用于聚合酶链式反应(PCR)引物设计的计算机程序

 本文报道了两个用于PCR引物设计的计算机程序PCRDESN和PCRDESNA。PCRDESN程序主要从以下4个方面评价用户自己设计的一对引物的质量:(1)引物内的碱基反向重复或发夹结构,(2)两个引物之间的碱基互补配对,(3)两个引物之间的同源性,(4)引物的碱基组成及特点和T_m值计算。通过用多例文献发表的及本院有关实验室提供的引物对序列的验证,确定了程序的运算参数,证明该程序能较好地检验引物对的质量和解释某些PCR实验失败的原因。PCRDESNA程序采用逐级优化的方法和比PCRDESN所选用的更严紧的引物选择参数对用户提供的核酸序列进行快速检索,以确定所有可能的和合适的引物对。     

兔防御素(MCP-1)cDNA的克隆与序列分析

从兔脾脏细胞中分离提取总RNA,经反转录PCP(RT-PCR)扩增出兔巨嗜细胞阳离子多肽(MCP-1)cDNA,插入经EcoRI和XbaI双酶切的pUC19中,构建了克隆质粒pUCDEF.进行了限制性酶切鉴定和序列分析,结果在扩增出的cDNA288个碱基中,在前片段中有一个碱基与发表的兔MCP-1cDNA序列不同,即第157位碱基由G变为A,导致编码的氨基酸由丙氨酸变为苏氨酸.该cDNA全长288bp,编码94个氨基酸,由编码信号肽,前片段及成熟肽片段的序列组成.     

汉坦病毒HTN261株S基因片段的核苷酸序列测定及分析

黑龙江省是肾综合征出血热(HFRS)的重疫区。近年来HFRS年的发病人数曾超过万人。流行病学和血清学研究表明黑龙江省HFRS疫区主要是姬鼠型,但目前尚缺乏病毒的分子生物学资料。我们对从疫区捕获的宿主动物-黑线姬鼠肺中分离的汉坦病毒HTN261株的S基因片段的全基因序列进行了测定和初步分析。结果如下,HTN261株的S基因片段的全序列长为1697nt。只有一个主要的编码N蛋白的ORF,起始位置为第37nt,终止于1326nt,编码的蛋白长为429aa。没有发现存在ORF2。HTN261株的S基因片段核苷酸序列与HTN型中的病毒株的同源性很高,而与汉坦病毒其他型的同源性较差。从种系发生树分析来看,HTN261株归结于汉坦病毒的HTN型。在HTN型之内,HTN261株和HTN76-118株在一个分枝内。就其核苷酸和蛋白的同源性来说,HTN261株和HTN76-118株的同源性分别是89%(全S基因)和98%(蛋白)。而与中国境内发现的其他汉坦病毒株Z10,HU,Chen4,NC167等基因和蛋白的同源性相对较差。汉坦病毒除具有其宿主的依赖性外,还具有其地理的簇集性。HTN261株和HTN76-118株之间S基因和N蛋白序列的变异性的差异分别为11%和2%,表明HTN261株和HTN76-118株还有不同,可能是不同的亚型。不过,尚有待于进一步研究证明。     

大鼠脑α_1型甲状腺激素受体基因的cDNA克隆及序列分析

使用ＲＴ－ＰＣＲ方法克隆了Ｗｉｓｔａｒ大鼠脑α_１型甲状腺激素受体的ｃＤＮＡ,得到包含起始及终止密码子共１２３３ｂｐ、编码４０９个氨基酸的受体全长编码序列．酶切分析后,将此特异ＤＮＡ片段重组入质粒ｐＵＣ系统,得重组质粒ｐＴＲＡ．双脱氧末端终止法测定了全部核苷酸顺序,结果与文献报导的ＳＤ大鼠的结果一致,同时对长片段ＤＮＡ的ＲＴ－ＰＣＲ扩增进行了方法学的探讨。     

HGV广东株和香港株NS5区部分基因的克隆及序列分析

采用ＨＧＶＮＳ５特异的２对引物,对两个香港株和一个广东株ＨＧＶＲＮＡ进行逆转录套式ＰＣＲ扩增,ＰＣＲ产物克隆入ｐＵＣ１９,重组质粒转化ＤＨ５α和ＪＭ１０９菌株。ＰＣＲ和酶切法鉴定阳性克隆,双脱氧链末端终止法测定核苷酸序列并进行同源性分析。结果发现核苷酸变异呈散在分布,三株间核苷酸和氨基酸序列同源性分别为９３．３％～９４％及９７％～９９．２％,与已报道的中国株（ＣＮ）相比,则同源性分别为９０％～９１．２％和９４％～９６．３％,与美国株（ＰＮＦ２１６１及Ｒ１０２９１）相比,为８７．１％～８９．５％和９５．２％～９７％,而与西非株（ＧＢＶＣ）相比,则达９１．４％～９３．８％和９７％～９７．９％。提示ＨＧＶＮＳ５区核苷酸和氨基酸序列相对保守,不同ＨＧＶ株存在一定的地区差异。     

HGV广东株和香港株NS5区部分基因的克隆及序列分析

采用HGV NS5特异的2对引物,对两个香港株和一个广东株HGV-RNA进行逆转录套式PCR扩增,PCR产物克隆入pUC19,重组质粒转化DH5α和JM109菌株.PCR和酶切法鉴定阳性克隆,双脱氧链末端终止法测定核苷酸序列并进行同源性分析.结果发现核苷酸变异呈散在分布,三株间核苷酸和氨基酸序列同源性分别为93.3%～94%及97%～99.2%,与已报道的中国株(CN)相比,则同源性分别为90%～91.2%和94%～96.3%,与美国株(PNF2161及R10291)相比,为87.1%～89.5%和95.2%～97%,而与西非株(GBV-C)相比,则达91.4%～93.8%和97%～97.9%.提示HGV NS5区核苷酸和氨基酸序列相对保守,不同HGV株存在一定的地区差异.     

小菜蛾化学感受蛋白基因PxylCSP1的克隆和表达

利用RT-PCR和RACE技术克隆到小菜蛾Plutella xylostella 化学感受蛋白（CSP）基因PxylCSP1（GenBank登录号: FJ361903）,其核苷酸序列全长405 bp,编码134个氨基酸残基,预测N-末端包含19个氨基酸组成的信号肽序列,估测其成熟蛋白分子量为13.56 kD,等电点为6.12。该基因编码氨基酸序列和其他鳞翅目昆虫CSP的氨基酸序列比对同源性较高（70%~80%）。RT-PCR结果表明PxylCSP1不仅存在于小菜蛾的触角中,还存在于头、足、腹和翅中。Real-time PCR结果表明PxylCSP1的表达水平因被测小菜蛾的性别、日龄、组织不同和交配与否而异。     

蛇毒类凝血酶Calobin cDNA的设计合成与克隆

人工合成了朝鲜蝮蛇(Agkistrodon cnliginosus)类凝血酶Calobin的编码序列。在设计引物时选择大肠杆菌和酵母茵的偏爱密码子,通过相互搭桥的方式,将全长为789bp的编码序列分成14个片段,每个片段长度为78个碱基左右,采用“核心模板法”并加以改进,通过4次PCR延伸反应,得到了全长基因。经扩增后回收目的片段,双酶切后连接到克隆载体,将得到的转化子酶切鉴定后,任意选择6个克隆进行双向测序,得到了全序列正确的3个克隆。本工作为类凝血酶在大肠杆菌和酵母系统中的高效表达奠定了基础,也为长度为700-900bp片段的合成提供了帮助。     

甘蓝夜蛾几丁质酶基因cDNA序列的克隆与序列分析

昆虫几丁质酶在害虫生物防治中具有很大的发展潜力。以甘蓝夜蛾Mamestra brassicae L.预蛹期幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),扩增得到其几丁质酶的cDNA序列。该序列含有2826个碱基,包括1个1689个碱基的开放阅读框,预测编码1个含562个氨基酸的多肽,分子量约为62.6kDa,等电点为5.30。推导得到的氨基酸序列含有2个N-位糖基化位点,22个O-位糖基化位点,氨基酸序列与其他昆虫,尤其是鳞翅目昆虫的几丁质酶高度同源。获得的甘蓝夜蛾几丁质酶基因cDNA序列已经登录GenBank并获得登录号FJ436415。     

人干细胞因子cDNA的设计、合成与克隆

选择大肠杆菌的偏爱密码子,分成18个长约60个碱基且相互配对的寡核苷酸片段,合成了可溶形式的人干细胞因子（hSCF)的cDNA,合成片段经纯化、5′-端磷酸化后,通过PCR进行合成hSCF的cDNA一次性组装与扩增,得到了含有可溶形式的hSCF的全部编码区及EcoRⅠ、SalⅠ位点在内的全长共515个碱基对的DNA片段,回收该DNA片段,酶切消化,定向克隆进质粒pUC19。经酶切鉴定以及序列测定,得到了全序列正确的克隆株,为下一步的工作奠定了基础。     

Determination of RAPD Markers in Rice and their Conversion into Sequence Tagged Sites (STSs) and STS-Specific Primers

We produced 102 randomly amplified polymorphic DNA (RAPD) markersmapped on all 12 chromosomes of rice using DNAs of cultivarsNipponbare (japonica) and Kasalath (indica) and of F2 populationgenerated by a single cross of these parents. Sixty random primers10 nucleotides long were used both singly and in random pairsand about 1,400 primer-pairs were tested. Using both agarosegel and polyacrylamide gel electrophoresis enabled us to detectpolymorphisms appearing in the range from <100 bp to 2 kb.The loci of the RAPD markers were determined onto the frameworkof our RFLP linkage map and some of these markers were mappedto regions with few markers. Out of the 102 RAPD markers, 20STSs (sequence-tagged sites) and STS-specific primer pairs weredetermined by cloning, identifying and sequencing of the mappedpolymorphic fragments.     

HCV基因组NS1区的分子克隆及序列测定

对广东省一名慢性丙型肝炎病人血清中的ＨＣＶ基因组ＮＳ１区进行分子克隆及序列测定。采用微粒吸附法提取ＨＣＶ ＲＮＡ,随机引物逆转录后进行聚合酶链反应。所用引物位于ＮＳ１区,扩增产物７８０ｂｐ在低熔点琼脂糖中电泳,加嘏相应条带处凝胶,与ｐＵＣ１８的连接批应直接在低熔点琼脂糖中完成。重组体转化ＪＭ１０９,挑取菌落增殖后提取的质粒采用ＰＣＲ和酶切法鉴定阳性克隆。将其中３２０ｂｐ的片段亚克隆到ｐＵＣ１８和ｐ     

Restriction Site-dependent PCR: An Efficient Technique for Fast Cloning of New Genes of Microorganisms

New bioactive proteins need to be screened from various microorganismsfor the increasing need for industrial and pharmaceutical peptide,proteins, or enzymes. A novel polymerase chain reaction (PCR)method, restriction site-dependent PCR (RSD-PCR), was designedfor rapid new genes cloning from genomic DNA. RSD-PCR strategyis based on these principles: (i) restriction sites dispersethroughout genomes are candidacy for universal pairing; (ii)a universal primer is a combination of a 3'-end of selectedrestriction sites, and a 5'-end of degenerated sequence. A two-roundPCR protocol was designed and optimized for the RSD-PCR: amplifythe single strand target template from genomic DNA by a specificprimer and amplify the target gene by using the specific primerand one of the universal RSD-primers. The optimized RSD-PCRwas successfully applied in chromosome walking using specificinternal primers, and cloning of new genes using degeneratedprimers derived from NH₂-terminal amino acid sequence of protein.     

A PCR-Mediated Method for Cloning Spot DNA on Restriction Landmark Genomic Scanning (RLGS) Gel

A PCR-mediated direct cloning for target spot DNA from RLGSgel has been established. The method consists of PCR amplificationof adaptor-ligated spot DNA fragments without excluding similar-sizedDNA fragments co-localized on RLGS gel, and following selectiveligation with the NotI-dT vector. Applying this method, we havesuccessfully cloned several DNA fragments derived from targetspots whose intensities change developmentally due to DNA methylationin the telencephalon of C3H/HeN mice. Since only a few microgramsof total DNA is sufficient for our spot cloning, our methodmay be highly useful when the total DNA sample prepared forcloning is limited.     

合成生物学中不依赖限制性内切酶的分子克隆策略

随着合成生物学的兴起和发展,基因克隆和DNA大片段组装成为了常规操作。利用人工智能和液体操作机器人进行高通量的DNA组装和功能筛选已被广泛应用。传统的依赖于限制性内切酶识别位点的克隆技术对序列有选择性、步骤繁琐、实验人员的培训周期长,不利于以流水线形式进行工程化使用,已经逐步在生物工程领域内被淘汰。文中论述了一系列适于机械化操作的新一代分子克隆技术,即不依赖基因序列和连接反应克隆方法、Gibson组装、聚合酶环形延伸克隆、细胞裂解物体外无痕连接和细胞体内组装克隆。对这些方法的建立、基本原理及应用前景等方面进行了总结,并对其优缺点进行了比较。     

Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

Assessment and modification of degenerate qPCR primers that amplify functional genes from etheneotrophs and vinyl chloride-assimilators

Aims: Degenerate qPCR primer sets that target the functional genes etnC and etnE in etheneotrophs and vinyl chloride‐assimilating bacteria were assessed and modified in an effort to improve performance. Methods and Results: Functional gene abundance in four pure cultures was estimated by qPCR using novel (MRTC and MRTE) and existing (RTC and RTE) degenerate primer sets and compared to abundances estimated with nondegenerate gene‐specific primers (GSPs). Functional gene abundance in groundwater DNA extracted from several contaminated sites was also estimated with MRTC and MRTE primers. Conclusions: MRTC primers displayed significantly improved etnC quantification in both pure cultures and environmental samples. Significance and Impact of the Study: Application of MRTC and MRTE primer sets will enhance microbial ecology studies involving etheneotrophs and qPCR analyses that support vinyl chloride bioremediation strategies.     

The Sequence of the Hartmannella vermiformis Small Subunit rRNA Coding Region

ABSTRACT The Hartmannella vermiformis small-subunit rRNA coding region was amplified, and the amplified DNA was cloned and sequenced. The coding region is 1,840 nucleotides long, and is typical of eukaryotic rRNA genes in both size and composition. Different clones contained different nucleotides at three positions.