Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.     

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.     

利用DREAM设计和同源重组进行一步定点突变

目的：建立基于DREAM设计和同源重组的简便、快速定点突变方法。方法：设计两条包含突变的反向PCR（inverse PCR）引物,使其5'端互补从而产生同源重组,同时使用DREAM设计方案在上述引物中引入限制性内切酶位点以便突变子筛选。用能扩增长片段的高保真耐热 DNA聚合酶扩增全长的质粒DNA,直接转化大肠杆菌。转化到细菌中的全长质粒DNA PCR产物可利用其末端同源序列发生同源重组而环化。利用引入的酶切位点方便地进行突变子的筛选。结果：我们用该方法成功地对长度大于7 kb的质粒进行了定点突变。结论：本定点突变无需任何突变试剂盒和特殊的试剂,只需一步反应即可完成;利用DREAM设计使克隆筛选简便可靠,高保真耐热DNA聚合酶可保证多数突变子克隆不发生意外突变,而该酶扩增长片段的能力使该方法适合于大多数质粒不经亚克隆直接突变。     

A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis

A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli β-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification.     

An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal protein

A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx(2)SLYP and Kx(3)NSxYG (TPR-1) and motifs Kx(3)NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of phi29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in phi29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer.     

An in vitro approach to identifying specificity determinants of mutagenesis mediated by DNA misalignments

In vitro, misalignments of the newly synthesized (primer) strand during DNA polymerization lead to deletion and/or complex frameshift mutations. In vivo, similar misalignments of repeated and quasipalindromic DNA sequences are predicted to be intermediates of mutagenesis. The mutagenic misalignments are mediated by complementary pairing between the sequence at the 3'-OH end of the newly synthesized DNA strand and sequences in the template or in the newly synthesized DNA. Mutant sequences are produced when the misaligned primers act as substrates for DNA polymerization. The misalignments responsible for detected mutant sequences were compared to similar misalignments that were not implicated in mutagenesis, and all misalignment possibilities were compared to the position of pausing during polymerization by Escherichia coli polymerase I or its Klenow fragment. These comparisons revealed three characteristics of in vitro misalignment specificity. First, the termini produced by pausing are likely to be precursors to mutagenic misalignments. Second, the absence of some potential misalignments from the detected spectrum is explained well by the predicted undetectability of the mutant sequences they produce. Third, factors distinct from pausing and mutant detectability are responsible for differences in the specificity of misalignment mutagenesis mediated by E. coli DNA polymerase I and Klenow polymerase during in vitro synthesis.     

Primer extension mutagenesis powered by selective rolling circle amplification

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material.     

An efficient and site-specific gene trimming method

Trimming a DNA strand into a precisely determined fragment can be carried out efficiently by an improved method involving a site-specific trim-primer and a single-stranded DNA template which is generated from a multifunctional vector, pTZ18R, and linearized by using an Eco RI-pTZ18R splinter. A complementary DNA strand is synthesized by DNA polymerase I (Klenow), and the 3'-end of the template upstream from the annealed primer is trimmed by subsequent T4 DNA polymerase reaction. An ATG translation initiator codon or a termination codon can be incorporated into the trim-primer, providing versatility to this single-stranded DNA-initiated gene trimming method that can be applied to subcloning and expression of any DNA fragment with known terminal sequences.     

PFunkel: Efficient,Expansive, User-Defined Mutagenesis

We introduce PFunkel, a versatile method for extensive, researcher-defined DNA mutagenesis using a ssDNA or dsDNA template. Once the template DNA is prepared, the method can be completed in a single day in a single tube, and requires no intermediate DNA purification or sub-cloning. PFunkel can be used for site-directed mutagenesis at an efficiency approaching 100%. More importantly, PFunkel allows researchers the unparalleled ability to efficiently construct user-defined libraries. We demonstrate the creation of a library with site-saturation at four distal sites simultaneously at 70% efficiency. We also employ PFunkel to create a comprehensive codon mutagenesis library of the TEM-1 ß-lactamase gene. We designed this library to contain 18,081 members, one for each possible codon substitution in the gene (287 positions in TEM-1 x 63 possible codon substitutions). Deep sequencing revealed that ∼97% of the designed single codon substitutions are present in the library. From such a library we identified 18 previously unreported adaptive mutations that each confer resistance to the ß-lactamase inhibitor tazobactam. Three of these mutations confer resistance equal to or higher than that of the most resistant reported TEM-1 allele and have the potential to emerge clinically.     

Efficient site directed in vitro mutagenesis using ampicillin selection.

A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin. In tests of the vector, highly efficient (60-90%) mutagenesis was obtained.     

Creating randomized amino acid libraries with the QuikChange Multi Site-Directed Mutagenesis Kit

The QuikChange Multi Site-Directed Mutagenesis Kit is a simple and efficient method for introducing point mutations at up to five sites simultaneously in plasmid DNA templates. Here we used the QuikChange Multi kit with degenerate (one codon) primers to introduce all possible amino acids at selected sites in the lacZ gene. In reactions employing two or three degenerate primers, diverse libraries (10(4)-10(5) mutants/reaction) are created consisting of random combinations of mutations at two or three different sites. This method provides a one-day procedure for performing site-directed saturation mutagenesis and, when coupled with a suitable screening assay, should greatly facilitate the process of evaluating alternative amino acid chain substitutions at key residues and evolving protein function.     

A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles

Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase chain reaction was used to generate products that when combined, denatured and reannealed, form double-stranded DNA with discrete, cohesive single-stranded ends. The generation of these cohesive ends of DNA permits the formation of precise, directional DNA joints without dependence on enzyme restriction sites. The primers were designed such that these cohesive single-stranded ends annealed to form circular DNA. The recombinant of interest was generated following only 14 amplification cycles. These recombinant circles of DNA were directly transfected into E. coli. In the mutagenesis protocol, the desired mutant was obtained at 83%-100% efficiency. Unwanted mutations were not detected, indicating a less than 0.025% nucleotide misincorporation frequency. In the directional subcloning protocol, inserts were positioned precisely in the recipient plasmid and were in the correct orientation. One unwanted mutation was detected after sequencing 900 bases, indicating a 0.11% nucleotide misincorporation frequency. Each manipulation, from setting up for the DNA amplification to transfection into E. coli. can easily be accomplished in one day.     

Gene X of bacteriophage f1 is required for phage DNA synthesis. Mutagenesis of in-frame overlapping genes

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.     

Cellular transformation by adenovirus type 5 is influenced by the viral DNA polymerase.

Early region 2B (E2B) of the group C adenoviruses encodes a number of proteins, including the 140-kilodalton DNA polymerase, which plays a role in the initiation of viral DNA replication. Temperature-sensitive (ts) mutants with mutations mapping to E2B are conditionally defective for both DNA replication in human cells and transformation of rat cells. Nucleotide sequence analysis shows that the E2B mutant ts36 possesses a single point mutation specific to the viral DNA polymerase; this transition of a C to a T at position 7623 changes leucine residue 249 in the polymerase to a phenylalanine. A wild-type (ts+) revertant possesses a codon specifying the original leucine at position 249. Phenotypic analysis of revertant and wild-type viruses derived by marker rescue from ts36 shows that these variants are wild type for both viral DNA replication and transformation. Thus, the single point mutation in the polymerase gene of ts36 is responsible for both defects.     

金黄色葡萄球菌核酸酶基因的片段缺失与定点突变

金黄色葡萄球菌核酸酶R(SNase R)是金黄色葡萄球菌核酸酶(SNase A)的一种类似物,具有与SNaseA相同的酶活性,与SNase A唯一不同之处是在N端多出6个氨基酸残基。为了得到完整的SNase基因并使其在E.Coli中表达,我们利用单链U模板—单引物突变法,将为6个额外氨基酸残基编码的18个氨基酸残基删除,其突变率可达90％。进而,完整的SNase A基因被重组入表达载体pBV221。细菌表达产物的PAGE分析结果指出,SNase A在E.coli中得到高效表达。与此同时,我们利用两个不同的引物在单链U模板上同时介导两种不同类型的突变(片段缺失、碱基取代)其突变率可达83％以上,这为进行多种类型的高效突变提供一个有用的方法。本文也对影响突变率的某些实验因素进行了讨论。     

Mechanism of replication of bacteriophage phi X174. XXII. Site-specific mutagenesis of the A* gene reveals that A* protein is not essential for phi X174 DNA replication

The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.     

Efficient and erroneous incorporation of oxidized DNA precursors by human DNA polymerase eta

Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis.     

丙型肝炎病毒E2糖蛋白糖基化作用的研究

本研究构建丙型肝炎病毒(HCV)糖蛋白E2的N-糖基化位点定点突变体。采用高保真性的Pfx DNA聚合酶,设计两对引物,分别引入两个突变位点,通过PCR体外定点突变,使E2第535、583位核苷酸由A突变为T,从而使AAC编码的天冬酰氨突变为TAC编码的酪氨酸,使得N-糖苷化位点NNT、NST突变为YNT、YST.结果得到两个单位点以及一个双位点突变体,并将突变型E2连接到真核表达载体peDNA3．1(-)／Myc—HisB上。成功获得的3个HCVE2糖蛋白糖基化位点定点突变体,为进一步进行HCVE2糖蛋白糖基化位点与分子伴侣之间的相互关系以及突变体对机体的免疫功能的影响的研究奠定了基础。