* To whom correspondence should be addressed. Tel: +61 2 9209 4034; Fax: +61 2 9209 4084; Email: daniel@nucleics.com
ABSTRACT
Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.
INTRODUCTION
Numerous techniques have been developed to allow the introduction of site-directed mutations into cloned genes. These fall into two major categories: those based on primer extension on a plasmid template (such as the original Kunkel method), and a number of PCR-based methods .
Owing to their relative technical simplicity, the PCR-based site-directed mutagenesis approaches have proven highly popular (3–9). These techniques involve either DNA ligase-based circularization of inverse PCR products (with the desired mutations incorporated into the amplification primers) (4,7,9), or the use of multiple PCR products such as splicing overlap extension (10) and megaprimer approaches (3,5,6,8,11).
The inherent limitation of all current site-directed mutagenesis approaches is the background of non-mutated and/or incorrectly mutated clones. Typically, more than 50% of the clones will not contain the correct mutation making it necessary to sequence multiple clones each time a site-directed mutant is constructed (12–14).
For the PCR-based techniques, the primary causes of this background are (i) failure to completely remove the original, transformable, template, and (ii) the generation of incorrect, transformation-competent, fragments derived from PCR mis-priming events. While the original template can be removed by gel purification (4,15,16) or DpnI digestion (9,14), the cloning of mis-primed DNA fragments is problematic. These fragments, which contain the origin of replication and selection regions (e.g. the ori and antibiotic resistance cassettes), are generally smaller than the desired PCR fragment and hence clone preferentially to the desired full-length product. Such fragments result in clones containing unwanted deletions within the gene of interest.
A rapid universal mutagenesis approach for sequence insertion, deletion and substitution with simple steps and high mutagenic efficiency would be very useful. In this paper, we describe a simple, highly efficient, robust PCR-based, ligase-independent mutagenesis approach to insert, delete and substitute nucleotide sequences at any position of a plasmid in a single reaction tube. This method avoids problems arising from template carryover and PCR mis-priming events. Furthermore, the experimental design can be adapted to automated high-throughput applications allowing mass generation and screening of mutants.
MATERIALS AND METHODS
Site-directed, ligase-independent mutagenesis (SLIM) primer design
Primers were designed to amplify the entire pBAD vector carrying the Escherichia coli bacteriophage T7 DNA polymerase gene with six extra histidine codons at the 5' end of the gene 5 coding sequence (pT7Pol-thio; J. Chiu, D. Tillett and P. E. March, manuscript in preparation) (Table 1). This plasmid is 6.6 kb in length. Modifications to the gene sequence were introduced into the 5'-adapter tail (18 bases) of primers used in PCR amplification. Three classes of modifications were investigated: (i) insertion of histidine codons into pT7pol-thio, (ii) deletion of existing N-terminal six histidine codons from pT7pol-thio, and (iii) mutagenesis of codon Tyr326 to Cys.
Table 1. Oligonucleotides used in SLIM for gene engineering
SLIM PCR amplification
A single PCR was performed for each modification and contained the following components: 2.5 μl of 10x Pfx buffer, 200 μM each dNTP, 1 mM MgSO4, 100 mM betaine, 10 pmol of each primer (all four primers), 100 pg of the 6.6 kb plasmid template pT7Pol-thio, 0.5 U Taq DNA polymerase (New England Biolabs, Beverley, MA), 0.25 U Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA) and molecular biology grade water to a final volume of 25 μl. The reactions were hot-started by heating to 98°C for 2 min, then cooling to 85°C at which point the DNA polymerase was added. The PCRs were subjected to a further 25 cycles of 95°C for 15 s, 50°C (61°C for point mutation) for 20 s and 68°C for 3.5 min, with a final 7 min extension step at 68°C.
SLIM hybridization
The conditions for SLIM heteroduplex formation were adapted from Tillett and Neilan (17). The PCR mixture was diluted in 5 μl of D-Buffer (20 mM MgCl2, 20 mM Tris, pH 8.0 and 5 mM DTT) that contained 5 U DpnI. This mixture was incubated for 60 min at 37°C. The DpnI digestion was stopped by the addition of 30 μl of H-Buffer (300 mM NaCl, 50 mM Tris, pH 9.0, 20 mM EDTA, pH 8.0) and denaturation at 99°C for 3 min. Hybridization was performed using two cycles of 65°C for 5 min and 30°C for 15 min. An aliquot of 20 μl from each reaction was used to transform CaCl2 competent E.coli JM109 (18).
Screening of SLIM clones
Clones were screened by colony PCR for the desired mutations using SLIM detection primers (Table 2). Colonies were picked and lysed in 35 μl of lysis buffer (0.1% Triton X-100, 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, pH 8.0) at 100°C for 1 min. An aliquot of 2 μl of colony lysate was used as template in a PCR containing: 2.5 μl 10x PCR buffer (500 mM KCl, 100 mM Tris–HCl, pH 9.0, 0.1% Triton X-100), 200 μM each dNTP, 2 mM MgCl2, 100 mM betaine, 10 pmol of each forward and reverse primer, 1 U of Taq DNA polymerase and molecular biology grade water to a final volume of 25 μl. The reactions were hot-started by heating to 85°C at which point the Taq DNA polymerase was added. The PCRs were heated to 95°C for 2 min then subjected to a further 25 cycles of 95°C for 15 s, 54°C for 20 s and 72°C for 2.5 min, with a final 7 min extension step at 72°C. Positive clones were identified by electrophoresis on 1% (w/v) agarose gel (18). Positive clones were further screened for the correct mutation by DNA sequencing of the plasmid DNA template prepared using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany).
Table 2. Detection oligonucleotides used in PCR screening
RESULTS
Design of SLIM primers
The basic principle of SLIM is illustrated in Figure 1. In a manner similar to inverse PCR mutagenesis (4,7), the desired mutations were introduced by PCR amplification of the entire vector with the target modifications incorporated into the oligonucleotide primers. However, to generate clonable circular DNA, SLIM utilizes a ligation independent approach based on the formation of complementary 5' and 3' single-stranded overhangs following heteroduplex formation. The desired heteroduplexes are generated by performing a single PCR reaction using four primers (two tailed and two non-tailed). In the case of sequence insertion (Figure 1A), the short primers FS and RS hybridize on each strand of the template to gene-specific sequence immediately 5' and 3' to the site of insertion (Figure 1A, vertical dotted line). Similarly, the longer tailed primers FT and RT have the same 3' sequence as FS and RS (indicated by gray arrow in FT and FS, and black arrow for RT and RS) but contain the region to be inserted on their 5' ends. Four different PCR products are produced using these primers (Figure 1A, Products 1–4). Products 1 and 2 are identical, except for the position of the inserted sequence which is found on opposite termini (i.e. formed from either FT and RS, or FS and RT). Product 3 contains the inserted sequence on both termini (formed from FT and RT), while Product 4 does not contain any sequence insertion (formed from FS and RS). Denaturation and re-annealing of these four PCR products generates 16 different double-stranded heteroduplex DNA species. Two of the double-stranded heteroduplex hybrids (generated from a DNA strand from Product 1 and the corresponding strand from Product 2) create complementary 5' and 3' overhangs at opposite termini that can form stable, non-covalently joined, DNA circles. Upon transformation into E.coli, only the circular DNA molecules (containing the desired mutation) give rise to colonies on selective media.
Figure 1. Representation of SLIM. The gene-specific portion of each primer is denoted by arrows. Forward and reverse primers are shown in gray and black, respectively. The hash line represents the 5'-adapter of the tailed primer. (A) Sequence insertion. Template and four primers (FT, FS, RT, RS) were mixed and the entire plasmid template was amplified. Four PCR products were created (Products 1–4). The 5'-adapter sequences were incorporated at the opposite terminal ends of the Products 1 and 2. These are productive as they contribute to a hybrid that can form a non-covalent closed circle. Products 3 and 4 are not productive in this regard. The PCR fragments were then subjected to denaturation and re-annealing. Productive hybrids form when a strand from Product 1 forms a hybrid with a strand from Product 2 and creates a double-stranded fragment with complementary overhangs that can circularize. Fourteen non-clonable hybrids also form (data not shown). (B) Sequence deletion. As for sequence insertion, inverse PCR was performed on the circular plasmid template. To simplify the figure, only the initial primer binding sites and the final deleted product are shown. The region to be deleted is denoted by a gray rectangle. The 5' portion of the tailed forward primer (FT) contains sequence complementary to DNA sequence (hash-lined square) adjacent to the region to be deleted. The reverse tailed primer (RT) contains a 5' tail complementary to the same region adjacent to the deletion. The short gene-specific primers are located immediately 3' to the deletion (FS) and 5' to the adjacent region (RS). Four PCR products were created in the amplification step, two of which were productive products. Following denaturing and re-annealing, 16 possible hybrids form, two of which were productive hybrids. The final product with the gray rectangle deleted is obtained after transformation. (C) Sequence exchange (mutagenesis). Inverse PCR was performed on circular plasmid template. To simplify the figure, only the initial primer binding sites are shown. The region undergoing mutagenesis is represented by a hash-lined rectangle with the positions of mutation indicated by an asterisk. The 5' portion of each tailed primer (FT and RT) carries the mutation(s) to be made on the target sequence. The resulting linear PCR products carry the designed mutations on the adapters. PCR, hybridization and transformation were performed as described for sequence insertion.
Sequence deletion from a plasmid is accomplished by excluding the desired deletion region from the PCR amplification. In order to delete a DNA sequence from a cloned gene, three regions need to be considered in the design of PCR primers. The first is the complementary region recognized by the gene-specific portion of the primers. In Figure 1B, this region is shown by the gray arrow for FT and FS and black arrow for RT and RS. The second region to be considered is the region of DNA sequence that will make up the overhang (the hashed square in Figure 1B). This region must be adjacent to the deleted sequence and in the example shown in Figure 1B, is immediately 5' to the region deleted. The third region to be considered is the region to be deleted (gray rectangle in Figure 1B). The position of the deleted DNA sequence defines the placement of the other regions. The 5'-adapter of primers FT and RT both contain DNA sequence complementary to the hashed sequence (Figure 1B). Using this method, precisely defined deletions of any size can be created by the selection of appropriate amplification primers.
Similarly, substitution mutagenesis can be performed by the design of primers containing the desired changes in the 5' region of FT and RT (Figure 1C). Using this strategy, it is possible to replace one or more nucleotides simultaneously. We have used this approach to create a single point mutation (mutation of the T7 DNA Polymerase Tyr326 to Cys); however, the entire region of the adapter (Figure 1C, hashed rectangle) can be replaced by any sequence. In cases where one or two nucleotides are altered, shorter primers of 18–20 bases composed of just the 5'-adapter sequence could be used in conjunction with the gene-specific primers in the mutagenesis to generate the desired overhangs.
Mutagenic efficiency of SLIM
The efficiency of SLIM was assessed by constructing a series of insertion, deletion and substitution mutations of a 6.6 kb expression clone of T7 DNA polymerase (J. Chiu, D. Tillett and P. E. March, manuscript in preparation). The elements present on this plasmid are presented in Materials and Methods. The sequence deletion, insertions and alteration were at different positions of the gene 5 open reading frame as indicated in Tables 1 and 3. Upon transformation of the SLIM generated heteroduplex fragments, transformants were screened for the desired modification by PCR using specific detection primers. In total, 72 colonies were screened with 67 clones identified as putative positives. Since the PCR screening was performed directly from colonies growing on agar, it was possible that some of the negatives were actually positives that failed to amplify due to variability inherent to colony PCR screening. Therefore, all negatives were subjected to secondary screening by DNA sequencing. In addition, 37 of the PCR positive clones were subjected to DNA sequence analysis. All 37 of the PCR-positive candidates contained the correct mutation. Two of the negatives also contained the correct mutation, therefore 39 of these 42 clones were identified as containing the correct mutation, one was incorrectly modified and two were unmodified (Table 3). These data showed that 100% of clones that were positive by PCR screening contained the correct modification and, by extrapolation, the overall mutagenic efficiency of SLIM was 95.8% (69 out of 72 clones, Table 3). The minimum efficiency would be that actually confirmed by DNA sequencing (93%, 39 out of 42 clones sequenced). This high efficiency was observed even from poor PCR amplifications that contained extensive mis-primed fragments (seen as DNA smears) (Figure 2), demonstrating the extreme robustness of the SLIM technique. The transformation efficiency of the competent cells was 106 CFU/μg DNA determined using 0.1 ng pGEM3Z(f+), indicating that the method does not require highly competent cells.
Table 3. Efficiency of SLIM mutagenesis
Figure 2. SLIM pT7pol-thio PCR products. Lane 1, products from PCR amplification used to create an insertion of six histidine codons; lane 2, products from PCR amplification used to delete the N-terminal six histidine codons. Arrowhead indicates the position of migration of the 6.6 kb linear plasmid band in 1% agarose gel electrophoresis. Photograph of an ethidium bromide stained gel is shown.
DISCUSSION
Current PCR-mediated site-directed mutagenesis approaches, utilizing inverse PCR or megaprimer methodologies, can introduce mutations of all types (insertion, deletion and sequence alteration) into any amplifiable site of double-stranded DNA templates (4,7,19,20). However, all existing approaches require circularization of the PCR product by a DNA ligase. Apart from introducing an additional point of experimental failure, ligation does not allow discrimination against the cloning of PCR mis-priming events. This can lead to a significant background of incorrectly mutated clones requiring that multiple plasmids be screened for each mutagenesis experiment.
SLIM was designed to address the disadvantages of both PCR- and non-PCR-based mutagenesis approaches. By combining the technical simplicity of the PCR-based approaches, with a mechanism to avoid the cloning of PCR mis-primed events, the SLIM technique provides mutagenic efficiency of >95% in 4 h. The success of the technique depends on the generation of clonable heteroduplex DNA fragment created via mixed PCR amplification using four primers. The strict requirement for heteroduplex formation to generate complementary single-stranded overhangs ensures that only the correct full-length PCR fragments are clonable. For any gene modification by SLIM, the specificity and yield of the PCR affects the total number of transformants obtained. Heteroduplexes formed from PCR products derived from a single prominent band yield a high number of transformants. Heteroduplexes formed from poor quality PCR products (smears) yield a lower number of transformants. However, even with smeared PCR products the mutagenic efficiency remains >95%.
When inserting a sequence tag containing tandem codon repeats (e.g. histidine codons), it is recommended that a non-repetitive codon order is used in the adapter region to prevent misalignment of complementary overhangs during heteroduplex circularization. However, if a series of plasmid constructs varying in the number of tandem codon repeats is desirable, the use of repeated codons may be useful. This approach was employed to generate a number of clones varying in the number of histidine codons by using repeats of the histidine CAT codon in primer adapters (J. Chiu, D. Tillett and P. E. March, manuscript in preparation).
In the development of SLIM, DpnI digestion was identified as the most critical step for high mutagenic efficiency. Initially, when 1 U of DpnI was used, 12% of the examined transformants were found to be the unmodified template DNA. This led to re-examination of the template removal step, and when DpnI was increased to 5 U per reaction, the template clones were reduced to <3%.
The SLIM approach is highly flexible and can accommodate all types of modification and mutagenesis in any clonable template that can be PCR amplified. Adapters of length >24 bases can be designed to accommodate a variety of simultaneous modifications. Oligonucleotides of up to 250 bases are commercially available (Gene Link), alternatively, the required SLIM primers can be generated using sets of tailed primers in a manner analogous to Megaprimer PCR (11). SLIM can also be utilized to introduce modified base residues into a specific site of a gene by incorporating the modified bases into the tailing primers. SLIM requires minimal labor and can be readily adapted to robotic manipulation. It is therefore suitable for the construction of large-scale, high-throughput, mutagenesis investigations.
In comparison with other site-directed mutagenesis protocols, SLIM is rapid with all steps (amplification, DpnI digestion and hybridization) carried out in a single reaction tube in 4 h. Transformation of hybridized products does not require highly competent cells for recovery of the correctly mutated clones. In addition, as oligonucleotide synthesis has become very affordable, the scope of SLIM is greater than demonstrated here. One potential example is the use of SLIM in alanine-scanning mutagenesis to analyze the function(s) of particular amino residues on the surface of a protein. This involves the systematic replacement of charged amino acids with alanine (21,22). Using SLIM, a large number of variants could be created rapidly in a single experiment.
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* To whom correspondence should be addressed. Tel: +61 2 9209 4034; Fax: +61 2 9209 4084; Email: daniel@nucleics.com
ABSTRACT
Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.
INTRODUCTION
Numerous techniques have been developed to allow the introduction of site-directed mutations into cloned genes. These fall into two major categories: those based on primer extension on a plasmid template (such as the original Kunkel method), and a number of PCR-based methods .
Owing to their relative technical simplicity, the PCR-based site-directed mutagenesis approaches have proven highly popular (3–9). These techniques involve either DNA ligase-based circularization of inverse PCR products (with the desired mutations incorporated into the amplification primers) (4,7,9), or the use of multiple PCR products such as splicing overlap extension (10) and megaprimer approaches (3,5,6,8,11).
The inherent limitation of all current site-directed mutagenesis approaches is the background of non-mutated and/or incorrectly mutated clones. Typically, more than 50% of the clones will not contain the correct mutation making it necessary to sequence multiple clones each time a site-directed mutant is constructed (12–14).
For the PCR-based techniques, the primary causes of this background are (i) failure to completely remove the original, transformable, template, and (ii) the generation of incorrect, transformation-competent, fragments derived from PCR mis-priming events. While the original template can be removed by gel purification (4,15,16) or DpnI digestion (9,14), the cloning of mis-primed DNA fragments is problematic. These fragments, which contain the origin of replication and selection regions (e.g. the ori and antibiotic resistance cassettes), are generally smaller than the desired PCR fragment and hence clone preferentially to the desired full-length product. Such fragments result in clones containing unwanted deletions within the gene of interest.
A rapid universal mutagenesis approach for sequence insertion, deletion and substitution with simple steps and high mutagenic efficiency would be very useful. In this paper, we describe a simple, highly efficient, robust PCR-based, ligase-independent mutagenesis approach to insert, delete and substitute nucleotide sequences at any position of a plasmid in a single reaction tube. This method avoids problems arising from template carryover and PCR mis-priming events. Furthermore, the experimental design can be adapted to automated high-throughput applications allowing mass generation and screening of mutants.
MATERIALS AND METHODS
Site-directed, ligase-independent mutagenesis (SLIM) primer design
Primers were designed to amplify the entire pBAD vector carrying the Escherichia coli bacteriophage T7 DNA polymerase gene with six extra histidine codons at the 5' end of the gene 5 coding sequence (pT7Pol-thio; J. Chiu, D. Tillett and P. E. March, manuscript in preparation) (Table 1). This plasmid is 6.6 kb in length. Modifications to the gene sequence were introduced into the 5'-adapter tail (18 bases) of primers used in PCR amplification. Three classes of modifications were investigated: (i) insertion of histidine codons into pT7pol-thio, (ii) deletion of existing N-terminal six histidine codons from pT7pol-thio, and (iii) mutagenesis of codon Tyr326 to Cys.
Table 1. Oligonucleotides used in SLIM for gene engineering
SLIM PCR amplification
A single PCR was performed for each modification and contained the following components: 2.5 μl of 10x Pfx buffer, 200 μM each dNTP, 1 mM MgSO4, 100 mM betaine, 10 pmol of each primer (all four primers), 100 pg of the 6.6 kb plasmid template pT7Pol-thio, 0.5 U Taq DNA polymerase (New England Biolabs, Beverley, MA), 0.25 U Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA) and molecular biology grade water to a final volume of 25 μl. The reactions were hot-started by heating to 98°C for 2 min, then cooling to 85°C at which point the DNA polymerase was added. The PCRs were subjected to a further 25 cycles of 95°C for 15 s, 50°C (61°C for point mutation) for 20 s and 68°C for 3.5 min, with a final 7 min extension step at 68°C.
SLIM hybridization
The conditions for SLIM heteroduplex formation were adapted from Tillett and Neilan (17). The PCR mixture was diluted in 5 μl of D-Buffer (20 mM MgCl2, 20 mM Tris, pH 8.0 and 5 mM DTT) that contained 5 U DpnI. This mixture was incubated for 60 min at 37°C. The DpnI digestion was stopped by the addition of 30 μl of H-Buffer (300 mM NaCl, 50 mM Tris, pH 9.0, 20 mM EDTA, pH 8.0) and denaturation at 99°C for 3 min. Hybridization was performed using two cycles of 65°C for 5 min and 30°C for 15 min. An aliquot of 20 μl from each reaction was used to transform CaCl2 competent E.coli JM109 (18).
Screening of SLIM clones
Clones were screened by colony PCR for the desired mutations using SLIM detection primers (Table 2). Colonies were picked and lysed in 35 μl of lysis buffer (0.1% Triton X-100, 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, pH 8.0) at 100°C for 1 min. An aliquot of 2 μl of colony lysate was used as template in a PCR containing: 2.5 μl 10x PCR buffer (500 mM KCl, 100 mM Tris–HCl, pH 9.0, 0.1% Triton X-100), 200 μM each dNTP, 2 mM MgCl2, 100 mM betaine, 10 pmol of each forward and reverse primer, 1 U of Taq DNA polymerase and molecular biology grade water to a final volume of 25 μl. The reactions were hot-started by heating to 85°C at which point the Taq DNA polymerase was added. The PCRs were heated to 95°C for 2 min then subjected to a further 25 cycles of 95°C for 15 s, 54°C for 20 s and 72°C for 2.5 min, with a final 7 min extension step at 72°C. Positive clones were identified by electrophoresis on 1% (w/v) agarose gel (18). Positive clones were further screened for the correct mutation by DNA sequencing of the plasmid DNA template prepared using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany).
Table 2. Detection oligonucleotides used in PCR screening
RESULTS
Design of SLIM primers
The basic principle of SLIM is illustrated in Figure 1. In a manner similar to inverse PCR mutagenesis (4,7), the desired mutations were introduced by PCR amplification of the entire vector with the target modifications incorporated into the oligonucleotide primers. However, to generate clonable circular DNA, SLIM utilizes a ligation independent approach based on the formation of complementary 5' and 3' single-stranded overhangs following heteroduplex formation. The desired heteroduplexes are generated by performing a single PCR reaction using four primers (two tailed and two non-tailed). In the case of sequence insertion (Figure 1A), the short primers FS and RS hybridize on each strand of the template to gene-specific sequence immediately 5' and 3' to the site of insertion (Figure 1A, vertical dotted line). Similarly, the longer tailed primers FT and RT have the same 3' sequence as FS and RS (indicated by gray arrow in FT and FS, and black arrow for RT and RS) but contain the region to be inserted on their 5' ends. Four different PCR products are produced using these primers (Figure 1A, Products 1–4). Products 1 and 2 are identical, except for the position of the inserted sequence which is found on opposite termini (i.e. formed from either FT and RS, or FS and RT). Product 3 contains the inserted sequence on both termini (formed from FT and RT), while Product 4 does not contain any sequence insertion (formed from FS and RS). Denaturation and re-annealing of these four PCR products generates 16 different double-stranded heteroduplex DNA species. Two of the double-stranded heteroduplex hybrids (generated from a DNA strand from Product 1 and the corresponding strand from Product 2) create complementary 5' and 3' overhangs at opposite termini that can form stable, non-covalently joined, DNA circles. Upon transformation into E.coli, only the circular DNA molecules (containing the desired mutation) give rise to colonies on selective media.
Figure 1. Representation of SLIM. The gene-specific portion of each primer is denoted by arrows. Forward and reverse primers are shown in gray and black, respectively. The hash line represents the 5'-adapter of the tailed primer. (A) Sequence insertion. Template and four primers (FT, FS, RT, RS) were mixed and the entire plasmid template was amplified. Four PCR products were created (Products 1–4). The 5'-adapter sequences were incorporated at the opposite terminal ends of the Products 1 and 2. These are productive as they contribute to a hybrid that can form a non-covalent closed circle. Products 3 and 4 are not productive in this regard. The PCR fragments were then subjected to denaturation and re-annealing. Productive hybrids form when a strand from Product 1 forms a hybrid with a strand from Product 2 and creates a double-stranded fragment with complementary overhangs that can circularize. Fourteen non-clonable hybrids also form (data not shown). (B) Sequence deletion. As for sequence insertion, inverse PCR was performed on the circular plasmid template. To simplify the figure, only the initial primer binding sites and the final deleted product are shown. The region to be deleted is denoted by a gray rectangle. The 5' portion of the tailed forward primer (FT) contains sequence complementary to DNA sequence (hash-lined square) adjacent to the region to be deleted. The reverse tailed primer (RT) contains a 5' tail complementary to the same region adjacent to the deletion. The short gene-specific primers are located immediately 3' to the deletion (FS) and 5' to the adjacent region (RS). Four PCR products were created in the amplification step, two of which were productive products. Following denaturing and re-annealing, 16 possible hybrids form, two of which were productive hybrids. The final product with the gray rectangle deleted is obtained after transformation. (C) Sequence exchange (mutagenesis). Inverse PCR was performed on circular plasmid template. To simplify the figure, only the initial primer binding sites are shown. The region undergoing mutagenesis is represented by a hash-lined rectangle with the positions of mutation indicated by an asterisk. The 5' portion of each tailed primer (FT and RT) carries the mutation(s) to be made on the target sequence. The resulting linear PCR products carry the designed mutations on the adapters. PCR, hybridization and transformation were performed as described for sequence insertion.
Sequence deletion from a plasmid is accomplished by excluding the desired deletion region from the PCR amplification. In order to delete a DNA sequence from a cloned gene, three regions need to be considered in the design of PCR primers. The first is the complementary region recognized by the gene-specific portion of the primers. In Figure 1B, this region is shown by the gray arrow for FT and FS and black arrow for RT and RS. The second region to be considered is the region of DNA sequence that will make up the overhang (the hashed square in Figure 1B). This region must be adjacent to the deleted sequence and in the example shown in Figure 1B, is immediately 5' to the region deleted. The third region to be considered is the region to be deleted (gray rectangle in Figure 1B). The position of the deleted DNA sequence defines the placement of the other regions. The 5'-adapter of primers FT and RT both contain DNA sequence complementary to the hashed sequence (Figure 1B). Using this method, precisely defined deletions of any size can be created by the selection of appropriate amplification primers.
Similarly, substitution mutagenesis can be performed by the design of primers containing the desired changes in the 5' region of FT and RT (Figure 1C). Using this strategy, it is possible to replace one or more nucleotides simultaneously. We have used this approach to create a single point mutation (mutation of the T7 DNA Polymerase Tyr326 to Cys); however, the entire region of the adapter (Figure 1C, hashed rectangle) can be replaced by any sequence. In cases where one or two nucleotides are altered, shorter primers of 18–20 bases composed of just the 5'-adapter sequence could be used in conjunction with the gene-specific primers in the mutagenesis to generate the desired overhangs.
Mutagenic efficiency of SLIM
The efficiency of SLIM was assessed by constructing a series of insertion, deletion and substitution mutations of a 6.6 kb expression clone of T7 DNA polymerase (J. Chiu, D. Tillett and P. E. March, manuscript in preparation). The elements present on this plasmid are presented in Materials and Methods. The sequence deletion, insertions and alteration were at different positions of the gene 5 open reading frame as indicated in Tables 1 and 3. Upon transformation of the SLIM generated heteroduplex fragments, transformants were screened for the desired modification by PCR using specific detection primers. In total, 72 colonies were screened with 67 clones identified as putative positives. Since the PCR screening was performed directly from colonies growing on agar, it was possible that some of the negatives were actually positives that failed to amplify due to variability inherent to colony PCR screening. Therefore, all negatives were subjected to secondary screening by DNA sequencing. In addition, 37 of the PCR positive clones were subjected to DNA sequence analysis. All 37 of the PCR-positive candidates contained the correct mutation. Two of the negatives also contained the correct mutation, therefore 39 of these 42 clones were identified as containing the correct mutation, one was incorrectly modified and two were unmodified (Table 3). These data showed that 100% of clones that were positive by PCR screening contained the correct modification and, by extrapolation, the overall mutagenic efficiency of SLIM was 95.8% (69 out of 72 clones, Table 3). The minimum efficiency would be that actually confirmed by DNA sequencing (93%, 39 out of 42 clones sequenced). This high efficiency was observed even from poor PCR amplifications that contained extensive mis-primed fragments (seen as DNA smears) (Figure 2), demonstrating the extreme robustness of the SLIM technique. The transformation efficiency of the competent cells was 106 CFU/μg DNA determined using 0.1 ng pGEM3Z(f+), indicating that the method does not require highly competent cells.
Table 3. Efficiency of SLIM mutagenesis
Figure 2. SLIM pT7pol-thio PCR products. Lane 1, products from PCR amplification used to create an insertion of six histidine codons; lane 2, products from PCR amplification used to delete the N-terminal six histidine codons. Arrowhead indicates the position of migration of the 6.6 kb linear plasmid band in 1% agarose gel electrophoresis. Photograph of an ethidium bromide stained gel is shown.
DISCUSSION
Current PCR-mediated site-directed mutagenesis approaches, utilizing inverse PCR or megaprimer methodologies, can introduce mutations of all types (insertion, deletion and sequence alteration) into any amplifiable site of double-stranded DNA templates (4,7,19,20). However, all existing approaches require circularization of the PCR product by a DNA ligase. Apart from introducing an additional point of experimental failure, ligation does not allow discrimination against the cloning of PCR mis-priming events. This can lead to a significant background of incorrectly mutated clones requiring that multiple plasmids be screened for each mutagenesis experiment.
SLIM was designed to address the disadvantages of both PCR- and non-PCR-based mutagenesis approaches. By combining the technical simplicity of the PCR-based approaches, with a mechanism to avoid the cloning of PCR mis-primed events, the SLIM technique provides mutagenic efficiency of >95% in 4 h. The success of the technique depends on the generation of clonable heteroduplex DNA fragment created via mixed PCR amplification using four primers. The strict requirement for heteroduplex formation to generate complementary single-stranded overhangs ensures that only the correct full-length PCR fragments are clonable. For any gene modification by SLIM, the specificity and yield of the PCR affects the total number of transformants obtained. Heteroduplexes formed from PCR products derived from a single prominent band yield a high number of transformants. Heteroduplexes formed from poor quality PCR products (smears) yield a lower number of transformants. However, even with smeared PCR products the mutagenic efficiency remains >95%.
When inserting a sequence tag containing tandem codon repeats (e.g. histidine codons), it is recommended that a non-repetitive codon order is used in the adapter region to prevent misalignment of complementary overhangs during heteroduplex circularization. However, if a series of plasmid constructs varying in the number of tandem codon repeats is desirable, the use of repeated codons may be useful. This approach was employed to generate a number of clones varying in the number of histidine codons by using repeats of the histidine CAT codon in primer adapters (J. Chiu, D. Tillett and P. E. March, manuscript in preparation).
In the development of SLIM, DpnI digestion was identified as the most critical step for high mutagenic efficiency. Initially, when 1 U of DpnI was used, 12% of the examined transformants were found to be the unmodified template DNA. This led to re-examination of the template removal step, and when DpnI was increased to 5 U per reaction, the template clones were reduced to <3%.
The SLIM approach is highly flexible and can accommodate all types of modification and mutagenesis in any clonable template that can be PCR amplified. Adapters of length >24 bases can be designed to accommodate a variety of simultaneous modifications. Oligonucleotides of up to 250 bases are commercially available (Gene Link), alternatively, the required SLIM primers can be generated using sets of tailed primers in a manner analogous to Megaprimer PCR (11). SLIM can also be utilized to introduce modified base residues into a specific site of a gene by incorporating the modified bases into the tailing primers. SLIM requires minimal labor and can be readily adapted to robotic manipulation. It is therefore suitable for the construction of large-scale, high-throughput, mutagenesis investigations.
In comparison with other site-directed mutagenesis protocols, SLIM is rapid with all steps (amplification, DpnI digestion and hybridization) carried out in a single reaction tube in 4 h. Transformation of hybridized products does not require highly competent cells for recovery of the correctly mutated clones. In addition, as oligonucleotide synthesis has become very affordable, the scope of SLIM is greater than demonstrated here. One potential example is the use of SLIM in alanine-scanning mutagenesis to analyze the function(s) of particular amino residues on the surface of a protein. This involves the systematic replacement of charged amino acids with alanine (21,22). Using SLIM, a large number of variants could be created rapidly in a single experiment.
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