Site-directed mutagenesis of virtually any plasmid by eliminating a unique site.

We describe an efficient site-specific mutagenesis procedure that is effective with virtually any plasmid, requiring only that the target plasmid carry a unique, nonessential restriction site. The procedure employs two mutagenic oligonucleotide primers. One primer contains the desired mutation and the second contains a mutation in any unique, nonessential restriction site. The two primers are annealed to circular single-stranded DNA (produced by heating circular double-stranded DNA) and direct synthesis of a new second strand containing both primers. The resulting DNA is transformed into a mismatch repair defective (mut S) Escherichia coli strain, which increases the probability that the two mutations will cosegregate during the first round of DNA replication. Transformants are selected en masse in liquid medium containing an appropriate antibiotic and plasmid DNA is prepared, treated with the enzyme that recognizes the unique, nonessential restriction site, and retransformed into an appropriate host. Linearized parental molecules transform bacteria inefficiently. Plasmids with mutations in the unique restriction site are resistant to digestion, remain circular, and transform bacteria efficiently. By linking a selectable mutation in a unique restriction site to a nonselectable mutation, the latter can be recovered at frequencies of about 80%. Since most plasmids share common vector sequences, few primers, targeted to shared restriction sites, are needed for mutagenizing virtually any plasmid. The procedure employs simple procedures, common materials, and it can be performed in as little as 2 days.     

Site-directed mutagenesis by complementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates

An approach for generating structures capable of directing full-length complementary-strand synthesis for double-stranded plasmid DNA is described. The structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing" oligonucleotide. Consisting of a sequence complementary to the free ends of one of the two plasmid strands, the closing oligonucleotide functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently closed heteroduplex molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.     

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.     

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.     

Half-site editing: an in vitro mutagenesis procedure for truncating a DNA fragment and introducing a new restriction site

Half-site editing is an in vitro mutagenesis procedure designed for use in making precise plasmid constructions. Like many in vitro mutagenesis techniques, this procedure involves hybridization of a mutagenic oligonucleotide primer to single-stranded template DNA followed by polymerization with DNA polymerase I (Klenow). Half-site editing differs from other techniques in two main ways. First, T4 DNA polymerase treatment truncates the target DNA at a point determined by the primer and repairs any mismatches to the sequence specified by the primer. Second, a blunt-end ligation step is included. This ligation exploits the symmetry inherent in most restriction sites to create a desired restriction site at the truncated end of the target DNA fragment. Half-site editing has been used to place ClaI restriction sites at the 3' end of the yeast pyruvate kinase promoter and at two positions at the 5' end of the yeast acetolactate synthase coding sequence.     

Construction of recombinant DNA molecules by the use of a single stranded DNA generated by the polymerase chain reaction: its application to chimeric hepatitis A virus/poliovirus subgenomic cDNA.

In order to study the importance of VP4 in picornavirus replication and translation, we replaced the hepatitis A virus (HAV) VP4 with the poliovirus (PV1) VP4. Using a modification of oligonucleotide site directed mutagenesis and the polymerase chain reaction (PCR), we created a subgenomic cDNA chimera of hepatitis A virus in which the precise sequences coding for HAV VP4 capsid protein were replaced by the sequences coding for the poliovirus VP4 capsid protein. The method involved the use of PCR primers corresponding to the 3' and 5' ends of the poliovirus VP4 sequence and that had HAV VP4 3' and 5' flanking sequences on their 5'ends. Single stranded DNA of 240 and 242 nt containing the 204 nt coding for the complete poliovirus VP4 were produced by using a limiting amount of one of the primers in a PCR reaction. These single stranded PCR products were used like mutagenic oligonucleotides on a single stranded phagemid containing the first 2070 bases of the HAV genome. Using this technique, we precisely replaced the HAV VP4 gene by the poliovirus VP4 gene as determined by DNA sequencing. The cDNA was transcribed into RNA and translated in vitro. The resulting protein could be precipitated by antibody to poliovirus VP4 but not to HAV VP4.     

Application for PCR technology to subtractive cDNA cloning: identification of genes expressed specifically in murine plasmacytoma cells.

We describe a simple method for preparing a renewable source of subtractive cDNA which can be used as a hybridization probe or as insert which can be cloned into a variety of convenient vectors. This has been done by ligating a double-stranded oligonucleotide to each end of double-stranded subtractive cDNA, and then using this oligonucleotide sequence to amplify the heterogeneous population of cDNA molecules using the polymerase chain reaction and thermostable Taq DNA polymerase. This method improves the chances for identifying cDNA clones representing low abundance mRNAs that are expressed differentially. Using this approach, we have identified cDNA clones which detect three different low abundance mRNAs that are expressed in mouse plasmacytoma cell lines but not in mouse pre-B or B lymphoma cell lines.     

改造α乳清蛋白的定点突变研究

蛋白质结构与功能之间关系的研究一直是生命科学领域的焦点 .采用定点诱变技术在克隆 c DNA的预定位点导入突变 ,然后在适当的宿主细胞 -载体系统中表达已改变的基因 ,通过比较突变体蛋白与野生型蛋白的性质 ,往往可能鉴别出对蛋白质的结构完整性和生物学功能至关重要的结构域或氨基酸残基 [1,2 ] .α乳清蛋白是哺乳动物乳汁中的主要蛋白质 ,它和半乳糖苷转移酶一起形成复合物 ,称为乳糖合成酶 .C型溶菌酶的功能是催化裂解细菌细胞壁肽聚糖组分 NAM- NAG的 β- 1 ,4糖苷键 .早期的研究发现 ,虽然它们是两种功能截然不同的蛋白质 ,它们自…     

Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis

To develop a simple, speedy, economical and widely applicable method for multiple-site mutagenesis, we have substantially modified the Quik-Change™ Site-Directed Mutagenesis Kit protocol (Stratagene, La Jolla, CA). Our new protocol consists of (i) a PCR reaction using an in vitro technique, LDA (ligation-during-amplification), (ii) a DpnI treatment to digest parental DNA and to make megaprimers and (iii) a synthesis of double-stranded plasmid DNA for bacterial transformation. While the Quik Change™ Kit protocol introduces mutations at a single site, requiring two complementary mutagenic oligonucleotides, our new protocol requires only one mutagenic oligonucleotide for a mutation site, and can introduce mutations in a plasmid at multiple sites simultaneously. A targeting efficiency >70% was consistently achieved for multiple-site mutagenesis. Furthermore, the new protocol allows random mutagenesis with degenerative primers, because it does not use two complementary primers. Our mutagenesis strategy was successfully used to alter the fluorescence properties of green fluorescent protein (GFP), creating a new-color GFP mutant, cyan-green fluorescent protein (CGFP). An eminent feature of CGFP is its remarkable stability in a wide pH range (pH 4–12). The use of CGFP would allow us to monitor protein localization quantitatively in acidic organelles in secretory pathways.     

TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (eg. cDNA, genomic DNA and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3′ sequences of the primers match the template perfectly. When two outer mutagenic oligos are incorporated together with the desired mutagenic oligos into the newly synthesised mutant strand, they serve as anchors for PCR primers which have 3′ sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.     

Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis

The polymerase chain reaction catalyzed by Taq DNA polymerase has been used for site-specific mutagenesis. The amplification was primed by two oligodeoxyribonucleotides complementary to insulin receptor cDNA. To direct the synthesis of mutant DNA, mismatches were introduced into one of the primers. Six different mutations were constructed by this technique. Of twelve clones whose sequences were determined, ten (83%) had the correct sequence. This technique, which does not require the use of single-stranded DNA templates, provides a simple and efficient approach to site-specific mutagenesis.     

Oligonucleotide site-directed mutagenesis in Xenopus egg extracts.

Addition of M13mp18 single-stranded DNA annealed with an oligonucleotide to a Xenopus egg extract results in a rapid and efficient incorporation of the oligonucleotide in a complete double-stranded supercoiled molecule. Both the efficiency of DNA synthesis and the recovery of complete double-stranded molecules are increased relative to the reaction carried out by the classical technique using the E. coli Klenow DNA polymerase, DNA ligase, dNTPs, ATP and ions. Site specific mutagenesis was assayed by reverting a point mutation in the lacz region of M13mp18. The color assay described by Messing and sequencing of the DNA extracted from isolated plaques was used to check for the reversion. A 2 hr incubation of the heteroduplex carrying the mutagenic oligonucleotide in the Klenow-ligase-dNTP mixture allows a recovery of 6% mutant phage after transformation of competent cells with the reaction products. Using the Xenopus egg extract, 83% mutant phage were recovered after the same incubation time, in reactions entirely performed in parallel. The Xenopus extract is stable and contains all components required for the assay, including all ionic and protein factors; thus the only addition is the annealed DNA. Such an eukaryotic system is therefore an attractive alternative to the reconstituted prokaryotic DNA polymerase-DNA ligase system for site specific mutagenesis.     

寡聚核苷酸诱导突变法改造φ×174噬菌体A基因蛋白的DNA识别序列

 为了进一步研究φX174噬菌体A基因蛋白的复制功能与其所识别的30核苷酸保守序列的关系,我们采用寡聚核苷酸诱导的定点突变法成功地改造了这30核苷酸保守序列。将此保守序列重组到M_(13)mp9噬菌体后,以其单链为模板,在14或16寡聚核苷酸的诱导下,合成共价闭环DNA。经转化到E.coli JM103菌株,用点印迹(Dot blot)杂交法筛选,得到两种重组突变株。一种突变株其30核苷酸保守序列正链的第22碱基由A改为G。另一突变株为其第10碱基A改为C,第11碱基T改为A。突变效率约为5％。制备了此突变株单链及双链DNA,分别做了双脱氧末端终止法及Maxam和Gilbert法序列分析鉴定。     

Cloning of HSP60 (GroEL) operon from Clostridium perfringens using a polymerase chain reaction based approach.

Using degenerate oligonucleotide primers for conserved regions of HSP60, a 0.6 kilobase fragment of Clostridium perfringens DNA was amplified by the polymerase chain reaction. The amplified fragment was used as a probe to isolate a genomic clone containing the C. perfringens HSP60 operon. The clone contained two open reading frames homologous to the GroES and GroEL (or HSP60) family of bacterial and eukaryotic proteins as well as other upstream and downstream sequences. The approach described here, employing this set of degenerate oligonucleotide primers, could be used to clone HSP60 gene/cDNA from any species.     

Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.

We describe codon cassette mutagenesis, a simple method of mutagenesis that uses universal mutagenic cassettes to deposit single codons at specific sites in double-stranded DNA. A target molecule is first constructed that contains a blunt, double-strand break at the site targeted for mutagenesis. A double-stranded mutagenic codon cassette is then inserted at the target site. Each mutagenic codon cassette contains a three base pair direct terminal repeat and two head-to-head recognition sequences for the restriction endonuclease Sapl, an enzyme that cleaves outside of its recognition sequence. The intermediate molecule containing the mutagenic cassette is then digested with Sapl, thereby removing most of the mutagenic cassette, leaving only a three base cohesive overhang that is ligated to generate the final insertion or substitution mutation. A general method for constructing blunt-end target molecules suitable for this approach is also described. Because the mutagenic cassette is excised during this procedure and alters the target only by introducing the desired mutation, the same cassette can be used to introduce a particular codon at all target sites. Each cassette can deposit two different codons, depending on the orientation in which it is inserted into the target molecule. Therefore, a series of eleven cassettes is sufficient to insert all possible amino acids at any constructed target site. Thus codon cassettes are 'off-the-shelf' reagents, and this methodology should be a particularly useful and inexpensive approach for subjecting multiple different positions in a protein sequence to saturation mutagenesis.     

Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach.

Several improvements to the existing phosphorothioate-based site-directed mutagenesis methodology are reported, and here it is demonstrated that the new procedure is able to produce large deletions, insertions and point mutations rapidly and with very high efficiency. The time required for the polymerization step has been reduced by using T7 DNA polymerase to extend the mutant oligonucleotide primer-template. The reaction produces good yields of double-stranded closed-circular DNA and some partially polymerized template. The reaction was treated with T5 D15 exonuclease to selectively destroy partially polymerized single-stranded phage DNA that may otherwise contribute to an increased background of wild-type transformants. The use of these enzymes greatly facilitates the implementation of the phosphorothioate-based site-directed mutagenesis method by requiring less template DNA and by allowing all the in vitro manipulations to be completed in a day. In its present form the method may easily be automated, enabling large systematic site-directed mutagenesis projects to be undertaken.     

Site specific mutagenesis: insertion of single noncomplementary nucleotides at specified sites by error-directed DNA polymerization

We have utilized infidelity of DNA synthesis as a basis for site-directed mutagenesis. Both an endonuclease restriction fragment and a synthetic oligonucleotide were used as primers. DNA polymerase from bacteriophage T4 was used to elongate primer termini to a position immediately adjacent to two different preselected positions on phiX174 DNA templates. Then, the error-prone DNA polymerase from avian myeloblastosis virus was used to insert single non-complementary nucleotides at the designated positions at high efficiency. DNA sequence analysis confirmed that the mutant phage produced as a result of each site-specific mutagenesis reaction contained the nucleotide that was complementary to the one provided during the DNA copying reaction. The general applicability of this methodology to cloned DNAs will be discussed.     

The "megaprimer" method of site-directed mutagenesis

We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.     

用DREAM技术进行全长质粒快速定点突变

利用“设计限制酶辅助突变”(Designed Restriction Enzyme Assisted Mutagenesis, DREAM)进行全长质粒快速定点突变。根据突变位点附近氨基酸靶序列, 以简并密码子进行逆向推导, 这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体(Silent mutants), 这些突变体中包含大量的限制性酶切位点, 选择合适的酶切位点设计引物, 用Phusion超保真DNA聚合酶扩增全长质粒的DNA序列, 得到的PCR产物用T4多聚核苷酸激酶添加5￠磷酸基团后进行平末端连接, 转化大肠杆菌受体菌后用设计的酶切位点进行快速筛选。本研究用该方法成功地纠正了长约8 kb的质粒pcDNA3.1-pIgR中的突变碱基, 从而获得了多聚免疫球蛋白受体(pIgR)的野生型氨基酸序列。以上结果表明: 利用DREAM技术将限制性酶切位点引入目的基因而不改变目的蛋白质的氨基酸序列, 使突变体的筛选简单化; 配合使用高保真和高效率的Phusion DNA聚合酶可以进行长达8 kb的全长质粒的快速突变; 该方法无需使用定点突变试剂盒和特殊的受体菌, 同时避免了核酸杂交以及同位素的使用。     

Isolation and characterization of lamprey rhodopsin cDNA.

Genomic DNA fragments coding a visual pigment of the lamprey were amplified by polymerase chain reaction, using oligonucleotide mixtures as primers. The complete coding region of the cDNA was obtained by separate amplification of both cDNA ends. The deduced amino acid sequence of the coding region showed 78-82% identity with those of rhodopsins of higher vertebrates, but only 43-47% identity with those of human color pigments. The cloned DNA appears to be the cDNA of a lamprey rhodopsin, which is expressed in the "short" photoreceptor cell.