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。     

一步法定点突变技术快速构建bsh基因突变启动子

目的：建立一种高效便捷的定点突变方法,为基因表达调控以及蛋白质结构和功能的研究提供技术支撑。方法：以构建单核细胞增生李斯特菌（Listeria monocytogenes）中编码胆碱水解酶（bile salt hydrolase,BSH）的bsh基因突变启动子为例,采用一对完全互补并带有突变位点的引物扩增携带bsh基因启动子的重组质粒DNA全序列,通过DpnⅠ消化PCR产物中剩余的甲基化的模板DNA,酶切后的PCR产物直接转化大肠杆菌,从而获得含有突变启动子的重组质粒。结果：通过一步法定点突变技术成功构建了bsh基因的三种突变启动子。结论：该方法简单高效,只要把握好对引物设计,高保真的DNA聚合酶、模板DNA的浓度以及PCR扩增程序的选择,突变成功率可以达到100%。     

定点突变三种方法的比较研究

目的：通过使用优化后的定点突变三种方法分别对一个新基因重组载体进行定点突变研究,比较这几种定点突变方法的优缺点。方法：重叠延伸PCR法使用Stratagene在线定点突变引物设计程序从而使引物设计简化而可靠;MutaBEST定点突变试剂盒采用胶纯化试剂盒代替说明书推荐的酚-氯仿抽提质粒的方法以提高回收率;使用PrimeSTAR高保真DNA聚合酶和超级感受态试剂盒代替Quikchange定点突变试剂盒的相应组分可使产物不受影响同时降低试验费用。结果：三种方法都能够获得单碱基突变重组载体。结论：QuikChange突变策略通过改进是一种高效、简洁、经济和可靠的定点突变方法。     

人脑髓鞘碱性蛋白cDNA体外扩增、克隆和鉴定

采用聚合酶链反应(PCR)从人脑cDNA文库中扩增出600bp的髓鞘碱性蛋白(MBP)cDNA片段,与载体pGEM-3Zf(+)平端连接.重组质粒DNA转化宿主菌JM109,在含X-gal和IPTG的平板上直接筛选阳性克隆.限制性内切酶分析和成套引物扩增鉴定证明,该克隆含有7个外显子的21.5kD人脑MBP全长编码序列.     

单引物PCR法引入定点突变

目的:利用单个突变引物,在含人呼吸道合胞病毒F蛋白基因编码序列的pc DNA3.1(+)-F质粒中,通过单次环形PCR在特定序列位置引入定点突变。方法:以双链环状的pc DNA3.1(+)-F质粒DNA为模板,设计分别含有三种目的突变N70Q,I431N,Q270T的三条单引物,分别进行单次PCR。用甲基化DNA特异的限制性内切酶Dpn I处理PCR产物后转化大肠杆菌DH5α,进行克隆筛选,酶切鉴定和测序分析。结果:酶切鉴定结果和测序结果均符合预期,利用单引物PCR法成功在含人呼吸道合胞病毒F蛋白基因编码序列的pc DNA3.1(+)-F质粒DNA中引入了单碱基突变、两个间隔碱基突变及相邻三碱基突变三种目的突变。结论:单引物PCR法解决了常规定点突变方法中多个PCR反应,程序繁琐及突变效率低等问题,是一种简便、快速、有效的基因工程定点突变新方法。     

单引物PCR法引入定点突变 *

目的:利用单个突变引物,在含人呼吸道合胞病毒F蛋白基因编码序列的pcDNA3.1(+)-F质粒中,通过单次环形PCR在特定序列位置引入定点突变。 方法: 以双链环状的pcDNA3.1(+)-F质粒DNA为模板,设计分别含有三种目的突变N70Q, I431N, Q270T的三条单引物,分别进行单次PCR。用甲基化DNA特异的限制性内切酶Dpn I处理PCR产物后转化大肠杆菌DH5α,进行克隆筛选,酶切鉴定和测序分析。 结果: 酶切鉴定结果和测序结果均符合预期,利用单引物PCR法成功在含人呼吸道合胞病毒F蛋白基因编码序列的pcDNA3.1(+)-F质粒DNA 中引入了单碱基突变、两个间隔碱基突变及相邻三碱基突变三种目的突变。 结论: 单引物PCR法解决了常规定点突变方法中多个PCR反应,程序繁琐及突变效率低等问题,是一种简便、快速、有效的基因工程定点突变新方法。     

用DREAM技术纠正人工合成基因中的突变

目的：介绍一种简便、有效的定点突变技术。方法：根据突变位点附近的DNA序列推导出氨基酸序列,再以此氨基酸序列进行逆翻译,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内切酶位点,选择合适的酶切位点设计引物用PCR技术扩增两侧DNA片段,然后以相应酶切融合这两个片段即可完成定点突变。结果：用该方法成功地在人工合成的含有缺失的可溶性组织因子基因的472位插入C,T两个碱基,校正了阅读框架,获得了预期的目的基因。结论：该方法简便、有效, 避免了多轮PCR和合成长引物导致突变的可能性,这种改进的PCR 定点诱变技术我们称之为“设计限制酶辅助突变”（Designed Restriction Enzyme Assisted Mutagenesis, DREAM）。此技术简单方便, 诱变的成功率高, 适于实验室常规应用。     

一种通用高效的复杂载体构建的新方法

文章报道了一种简便、通用、高效的复杂载体构建方法。此法是在PCR引物设计时,在目的片段5′端加上随机设计的接头,利用PCR克隆目的片段,再用T4 DNA聚合酶3′→5′外切酶活性处理PCR克隆目的片段,产生多个首尾相匹配的粘性末端,进行多片段定向连接、转化、重组子鉴定。以7片段拼接的水稻单交换质体定点整合表达载体pRSMGA的构建为例,应用上述方法构建只需做两次连接、转化,且其重组、转化效率高。数10次实验证明：这是一种简便、通用、高效的复杂构建载体的方法,该法至今尚未见报道。      

结合单酶切位点的融合PCR技术对SCN1A基因的定点诱变

目的:利用结合单酶切位点的融合PCR技术对癫痫相关基因SCN1A进行定点突变。方法:首先设计两对引物PF1/PR1和PF2/PR2,PF1和PR2均位于突变位点最近的单酶切位点处,而突变位点设计在第一对反向引物(PR1)和第二对正向引物上(PF2)。通过重叠延伸法两次PCR扩增:第一次用PF1/PR1和PF2/PR2分开扩增,以扩增产物作模板,PF1/PR2作引物进行融合PCR,得到的扩增产物即含有所需要的突变位点,最后将扩增片段克隆入pMD18-T载体,经测序筛选阳性克隆。结果:DNA测序表明SCN1A基因所编码的第946位密码子由精氨酸(Arg)突变为组氨酸(His),再通过酶切和连接反应将重组质粒上的突变片段替换SCN1A表达质粒上的对应片段,成功构建了SCN1A突变载体。结论:与现在常用的长距离PCR定点诱导突变相比较,结合单酶切位点的融合PCR定点突变技术具备扩增距离短的优点,大大降低了自发突变的概率,适合于大肠杆菌中易自发突变的较大载体的定点诱变。     

长片段融合PCR在构建烟曲霉rho1基因回补株中的应用

目的融合PCR是一种常用的构建重组片段或重组质粒的手段,但长片段融合PCR的难度较大。文中将探讨长片段融合PCR过程中引物设计及扩增条件对产物的影响。方法以构建烟曲霉rho 1基因回补株为例,采用融合PCR的方法扩增重组片段(长达6.5 kb),在引物设计时引入不同大小的同源区,并设置不同的扩增体系。结果当设计引物的同源区为35 bp,选用具有高扩增效率、高保真性的DNA聚合酶,以及各片段在融合PCR反应体系中的浓度为15 ng/μL时,实现了长达6.5 kb的片段扩增并完成了烟曲霉rho 1回补株的构建。结论在合适的PCR引物设计、片段浓度配比及聚合酶条件下,长片段融合PCR在丝状真菌的基因敲除及回补株的构建中是一种非常有效的工具。     

Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli

A rapid site-directed mutagenesis strategy using homologous recombination and DpnI digestion of the template in Escherichia coli is described. Briefly, inverse polymerase chain reaction amplification of the entire circular plasmid was performed by mutagenic primers with overlapping sequences ( approximately 15 bp) for generating PCR products with approximately 15 bp of homology on the terminal ends. On direct transformation of the amplified PCR products into restriction endonuclease DpnI-expressing E. coli BUNDpnI, homologous recombination occurs in E. coli while the original templates are removed via DpnI digestion in vivo, thus yielding clones harboring mutated circular plasmids. Nearly 100% efficiency was attained when this strategy was used to modify DNA sequences.     

New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis

The QuikChange^TM site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange^TM method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5′-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that the PCR enzyme fills the 5′-overhangs in the early cycles, and the product is then used as the template for exponential amplification. The linear DNA molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in E. coli. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and Phusion DNA polymerase for site-directed mutagenesis. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear DNA molecules with homologous ends for site-directed mutagenesis.     

QuikChange shuffling: a convenient and robust method for site-directed mutagenesis and random recombination of homologous genes

Here we describe a robust method, termed QuikChange shuffling, for efficient site-directed mutagenesis and random recombination of homologous genes. The homologous genes are fragmented, and the random fragments are reassembled in a self-priming polymerase reaction to obtain chimeric genes. The product is then mixed with linearized vector and two pairs of complementary mutagenic primers, followed by assembly of the chimeric genes and linearized vector through QuikChange-like amplification to introduce recombinant plasmids with a site-directed mutation. The method, which can yield 100% chimeric genes after library construction, is more convenient and efficient than current DNA shuffling methods.     

异形胞分化蓝细菌dnaA温度敏感型突变体的构建

以能分化异形胞的蓝细菌(Anabaenasp.PCC7120)为材料,采用重组PCR在体外对控制DNA复制起始的dnaA基因进行定点突变后克隆到整合质粒中,再通过三亲本杂交将整合质粒转移到Anabaena PCC7120中,以分离和筛选温度敏感型突变体。结果成功获得Anabaena PCC 7120 dnaA高温敏感性突变体。研究表明,利用重组PCR技术可在体外实现对Anabaena PCC 7120的dnaA的定点突变,并可通过同源重组双交换成功实行整合质粒中突变基因对野生型基因的置换,使突变基因插入到细胞染色体中,进而成功构建温度敏感型突变菌株。     

A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction

We have developed a novel polymerase chain reaction (PCR) method that permits the rapid generation of site-specific mutants and recombinant DNA constructs with a minimum number of steps and primers. DNA segments are modified by using amplifying primers that add homologous ends to the polymerase chain reaction product(s). These homologous ends undergo recombination in vivo following transformation of recA-E. coli strains used routinely in cloning. In vivo circularization of PCR products containing plasmid sequences with a selective marker permits the rapid cloning of the desired mutant or recombinant. In the mutagenesis protocol, 7 of the 12 clones contained the product of interest, and 6 of these clones had no detected error (50% of the clones without detected errors). In each of several recombination protocols, at least 50% of the clones tested contained the insert of interest without detected errors.     

Optimization of DNA shuffling for high fidelity recombination.

A convenient 'DNA shuffling' protocol for random recombination of homologous genes in vitro with a very low rate of associated point mutagenesis (0.05%) is described. In addition, the mutagenesis rate can be controlled over a wide range by the inclusion of Mn2+or Mg2+during DNase I digestion, by choice of DNA polymerase used during gene reassembly as well as how the genes are prepared for shuffling (PCR amplification versus restriction enzyme digestion of plasmid DNA). These protocols should be useful for in vitro protein evolution, for DNA based computing and for structure-function studies of evolutionarily related genes.     

Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3′ end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.