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。     

Primer Spanner:一个高效的定点突变PCR引物设计在线工具

基于PCR的实验策略在生物工程研究中具有广泛应用,如定点突变(site-directed mutagenesis,SDM),DNA拼接和载体构建。引物设计是这类实验技术中的关键一环,因其直接影响扩增效率和PCR产物的拼合。在嵌合式引物设计方法(一对突变引物在5'端具有互补序列)的基础上,开发了一个在线工具Primer Spanner(PS),可简单高效获得设计定点突变引物。PS可应用于单碱基或连续多碱基替换、插入、敲除等突变形式。通过大量突变实验与测序验证,结果表明该工具设计的引物进行的定点突变效果良好1)。     

改进重叠延伸PCR技术构建定点双突变

目的:目的DNA片段中快速构建位点不同的定点双突变体。方法: 借鉴DNA shuffling技术中DNA小片段延伸扩增获得全长DNA片段的工作原理,与常规基因定点突变技术相结合,改进重叠延伸PCR技术构建定点双突变。结果:对嗜酸热脂肪杆菌(Alicyclobacillus acidocaldarius)Tc-12-31的甘露聚糖酶基因AamanA中两个可能的活性位点E151和E231进行双点突变,先后经过无引物和有引物两步PCR,扩增获得全长DNA,测序结果表明得到预期的定点双突变体;酶活性检测和薄层层析结果表明双点突变体丧失了酶的活性。结论: 改良的重叠PCR技术,能经济、简便、高效地获得双点定点突变体,在酶的催化机理的阐述、蛋白质结构改造等分子生物学领域中具有较高的应用价值。     

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。     

PCR技术对人IL-3 cDNA进行定点突变的研究

本文利用PCR技术对人IL-3cDNA体外进行定点突变,将人IL-3cDNA第3位Met,第116位Lys密码子突变为Val密码子GTT。PCR扩增片段核苷酸序列与引物设计相应的cDNA突变体序列完全一致。结果证实此方法比经典寡聚核苷酸方法简单、迅速、成本低、效率高,也为基因的修饰,蛋白质工程研究提供了简便、稳定的方法。     

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。     

蛇毒锯鳞蝰素基因Leu14—Lys15—Glu16的定点突变

本研究的目是的利用蛋白质工程定点突变的方法,在蛇毒锯鳞蝰素基因分子上增加另一个保守序列ＲＧＤ（１４位精氨酸残基,１５位甘氨酸残基,１６位天氨酸残基）,以其增加该分子的生物活性,并探讨蛋白质一级结构,空间结构和功能的关系,在质粒ｐＪＣ６４的基础上,利用ＰＣＲ定点突变方法,对蛇毒锯鳞蝰素基因Ｌｅｕ１４－Ｌｙｓ１５－Ｇｌｕ１６进行定点突变,使相应的ＰＣＲ定点突变方法,对蛇毒锯鳞蝰素基因Ｌｅｕ１４－Ｌｙｓ     

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%。     

PSS定点突变及酶活研究

磷脂酰丝氨酸可在磷脂酰丝氨酸合成酶(PSS)催化下由磷脂酰胆碱和L-丝氨酸生成。通过生物信息学手段对磷脂酰丝氨酸合成酶蛋白质结构进行解析和研究分析,获得2个可突变位点F139和P272,通过Overlap PCR法进行定点突变,测定突变序列,并进行酶活测定。结果显示,PSS位点F139突变为L139、M139,位点P272突变为A272,能提高酶活力,说明蛋白质结构解析结合氨基酸定点突变技术,可以改善重组酶的活力。     

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。     

SORS: A Universal One-Round PCR-Based Method for Site-Directed Mutagenesis

We have developed a novel protocol for site-directed mutagenesis of double-stranded DNA. The procedure, termed SORS (named because it undergoes the sequential procedure of segmentation-overhang creating PCR-reannealing-splicing) mutagenesis, is exemplified by a substitution, a deletion, and an insertion of nucleotide(s) in target genes. The template DNA is PCR-amplified into two separate segments divided at the prospective mutation site, and each segment is amplified in two parallel PCRs using primers introducing the mutation. The primers are designed to be able to create protruding bases upon pooling, denaturing, and reannealing the two parallel reactions. The protruding bases at the prospective junction of the two segments are mutually complementary; therefore, the two segments can be re-spliced together to generate the mutated gene. Compared to previously published protocols, this procedure is rapid, restriction-independent and ensures higher success rate and lower potential to produce second-site mutations.     

病原菌TAL效应子与寄主靶基因相互识别的分子密码

黄单胞杆菌属TAL效应子类蛋白作为病原菌的毒性因子或无毒因子,能够与寄主靶基因DNA的启动子进行特异性识别,调控寄主的基因表达,引起致病或抗病反应。TAL效应子类蛋白识别靶基因DNA的模式,是2个氨基酸决定1个核苷酸的识别。这种新型的蛋白质-DNA互作方式有可能在基因治疗、植物抗病基因发掘、广谱抗病基因构建等生物医学工程和农业工程方面得到广泛应用。文中综述了TAL效应子类蛋白的发现及功能,TAL效应子与寄主靶基因识别的专一性及分子密码,并对该分子密码当前的应用现状及前景进行了讨论和展望。     

增强型青色荧光蛋白慢病毒表达载体的构建

目的：通过多点突变构建增强型青色荧光蛋白（ECFP）慢病毒表达载体。方法与结果：根据增强型绿色荧光蛋白（EGFP）和ECFP基因序列的差异设计3对引物,以pLentiLox3.7-EGFP为模板进行分段PCR扩增,再以分段PCR扩增产物为模板扩增出突变的ECFP基因片段,将其与载体连接,得到ECFP慢病毒表达载体pLentiLox3.7-ECFP,测序结果证实经过多点突变扩增的ECFP片段基因序列完全正确;磷酸钙介导pLentiLox3.7-ECFP在293T细胞中表达,48h后在荧光显微镜下观察到青色荧光蛋白。结论：通过多点突变的方法得到了ECFP慢病毒表达载体。     

K-Rasa^G12D基因突变体慢病毒载体的构建及其鉴定

目的：构建K-RasGl2D基因突变体慢病毒载体。方法：从病人组织中提取RNA通过RT—PCR反转录获得cDNA作为K-RasGl2D基因模板,通过PCR法扩增出K-RasGl2D基因突变体片段。将酶切的片段克隆入真核表达载体pCDH-CMV—MCS—EF1-RFP中,构建K-RasG12D基因突变体逆转录病毒真核表达载体。将连接产物转化至感受态大肠埃希菌DH5a,挑取转化平板上的细菌克隆,在抗生素培养液中培养过夜后进行PCR鉴定。经测序正确后转染293T细胞系,利用重组质粒PCR及串联基因表达的检测等方法对目的基因的转录与表达进行分析与鉴定。结果：所构建的K-RasGl2D突变体基因逆转录病毒真核表达载体经PCR鉴定和测序鉴定正确,转染293T细胞后可以观察到可检测到高强度表达的RFP荧光信号。结论：成功构建了重组真核表达载体,为下一步建立稳定转染细胞系及进一步研究K-Ras突变在癌症发病中的作用奠定了基础。     

牦牛病毒性腹泻病毒E2基因的克隆鉴定及原核表达

运用聚合酶链式反应,以牦牛BVDV基因组DNA为模板扩增出牦牛BVDV E2基因.为研究E2蛋白的抗原性,将E2基因插入到pET-32a原核表达载体,构建重组表达质粒pET-32a-E2,并转化至BL21(DE3)宿主菌中,利用IPTG诱导表达.经SDS-PAGE检测,pET-32a-E2在宿主菌BL21(DE3)中表...     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Red体内同源重组介导的egfp、kan基因融合和重组质粒构建

重组工程是近年来建立的一种基于高效率体内同源重组的新型遗传工程技术,可应用于靶DNA序列的敲入、敲除和基因克隆等。在应用重组工程技术进行基因亚克隆时发现,体外重叠PCR法难以获得高质量的目的DNA打靶片段,严重影响重组效率。为了解决上述问题,根据Red重组酶介导的体内同源重组工作原理进行了技术改进。先用PCR方法合成egfp和kan两条末端互补的线性DNA片段,然后将其电击共转化进入携带Red重组酶和pcDNA3．1载体DNA的大肠杆菌DY331菌株内,经体内同源重组直接产生的pcDNA3.1—egfp-kan环状重组质粒DNA分子可通过抗生素标记筛选获得,阳性率可达到45％。瞬时转染pcDNA3．1-egfp-kan可获得绿色荧光蛋白在293细胞中的表达。     

A genetic enrichment for mutations constructed by oligodeoxynucleotide-directed mutagenesis

A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free (sup degrees) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (-)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup degrees host we promote the propagation of heteroduplexes with the oligo-containing (-)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2-20% for single base changes and from 0.3-9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (-)strands.     

Recognition and cleavage of the bacteriophage P1 packaging site (pac). I. Differential processing of the cleaved ends in vivo

The packaging of bacteriophage P1 DNA into viral capsids is initiated at a specific DNA site called pac. During packaging, that site is cleaved and at least one of the resulting ends is encapsidated into a P1 virion. We show here that pac is located on a 620 base-pair fragment of P1 DNA (EcoRI-20). When that fragment is inserted into the chromosome of cells that are then infected with P1, packaging of host DNA into phage particles is initiated at pac and proceeds down the chromosome, unidirectionally, for about five to ten P1 "headfuls" (about 5 X 10(5) to 10 X 10(5) bases of DNA). Using an assay for pac cleavage that does not depend on DNA packaging, we have identified a set of five amber mutations that are mapped adjacent to pac, and that define a gene (gene 9) essential for pac cleavage. Amber mutations that are located in genes necessary for viral capsid formation (genes 4, 8 and 23), or in a gene necessary for "late" protein synthesis (gene 10), do not affect pac cleavage. The latter result suggests that the synthesis of the pac cleavage protein is not regulated co-ordinately with other phage morphogenesis proteins. The products of pac cleavage were analyzed using two different DNA substrates. In one case, a single copy of pac was placed in the chromosome of P1-sensitive cells. When those cells were infected with P1, we could detect the cleavage of as much as 70% of the pac-containing DNA. The pac end destined to be packaged in the virion was detected five to 20 times more efficiently than was the other end. Since this result is obtained whether or not the infecting P1 phage can encapsidate the cut pac site, the differential detection of pac ends is not simply a consequence of one end being packaged and the other not. In a second case, pac was located in cells on a small (5 X 10(3) bases) multicopy plasmid. When those cells were infected with P1, neither pac end was detected efficiently after P1 infection, unless the cells carried a recBCD- mutation. In recBCD- cells, the results with plasmid-pac substrates were similar to those obtained with chromosomally integrated pac substrates. We interpret these results to mean that, following pac cleavage, the end destined to be packaged is protected from cellular nucleases while the other end is degraded by the action of at least two nucleases, one of which is the product of the host recBCD gene.(ABSTRACT TRUNCATED AT 400 WORDS)