一种简易的利用Pfu—DNA聚合酶进行反向长距离PCR获得定点突变的 …

定点突变是一种常用的分子生物学方法。利用Ｐｆｕ－ＤＮＡ聚酶进行反向长距离Ｐ衣效地获得定点突变。方法担任简易、突变效率高于９０％。整个操作过程可在一个试管中完成,不需亚克隆。对克隆在大所粒（如转化植物的Ｔｉ－类型质粒）尤为实用。     

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。     

用改进的重叠延伸PCR技术构建三位点突变载体

目的:改进传统重叠延伸PCR方法,实现引入3个不同DNA突变位点的简便的多位点定点突变。方法:根据前期构建的包含人线粒体12S rRNA(NC 01290)3个热点突变位点的野生型质粒序列,利用Muta Primer 2.0软件设计针对3个热点突变位点的3对互补的定点突变引物,以野生型质粒为模板,结合重叠延伸PCR反应和冷冻析出法,产生同时包含3个突变位点的突变目的片段,酶切后克隆到载体中,测序确证是否突变成功。结果:DNA测序证实3个不同突变位点同时成功引入,定点突变载体构建成功。结论:用改进的重叠延伸PCR技术能简便、高效地获得多位点定点突变载体,在分子生物学领域有较高的使用价值。     

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。     

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。     

部分扩增与双片段连接相结合制作长基因定点突变

重叠延伸PCR是基因定点突变的主要方法,但是以该方法制作长基因定点突变时,往往遇到难以获得第二轮PCR产物或容易引入新的非预期突变等问题。此时,可先以重叠延伸PCR扩增含突变位点的部分基因片段,再将其连入适当载体获得重组质粒。若该扩增片段两侧的酶切位点在质粒载体上不单一,则可采用双片段连接法构建完整质粒。以制作视网膜母细胞瘤基因S780E定点突变为例,直接以重叠延伸PCR扩增全长基因时未能得到理想的目标产物。故先扩增含点突变的F3片段,再将其与源自原始质粒的F2片段一起连入含F1片段的质粒载体而构建完整质粒。两个筛选出的重组质粒经序列检测完全符合目标突变序列特征,验证了该方案的可行性。该方法作为重叠延伸PCR的补充,可为许多长基因定点突变提供解决方案。     

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。     

鹅源新城疫病毒ZJI株基因组cDNA克隆的序列修饰

将鹅源新城疫病毒ZJI株全基因组cDNA克隆通过酶切切下包含T7启动子区域和转录载体的片段,将其自身环化后获得约6．5kb的质粒。设计引物,利用基因定点突变技术,在此质粒上T7启动子与NDV Leader序列之间突变插入额外的3个G碱基,将此突变最终引入到原基因组cDNA克隆中。应用RT—PCR技术从尿囊液中扩增NDV基因组F／HN基因区域部分片段,利用限制性内切酶BsmBI将扩增片段连接,最终将原cDNA克隆中相应片段替换下。测序结果表明,原基因组cDNA克隆中特定位置碱基插入突变成功,F／HN基因区域碱基突变均得以纠正。以上cDNA克隆的修饰与替换为该毒株的反向遗传研究打下了基础。     

BRAF和TP53基因热点突变的构建

目的:构建BRAF和TP53基因多个位点的点突变质粒,为进一步研究其在肿瘤形成中的作用奠定基础。方法:以HEK-293T基因组DNA为模板构建含BRAF600、TP53175和TP53248位点的野生型质粒,利用MutanBESTTM定点突变方法获得含BRAFV600E、TP53R175C和TP53R248W等突变位点的突变型质粒,并进行DNA测序鉴定。结果:DNA测序结果显示构建的三个点突变与实验设计完全一致。结论:成功构建了BRAFV600E、TP53R175C和TP53R248W三个具有不同突变位点的突变型质粒,MutanBESTTM定点突变技术是一种简单、快速、高效的基因定点突变方法。     

多片段扩增结合Gibson组装法克隆基因定点突变北大核心CSCD

为寻找一种简洁高效的基因定点突变方案,利用Gibson组装技术对重叠延伸PCR法进行简化,并以克隆细胞周期蛋白依赖性激酶4基因单位点与双位点突变为例进行验证。采用与重叠延伸PCR相似的策略扩增含点突变的基因片段,同时采用双酶切制备线性质粒载体,保证质粒载体与基因片段含有一小段重叠序列作为接头。直接将基因片段与线性载体通过Gibson组装法拼接成完整质粒。经转化、筛选与检测,成功得到数个单位点与双位点目标突变体克隆,且阳性率均为100%。由于没有繁琐的多轮PCR扩增和频繁的DNA回收操作,也无需消化原始质粒,该方案避免了很多干扰定点突变的因素,能简便、高效地克隆基因单位点与多位点突变。对比而言,该方案规避了重叠延伸PCR与滚环扩增法的主要缺陷,是一种基因定点突变的良好解决方案。     

一种简易的利用Pfu_DNA聚合酶进行反向长距离PCR获得定点突变的方法

Site-specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site-specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu-DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.     

Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.     

Parallel assembly for multiple site-directed mutagenesis of plasmids

A parallel assembly method for multiple site-directed mutagenesis of plasmids was developed here based on Golden Gate cloning. It takes advantage of type IIs restriction enzymes and T4 DNA ligase to assemble multiple DNA fragments into a plasmid by a defined order. This method can accommodate multiple plasmid mutagenesis at any desired position with all three sequence modification types (substitution, deletion, and insertion) simultaneously. Furthermore, it can be used to create otherwise difficult-to-make mutants-larger deletions and insertions and mutagenesis on larger plasmids. The processes of mutagenesis can be completed quickly by a single restriction-ligation reaction.     

Rapid physical mapping by transposon Tn5 mutagenesis to localize the cloned yeast ILV2 gene

A rapid method for Tn5 mutagenesis of cloned genes on multicopy plasmids was used to map a yeast ILV2 mutant allele encoding a sulfometuron methyl-resistant acetolactate synthase. Twenty-one of 40 independent Tn5 insertions were within the 5.6-kilobase-pair cloned segment. Of these, seven adjacent transposition events inactivated the sulfometuron methyl resistance determinant, localizing the ILV2 gene to a minimum 1.4-kilobase-pair region.     

Plasmid-mediated expression of the UmuDC mutagenesis proteins in an Escherichia coli strain engineered for human cytochrome P450 1A2-catalyzed activation of aromatic amines.

The mutagenic actions of many chemicals depend on the activities of bacterial "mutagenesis proteins", which allow replicative bypass of DNA lesions. Genes encoding these proteins occur on bacterial chromosomes and plasmids, often in the form of an operon (such as umuDC or mucAB) encoding two proteins. Many bacterial strains used in mutagenicity testing carry mutagenesis protein genes borne on plasmids, such as pKM101. Our objective was to introduce mutagenesis protein function into Escherichia coli strain DJ4309. This strain expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase and carries out the metabolic conversion of aromatic and heterocyclic amines into DNA-reactive mutagens. We discovered that many mutagenesis-protein plasmids severely inhibit the response of strain DJ4309 to 2-amino-3,4-dimethylimid-azo[4,5-f]quinoline (MeIQ), a typical heterocyclic amine mutagen. Among many plasmids examined, one, pGY8294, a pSC101 derivative carrying the umuDC operon, did not inhibit MeIQ mutagenesis. Strain DJ4309 pGY8294 expresses active mutagenesis proteins, as shown by its response to mutagens such as 1-nitropyrene and 4-nitroquinoline 1-oxide (4-NQO), and is as sensitive as the parent strain DJ4309 to P450-dependent mutagens, such as MeIQ and 1-aminopyrene.     

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.     

Genetic analysis of an Agrobacterium tumefaciens strain producing an agrocin active against biotype 3 pathogens

Summary The successful biocontrol agent for crown gall disease, Agrobacterium radiobacter strain K84, is unable to protect grapevines from infection. We have identified a strain of Agrobacterium tumefaciens, J73, which produces an agrocin active both in vitro and in vivo against grapevine pathogens (Webster et al. 1986). We now report on the curing of this strain of its nopaline-type Ti plasmid and the location, by transposon mutagenesis, of the genes involved in the production of the agrocin. The Ti plasmid was cured by the introduction of selectable plasmids carrying the origins of replication of either the nopaline Ti plasmid, pTiC58, or the octopine Ti plasmid, pTi15955. Tn5 mutagenesis indicated that the genes responsible for agrocin production and/or export are located both on the chromosome and on a plasmid, pAgJ73, which co-migrated in agarose gels with pTiJ73. As the two plasmids were separable after transposon mutagenesis, we postulate that during or after mutagenesis of the agrocin plasmid, DNA rearrangements occurred between it and pTiJ73, resulting in an increase in size of pAgJ73. We provide evidence that the rearrangements involved the duplication of nopaline catabolism genes from pTiJ73 and their insertion into pAgJ73, which facilitated the resolution of the two plasmids. As expected pTiJ73 has homology with the nopaline Ti plasmid, pTiC58.