Splicing together different regions of a gene by modified polymerase chain reaction-based site-directed mutagenesis

A modified polymerase chain reaction (PCR)-based site-directed mutagenesis method used to splice together different regions of a gene by deleting hundreds of nucleotides of undesired sequences is described. This method was inspired by a PCR-based site-directed mutagenesis method developed by Stratagene (La Jolla, CA, USA); the procedure and primer design were modified to enable the method to generate deletions several hundreds of nucleotides in length with an efficiency of 80-100%, and to delete two DNA fragments simultaneously in a single PCR. This method should be useful for deletion of large DNA fragments from a gene.     

An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes

Site-directed mutagenesis (SDM) methods are very important in modern molecular biology, biochemistry, and protein engineering. Here, we present a novel SDM method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction (PCR) method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly. We illustrate the usefulness of our method by introducing three mutations into the bacterial Streptococcus thermophilus Cas9 (bStCas9) gene, converting the humanized S. thermophilus Cas9 (hStCas9) gene into nuclease dead or H847A nickase mutants and generating sunnyTALEN mutagenesis from a wild-type TALEN backbone.     

A marker-coupled method for site-directed mutagenesis

A marker-coupled method for site-directed mutagenesis (SDM) has been developed. In this method, target DNA is first cloned into a plasmid vector which carries an inactivated tetracycline-resistance (TcR)-encoding tet gene. Using this cloned plasmid as template, polymerase chain reaction (PCR) is performed with a mutagenic primer and a marker primer. The mutagenic primer contains the desired mutations to be introduced into the target DNA, and the marker primer contains a mutation for restoring the activity of the inactivated tet gene. The PCR product is annealed with a gapped duplex plasmid template, extended and ligated in vitro. The resulting uni-strand-mutated plasmid is converted into the gapped duplex form, transformed into Escherichia coli JM109 and spread on yeast extract/tryptone culture medium + Tc plates. The TcR colonies grown on these plates all carry active tet genes. Due to the 'tight coupling' between the marker primer and the mutagenic primer formed in the PCR product, these TcR colonies should also carry the mutagenic primer, e.g., the desired mutations in the target DNA. In fact, practically all of the TcR colonies have been found to be the desired mutants in the present experiments. Therefore, this method provides a very efficient approach for SDM.     

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.     

A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction

A simple and fast method for introducing a series of mutations in cloned DNA has been developed. The polymerase chain reaction (PCR) has been used for site-directed mutagenesis. Because mutations can be introduced only within the primer sequences used for PCR, a suitable restriction site in the vicinity of the mutated nucleotide is required to permit recloning. Several methods have been devised to overcome this limitation. Our present method is a modification of the overlap extension method [Ho et al., Gene 77 (1989) 51-57], and has some advantages over this and other published methods. In our method, three common primers and a series of primers specific for various mutations are chemically synthesized. Once the proper oligodeoxyribonucleotides are selected as common primers, each mutation requires only one additional primer. Therefore, this method is very useful for introducing many mutations in various sites of the target DNA. We describe our protocol for the site-directed mutagenesis and an example of the introduction of several mutations in the hen egg-white lysozyme-encoding gene.     

Rapid and Efficient Gene Splicing Using Megaprimer-Based Protocol

Megaprimer-based methodology has been widely applied in site-directed mutagenesis, but rarely used in gene splicing. In this article, we describe a modification of the megaprimer PCR method, which can efficiently create and amplify a specific ligated chimeric gene segment in a PCR reaction and under a common PCR program that is widely used by researchers. More importantly, this modified method for splicing two or more gene fragments together revealed the mechanism of the megaprimer PCR method, by elucidating the key factor in the megaprimer-based protocol. In this method, the denatured megaprimer divided into two strands. One strand was used as template DNA to regenerate megaprimer and the other strand was used as an oligonucleotide primer to create a ligated chimeric gene product. In this article, we detail the modified megaprimer protocol for creating and amplifying these chimeric gene products, including a specific protocol for large chimeric gene products. We also provide additional tips to increase specificity and efficiency of the protocols. In conclusion, the improved megaprimer PCR protocol is a simple, broadly applicable protocol for splicing two different gene fragments together without relying on restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study

PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology. Type IIs restriction enzyme; enhanced green fluorescent protein; Bcl-2     

Efficient isolation of targeted Caenorhabditis elegans deletion strains using highly thermostable restriction endonucleases and PCR

Reverse genetic approaches to understanding gene function would be greatly facilitated by increasing the efficiency of methods for isolating mutants without the reliance on a predicted phenotype. Established PCR-based methods of isolating deletion mutants are widely used for this purpose in Caenorhabditis elegans. However, these methods are inefficient at isolating small deletions. We report here a novel modification of PCR-based methods, employing thermostable restriction enzymes to block the synthesis of wild-type PCR product, so that only the deletion PCR product is amplified. This modification greatly increases the efficiency of isolating small targeted deletions in C.elegans. Using this method six new deletion strains were isolated from a small screen of approximately 400000 haploid genomes, most with deletions <1.0 kb. Greater PCR detection sensitivity by this modification permitted ~10-fold greater pooling of DNA samples, reducing the effort and reagents required for screens. In addition, effective suppression of non-specific amplification allowed multiplexing with several independent primer pairs. The increased efficiency of this technique makes it more practical for small laboratories to undertake gene knock-out screens.     

A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method

In this study, we report a novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method modified from the QuikChange site-directed mutagenesis (QCM). One mutagenic oligonucleotide and one universal flanking primer were used to produce the complementary megaprimers that were then used to amplify the whole plasmid template. This method yields a mutagenesis efficiency ( approximately 90%) similar to that of QCM but uses only one mutagenic oligonucleotide instead of two of them, and the length of the oligonucleotide could be shorter. This method can be further extended to double mutations that are located at distant sites by using two mutagenic oligonucleotides and even to site saturation mutagenesis by introducing randomized codons.     

快捷的定点突变效率近100%的大引物PCR方法

报道了一种新的PCR突变方法,它不需要纯化大引物或设计特别的旁侧引物．利用一个诱变引物和两个测序引物(T_m≤58℃)作为旁侧引物．第一轮PCR产物12.5 μl直接加入到50 μl的第二轮PCR反应体系作为模板和大引物,在开始第二轮PCR反应时,增加在68℃退火温度下进行10个循环的不对称PCR,这一步骤大大提高了通过600 bp或800 bp大引物所导致的突变效率．结果表明,该方法的产物能够达到高保真、97%～98%的突变效率和高产率．     

Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA

This report describes the amplification of upstream genomic sequences using the polymerase chain reaction (PCR) based solely on downstream DNA information from a cDNA clone. In this novel and rapid technique, genomic DNA (gDNA) is first incubated with a restriction enzyme that recognizes a site within the 5′ end of a gene, followed by denaturation and polyadenylation of its free 3′ ends with terminal transferase. The modified gDNA is then used as template for PCR using a gene-specific primer complementary to a sequence in the 3′ end of its cDNA and an anchored deoxyoligothymidine primer. A second round of PCR is then performed with a second, nested gene-specific primer and the anchor sequence primer. The resulting PCR product is cloned and its sequence determined. Three independent plant genomic clones were isolated using this method that exhibited complete sequence identity to their cDNAs and to the primers used in the amplification.     

Efficient procedure for site-directed mutagenesis mediated by PCR insertion of a novel restriction site

Better understanding of proteins'' structure/function relationship and dissecting their functional domains are still challenges yet to be mastered. Site-directed mutagenesis approaches that can alter bases at precise positions on the gene sequence can help to reach this goal. This article describes an efficient strategy that can be applied not only for both deletion and substitution of target amino acids, but also for insertion of point mutations in promoter regions to study cis-regulating elements. This method takes advantage of the plasticity of the genetic code and the use of compatible restriction sites.Key words: site-directed mutagenesis, restriction site, cloning, PCRUnderstanding the proteins structure/function relationship and dissecting their functional domains is one of the biggest challenges to current proteomic studies.¹ This is mainly achieved by site-directed mutagenesis experiments that can alter bases at precise positions on the gene sequence.² Modifying DNA sequences has become feasible with PCR amplification.³ During the last decade, several strategies have been developed to simplify this approach and increase its efficiency.⁴ The introduction of a site-directed mutation can be realized by one or more PCR reactions. Most of the strategies used in site-directed mutagenesis are based on a substitution of a single base, which leads to a change in one amino acid. This article describes an efficient strategy that can be applied for either deletion or substitution of target amino acids. This strategy is based on performing PCR reactions to create a new restriction site in the sequence of origin, corresponding to the desired mutation. The choice of the restriction site to be created depends on the nature of the amino acid that one desires to introduce in the protein sequence. Since such restriction sites may extend beyond the mutated codon. The preservation of the other codon is done by taking advantage of the plasticity of the genetic code where one amino acid can be encoded by multiple codons.This method was performed in two steps (Fig. 1). In the first step, the DNA sequence of interest, cloned in a plasmid, served as a template for two PCR reactions. Two PCR products are generated. The first one consists of the beginning of the sequence, from the start codon to the mutagenized amino acid codon, where the forward primer bears the start codon region and the reverse primer bears the newly introduced restriction site at the same location of the mutagenized codon. The second PCR product consists of the end of the coding sequence, from the mutagenized amino acid codon to the stop codon. This fragment is generated using a forward primer bearing the same new restriction site as the first PCR product''s reverse primer, and a reverse primer bearing the stop codon region. The two PCR products were cloned separately into a vector in the appropriate orientation. In the second step, the cloning vector bearing the first PCR product was digested with a restriction enzyme site in the vector, and by the restriction enzyme corresponding to the restriction site created by the reverse primer used in the PCR reaction. The resulting fragment was cloned into the vector containing the second PCR fragment, predigested with same two restriction enzymes. The whole mutagenized coding sequence is reassembled by in-frame subcloning of the 3′ end of the coding sequence downstream the 5′ end. All the PCR products were generated using the high fidelity Pfu DNA Polymerase (Promega, Madison, WI USA). For any site-directed mutagenesis experiment, this two-step cloning procedure requires the use of appropriate PCR primers that harbor the desired mutation of the target amino acid. These primers are partially overlapping and contain a common or complementary restriction site enabling the in-frame assembly of the whole coding sequence.Open in a separate windowFigure 1Mutagenesis strategy by restriction enzyme site insertion. (A) In the first step, two PCR products were generated using the full length coding sequence as template. The mutation is carried by the two primers b and c, which are flanked by the same or compatible restriction enzyme''s site (white segment). Both PCR products are separately cloned in the cloning vector in the appropriate orientation. In the second step, the whole mutagenized coding sequence is reassembled by in-frame sub cloning of the 3′ end of the coding sequence downstream the 5′ end. (B) Substitution of threonine by arginine as a result of the insertion of a BglII restriction site. DNA sequencing is carried out to make sure that only the desired change is introduced in the coding sequence. (B-1) The sequence of the native cDNA. (B-2) the sequence of the mutagenized cDNA included BglII restriction site sequence.This approach has been used in a recent study to address the structure/function relationship of the STAS domain of the Arabidopsis thaliana Sultr1;2 sulfate transporter.⁵ A good example of this approach is the replacement of the threonine-serine couple at position 587–588 with an arginine-serine couple. The codon for threonine is: TGT, and that for arginine is: TCT. Serine can be encoded by both TCA and AGA codons. The chosen restriction site used for the reassembly of the whole coding sequence is that of the BglII enzyme: TCT AGA. The insertion of this restriction site enables the substitution of the Thr in position 587 with an Arg while preserving the serine residue in position 588. The BglII restriction site is introduced in the reverse primer and the forward primer used to generate the first and second PCR products respectively. The DNA sequence of the reassembled mutagenized cDNA was checked by sequencing. Than it was expressed, under pGAL1O promoter bearing by pYES2 vector, in yeast mutant deficient in sulfate transporter and the mutagenic protein was detected by imunodetection.Bioinformatic study reveals that this method can be applied to checked a large number of substitutions, insertions or deletions and that finding the right restriction site is not a limiting factor (data no shown).In conclusion, this article describes an efficient two-step procedure for site-directed mutagenesis using primers bearing a restriction site, which is absent from the sequence of origin. The primers flanked by sequences introducing the same or compatible restriction sites mediate the incorporation of the mutation at the selection site. The choice of the restriction site depends on the nature of the desired mutation: insertion, substitution or deletion of an amino acid in a particular position. This strategy can be also used to insert point mutations in promoter regions to study cis-regulating elements.     

Rapid amplification of genomic DNA ends bynla III partial digestion and polynucleotide tailing

We report a simple and efficient method, which combines restriction endonuclease digestion and deoxynucleotide tailing, for cloning unknown genomic sequences adjacent to a known sequence. Total genomic DNA is partially digested with the frequent-cutting restriction enzymeNla III. A homo-oligomeric cytosine tail is added by terminal transferase. The tailed DNA fragments are used as the template for cloning flanking regions from all sequences of interest. A first round PCR amplification is performed with a gene-specific primer and the selective (modified polyguanine) anchor primer complementary to the cytosine tail and theNla III recognition site, with a universal amplification primer sequence at its 5′ end. This is followed by another PCR amplification with a nested gene-specific primer and the universal amplification primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the upstream region of a salt-induced gene based upon a partial cDNA clone (RSC5-U) obtained from sunflower (Helianthus annuus L.).     

Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species

Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky-and blunt-end restriction enzyme recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used to design a variety of cassette-specific primers. The dephosphorylation prevents cassette self-ligation and creates a nick at the cassette: target genome DNA ligation site suppressing unspecific PCR amplifications. We introduce the single-strand amplification PCR (SSA-PCR) technique where a lone known locus-specific primer is firstly used to enrich the targeted template DNA strand resulting in significant PCR product specificity during the second round conventional nested PCR. The distance between the known locus-specific primer and the nearest location of the restriction enzyme used determined the length of the obtained PCR product. We used this technique to walk downstream into the isochorismatase and upstream into the hypothetical conserved genes flanking the mature extracellular lipase gene from Bacillus licheniformis. We further demonstrated the potential of the technique as a cost-effective method during PCR-based prospecting for novel genes by designing "universal" degenerate primers that detected homologues of Family VII bacterial lipolytic genes in Bacillus species. The cassette ligation-mediated PCR was used to clone complete nucleotide sequences encoding functional lipolytic genes from B. licheniformis and Bacillus pumilus.     

Template-blocking PCR: An advanced PCR technique for genome walking

This article describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette ligation-mediated polymerase chain reaction (PCR) technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3′ ends of the restriction enzyme-digested genomic DNA fragments were blocked with dideoxynucleoside triphosphate (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and a cassette primer (CP). The ddNTP blocking of the genomic DNA ends significantly reduced the nonspecific amplification and resulted in a simple and rapid walking along the genome. The efficiency of the template-blocking PCR method was confirmed by a carefully designed control experiment. The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp.     

A novel simple and rapid PCR-based site-directed mutagenesis method

Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector. In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5′ end of one of the primers, whereas the other primer starts with the nucleotide at position (−1) of the one to be modified. The PCR is carried out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat α integrin CD11b A-domain and the human CD8β cloned in pPICZαB, pGEX-2T, and CDM8 expression vectors, respectively.     

Efficient amplification of multiple transposon-flanking sequences

Transposon mutagenesis is a very useful tool for gene identification in bacteria. Once the transposon mutants of interest are isolated, it is often necessary to identify the sequences that flank the transposon insertions. We devised an efficient method for specific amplification of transposon-flanking sequences that requires the sequence information of only transposon-specific sequences. The basic steps for this method consists of (1) digestion with a restriction enzyme, (2) ligation with a Y-shaped linker and (3) polymerase chain reaction amplification using a transposon-specific primer and a primer specific to the Y-shaped linker. The feasibility of this method was demonstrated with mini-Tn5 mutants of Salmonella typhimurium. We also found that this method can be used for simultaneous amplification of multiple transposon-flanking sequences.     

Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences

A novel thermodynamically-balanced inside-out (TBIO) method of primer design was developed and compared with a thermodynamically-balanced conventional (TBC) method of primer design for PCR-based gene synthesis of codon-optimized gene sequences for the human protein kinase B-2 (PKB2; 1494 bp), p70 ribosomal S6 subunit protein kinase-1 (S6K1; 1622 bp) and phosphoinositide-dependent protein kinase-1 (PDK1; 1712 bp). Each of the 60mer TBIO primers coded for identical nucleotide regions that the 60mer TBC primers covered, except that half of the TBIO primers were reverse complement sequences. In addition, the TBIO and TBC primers contained identical regions of temperature- optimized primer overlaps. The TBC method was optimized to generate sequential overlapping fragments (~0.4–0.5 kb) for each of the gene sequences, and simultaneous and sequential combinations of overlapping fragments were tested for their ability to be assembled under an array of PCR conditions. However, no fully synthesized gene sequences could be obtained by this approach. In contrast, the TBIO method generated an initial central fragment (~0.4–0.5 kb), which could be gel purified and used for further inside-out bidirectional elongation by additional increments of 0.4–0.5 kb. By using the newly developed TBIO method of PCR-based gene synthesis, error-free synthetic genes for the human protein kinases PKB2, S6K1 and PDK1 were obtained with little or no corrective mutagenesis.