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。     

部分扩增结合双片段连接法克隆系列截短基因突变体

体外合成DNA伴随的随机突变是制约基因定点突变效率的重要因素。以克隆周期蛋白E（cyclin E）及其截短基因的激酶活性缺失突变体为例,对传统重叠延伸PCR（overlap extension PCR,OE-PCR）作适当改进,提出一种系列相关基因定点突变的优化方案。前期研究中已克隆了cyclin E基因及其2个截短基因T1、T2,并通过EcoRⅠ/SalⅠ双酶切插入pEGFP_C2载体。限制性酶切分析发现cyclin E基因序列中含有1个AgeⅠ位点将其分为F1、F2两段,目标突变位点KD位于F2段。对于cyclin E及其截短基因,F2段是完全一致的。因此,通过重叠延伸PCR扩增含突变位点的共有片段F2,从C2-cyclin E、C2-cyclin E_T1和C2-cyclin E_T2这3个原始质粒中切取相应的F1片段,再将F1与酶切的F2一起连接插入载体以重构完整突变体。对比检测发现OE-PCR扩增较短DNA片段更易成功。C2-cyclin E、C2-cyclin E_T1和C2-cyclin E_T2这3组均能筛选出一定数量克隆,经检测和序列鉴定,每组各得到1个序列完全准确的目标突变体。研究表明,采用部分扩增可以缩短DNA合成长度,避免了目标基因反复扩增等不利因素,从而减少随机突变;双片段连接避免了双AgeⅠ位点对常规酶切-连接的限制。两者相结合,可作为其他系列相关基因定点突变的优化方案。     

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。     

改进的多片段重叠延伸PCR制作基因多位点突变

为在研究工作中提高制作目标基因多位点突变体的效率,对常规重叠延伸PCR进行适当改进:对于相距较近的两个突变位点,只需设计一对突变引物各自涵盖其中一个位点即可一次突变;对于相距较远的两个位点,可以采用三片段重叠延伸PCR的办法解决;两者结合则可一次性突变多个位点。以制作RBCT的6位点突变体PSM6为例,利用上述策略,设计两对突变引物;采用OE-PCR法,第一轮PCR扩增出三个片段,第二轮PCR同时利用三片段重叠延伸产物作为模板扩增出目标基因突变体,再按常规分子克隆方法将其连入质粒载体。经测序检测,发现得到了预期的目标基因多位点突变体。因此,采用灵活的引物设计策略,结合多片段重叠延伸PCR即可一次性制作基因的多位点突变体,此方案可解决研究工作中大多数多位点突变问题。     

基于重叠延伸PCR法的定点突变技术

目的:建立一种高效而经济的定点突变方法。方法:采用重叠延伸PCR定点突变技术,引物设计时引入目的突变,以前两次PCR产物为模板,进行第三次PCR,即可获得突变后的目的DNA片段。将此片段连入pMDTM18-T载体后测序验证突变结果。结果:DNA测序表明,待突变位点已由ATTGG突变为ATTTT。结论:成功实现了目的位点的定点突变,重叠延伸PCR法是一种高效且经济的定点突变方法。     

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。     

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。     

一种快速获得基因密码子偏爱性改造的方法-SOE-PCR

SOE-PCR采用具有互补末端的引物,使PCR产物形成重叠链,在不需要内切酶消化和连接酶处理的条件下实现DNA片段的拼接,能够高效、快速地实现基因的定点突变。应用SOE-PCR原理成功地实现了对几丁质酶基因chi58的5个位点的突变,构建了几丁质酶基因密码子偏爱性改造突变体-chi58A。实验证明,SOE-PCR具有简捷、快速、高效的优点。扩增过程中未引入其他突变,获得的chi58A突变体基因片段可以用于后续的分子克隆。     

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。     

重叠延伸PCR对DNA片段进行定点双突变

探讨如何利用重叠延伸PCR对同一靶DNA片段中的两个不同位点实施联合突变。先用野生型DNA作模板,通过一轮重叠延伸PCR,获得突变一个预期位点的DNA片段,再用此突变DNA片段作模板,通过另一轮重叠延伸PCR获得两个预期位点均突变的DNA片段。重叠延伸PCR能对DNA片段进行双突变甚至多点突变,具有简便、快速、经济等特点,在阐明基因的调控机理、改造蛋白质结构等分子生物学领域中具有极大的应用价值。     

Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.     

Lambda Chops: Creation of Site-Directed Mutants in Insertable Fragments Utilizing Gateway<Superscript>®</Superscript> Technology

We describe a method to produce site-directed mutations anywhere within cDNA by assembling mutagenized PCR fragments in proper orientation using lambda integration in an extension of Gateway technology to yield a full-length mutated gene. This process exploits the directionality of lambda insertion sequences ensuring integration and directionality of PCR product into a cloning vector. The process requires only two sequential integration steps to yield a mutagenized expression vector. Mutagenized vasodilator associated phosphoprotein (VASP) was produced by generating two PCR fragments representing the upstream and downstream portions of the gene, substituting alanine or glutamate residues for VASP serine239. The upstream PCR was engineered with attB1 lambda integration sequences at the 5′ region and attB2 at the 3′ region of the downstream fragment to ensure correct orientation. The desired mutation was encoded by the forward primer of fragment 2. The reverse primer of the fragment 1 was phosphorylated for subsequent ligation. Vent polymerase provided sequence accuracy and blunt-ended product. The first integration into a donor vector, catalyzed by BP Clonase II created a linear product circularized by blunt end ligation, yielding hundreds of entry vectors containing the mutagenized VASP. A second integration into destination vector yielded plasmid expressing mutant VASP upon transfection.     

Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA.

This paper presents a versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA fragments cloned into M13 derived vectors. As an example, production of a transition mutation in a clone of the yeast MATa1 gene is described. The oligonucleotide is hybridized to the template DNA and covalently closed closed double stranded molecules are generated by extension of the oligonucleotide primer with E. coli DNA polymerase (large fragment) and ligation with T4 DNA ligase. The resulting double stranded closed circular DNA (CC-DNA) is separated from unligated and incompletely extended molecules by alkaline sucrose gradient centrifugation. This purification is essential for production of mutants at high efficiency. Competent E. coli JM101 cells are transformed with the CC-DNA fraction and single stranded DNA is isolated from individual plaques. The recombinants are screened for mutant molecules by 1) restriction endonuclease screening for the loss of the Hinf I site in the target region, and 2) by dot blot hybridization using the mutagenic oligonucleotide as probe. Double stranded DNA is isolated from the sequencing. Efficiency of mutant production is in the range of 10-45% and no precautions to prevent mismatch repair are required.     

Detection of nonsense and frameshift mutations in <Emphasis Type="Italic">BRCA1</Emphasis> gene using a new plasmid vector pPhoA-frame

The technique for detecting frameshift and nonsense mutations in the human BRCA1 gene has been suggested. The technique presumes the construction of recombinant plasmids where the tested DNA fragment is placed in frame with alkaline phosphatase gene of Escherichia coli (phoA). A special plasmid pPhoA-frame was constructed for this analysis, and the plasmid contained the DNA fragment that encodes alkaline phosphatase of E. coli. The synthetic DNA fragment with BglII, StuI, ApaI and SacII sites was inserted into the DNA fragment that encodes alkaline phosphatase of E. coli between Ala218 and Gly219 codons to facilitate the cloning of BRCA1 gene fragments. The occurrence of the frameshift or nonsense mutation in the tested DNA fragment can be detected after the transformation of E. coli by the recombinant plasmid that contains the tested fragment. E. coli colonies with newly constructed recombinant plasmids are plated out on the indicator agar. In the case of the frameshift or nonsense mutation, the colonies are not colored, and DNA fragments without these mutations result in the formation of blue colonies.     

Optimized PCR fragments for heteroduplex analysis of the whole human mitochondrial genome with denaturing HPLC

Denaturing high pressure liquid chromatography (dHPLC) is an efficient method for discovery of unknown mutations by heteroduplex analysis of PCR fragments. For comprehensive mutation scanning of the whole 16.569 bp human mitochondrial genome, we developed a set of 67 primer pairs defining overlapping PCR fragments that are well suited for heteroduplex analysis. The aim of our optimization efforts was to ensure that point mutations are detectable at every nucleotide position of each amplicon. Some GC-rich regions of mitochondrial DNA (mtDNA) were found to have unfavourable melting profiles in all possible amplicons, therefore requiring GC-clamps at the end of one or both oligonucleotide PCR primers. Following detection of a heteroduplex pattern by dHPLC, our primers can also be employed for DNA sequencing to identify the underlying mutation. In case of heteroplasmic mutations with a low proportion of mutant mtDNA, a fragment collector is useful to recover the heteroduplex peak, which contains mutant and wildtype DNA molecules in a 1:1 ratio.     

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.     

Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5′ end of the PCR primer and the extended newly synthesized DNA 3′ end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by ‘selection marker swapping’ upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.     

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR

Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair. In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy. We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer. This method is used for rapid analysis of mutational spectra generated in nuclease-defective strains of Saccharomyces cerevisiae to elucidate the role of RNase H(35) in RNA primer removal during DNA replication and in mutation avoidance. Strains deficient in RNH35 displayed a distinct spontaneous mutation spectrum of deletions characterized by a unique 4 bp deletion in a lys2-Bgl allele. This was in sharp contrast to strains deficient in rad27 that displayed duplication mutations. Further analysis of mutations in a rnh35/rad27 double mutant revealed a mixed spectrum. These results indicate that RNase H(35) may participate in a redundant pathway in Okazaki fragment processing and that mutational spectra caused by protein deficiencies may be more intermediate-specific than pathway-specific.