木瓜凝乳酶基因的克隆及序列分析

通过设计一对特异性的引物,采用RT－PCR方法从未成熟的木瓜组织中扩增得到木瓜凝乳酶基因,并将其重组到pPIC9K载体中,转化大肠杆菌并筛选阳性克隆,序列测定并利用BLAST软件进行核酸及氨基酸序列相似性分析,结果表明：通过序列组成及特征结构分析,扩增得到的基因为木瓜凝乳酶基因。     

猪FGL2基因cDNA末端序列检测及结构分析

检测猪FGL2基因cDNA末端序列并对该基因结构初步分析。α-32P dCTP放射性同位素标记cDNA探针筛选猪基因组DNA文库;cDNA末端快速扩增(rapid amplification of cDNA end,RACE)。以猪正常小肠及心脏组织提取新鲜总RNA,反转录后作为模板,设计基因特异性引物,采用Advantage 2 聚合酶混合物进行PCR扩增;依据猪与人FGL2基因3′端已知同源序列设计PCR上游引物,以人FGL2基因3′末端序列设计下游引物,以猪基因组DNA为模板采用Advantage 2 聚合酶混合物进行PCR反应;PCR载体重组质粒DNA亚克隆扩增。同位素探针未能筛选到特异阳性克隆,RACE反应检测到特异性转录起始位置及第一个转录终止位置,但仍未检测到第二个转录终止位置。猪基因组DNA行PCR扩增成功检测到猪FGL2基因3′末端未知序列及第二个转录终止位置。     

PCR-mtDNA技术鉴别检测不同动物肌肉组织和饲料中鸭源性成分

以鸭肌肉组织DNA为模板,利用PCR-mtDNA技术成功克隆出了鸭mtDNA COIII基因(GenBank Accession No.DQ655706).对所克隆的序列分析表明.其序列包括鸭细胞色素C氧化酶III(COIII)基因全序列784 bp,通过同源性分析可知,动物的线粒体DNA COIII基因是相对保守的,利用此特性设计PCR-mtDNA方法鉴别检测鸭源性成分的特异性引物;以各种动物肌肉组织及饲料DNA为模板进行PCR扩增、经反复验证筛选出只能扩增出鸭DNA的目的片段,而不能扩增出其他动物DNA片段的特异性强、稳定性好的引物P3、P4;利用此引物PCR扩增鸭DNA的特异性片段为226 bp,对PCR产物进行测序分析可知与已克隆的鸭mtDNA COIII基因同源性达到100%,证明了所筛选引物的准确性.通过对不同含量的DNA模板溶液进行PCR扩增的方法,对筛选出的特异性引物P3、P4进行灵敏度试验,结果分析表明灵敏度约为0.001%,证明该PCR方法具有特异性强、灵敏度高的特点,完全可作为鉴别不同动物肌肉组织和饲料中鸭源性成分的方法.     

华癸中生根瘤菌质粒复制基因repC的克隆与鉴定

摘要：【目的】repC为质粒复制必需的起始蛋白基因。本研究旨在对华癸中生根瘤菌菌株HN3015及其质粒消除突变株进行repC基因的克隆和鉴定。【方法】采用通用引物RC1和RC3进行repC基因的PCR扩增,扩增产物克隆到载体pMD-18T,然后测序。利用Southern 杂交对repC基因定位。利用在线软件分析基因的序列特征,BLAST 工具进行同源性搜索;ExPASy推断其氨基酸的序列;ClustalW进行同源核苷酸和氨基酸序列的多重比较分析;PredictProtein 进行蛋白二级结构分析。【结果】     

绵羊胚胎性别鉴定试剂盒的研究

根据绵羊Y-染色体的特异序列和常染色序列分别设计了确定公羊Y-染色体特异序列的3对特异性引物和内标基因的4对特异性引物。单重PCR扩增绵羊基因组DNA,筛选出了3对Y-染色体特异引物和3对羊DNA特异内标引物。将不同的绵羊Y-染色体特异引物与内标引物组合,利用多重PCR扩增绵羊基因组DNA,筛选出了1个可用于羊早期胚胎性别鉴定的PCR引物组合:A0/C1。按照最优PCR扩增DNA条件配制了绵羊PCR性别鉴定试剂盒并成功应用于绵羊血液、已知性别的绵羊成纤维细胞和胚胎,表明本研究建立的体系完全可用于绵羊早期的胚胎性别鉴定。     

基于RefSeq的人类基因荧光定量PCR引物库的构建

利用NCBI提供的RefSeq序列,通过BLAT、Sim4和自主开发的剪接比对程序Ealter1.0对人类RefSeq转录本进行外显子预测,根据预测的外显子信息,采用自主开发SYBR Green I Real Time PCR引物设计程序E-qPCR-Design1.0高通量设计21,118对SYBR Green I Real Time PCR引物,同时选取5000条基因进行SYBR Green I Real Time PCR引物验证,95.92%的基因引物取得良好效果,1.64%的基因引物产生引物二聚体,1.08%的基因引物有非特异性扩增,通过生物信息技术分析与实验验证,建立了基于RefSeq的人类基因荧光定量PCR引物库。     

中国农大小型猪胰淀素基因部分未知序列扩增与分析

目的扩增中国农大小型猪IAPP基因序列,分析其序列结构特点。方法提取中国农大小型猪血液基因组DNA,设计3对特异性引物进行PCR扩增,产物鉴定、测序和同源性比对分析。结果成功扩增出中国农大小型猪IAPP基因的1028bp的序列。结论经同源性比对分析显示,中国农大小型猪与人的IAPP基因的同源性为77%。     

内含子中正筛选标记neo基因在转录中的剪切研究

目的:研究插入内含子中的正筛选标记基因neo在转录中的剪切情况。方法:克隆了猪血清白蛋白基因5'端调控序列,以猪基因组DNA为模板,P10/P11为引物,PCR扩增猪血清白蛋白基因翻译终止密码子后2.9kb的3'端调控序列;以pEGFP-1为模板,P400/P401为引物PCR扩增绿色荧光蛋白(EGFP)基因,插入猪血清白蛋白基因5'端调控区之后,在3'端调控序列的内含子中靠近N端的序列中插入正筛选标记基因neo,构建了表达EGFP的真核表达载体pEXp11。转染人肝癌细胞系HepG2,通过G418药物筛选获得稳定转染的抗药性细胞克隆。提取抗性细胞克隆基因组RNA并进行反转录,获得cDNA序列。结果:用引物D400/D401及分别位于neo基因和3'端调控序列上的一对引物D394/D357进行PCR检测,其中D394/D357并未扩增出目的条带。结论:插入内含子中的neo基因在转录过程中可随内含子一起被剪切。     

小菜蛾酯酶基因的克隆

根据已知的酯酶基因的保守性氨基酸序列设计简并引物 ,通过逆转录 -聚合酶链反应 (RT PCR)扩增出小菜蛾PlutellaxylostellaL .酯酶基因片段 ,然后按照测序结果再设计 1对特异引物 ,利用PCR方法 ,筛选小菜蛾的cDNA文库。将RT PCR获得的 1条长度为 3 3 0bp的目的条带 ,亚克隆入T -载体 ,测序结果表明共得到了 1 0个不同的酯酶基因片段。利用特异引物对小菜蛾的cDNA文库进行初筛 ,显示文库中存在有小菜蛾的酯酶基因。     

链霉菌看家基因引物的设计与验证

看家基因的扩增与测序是进行多基因系统进化分析首先需要解决的问题。针对链霉菌这一群高(G C)mol%革兰氏阳性细菌,选定4个看家基因:atpD、recA、rpoB和trpB,利用NCBI数据库中已有的2个链霉菌和3个分枝杆菌的全基因组序列,以及另两个链霉菌的recA基因序列,通过软件分析设计了各基因的扩增和测序引物,并优化了扩增反应条件。从所试验的55株链霉菌中,均特异地扩增出了上述4个基因的片段,并成功进行了序列测定,验证了所设计引物的实用性。所归纳的引物设计方法可用于高(G C)mol%革兰氏阳性细菌的其它看家基因,以促进多基因系统进化研究的开展。     

人肾高亲和力钠离子依赖性二羧酸转运蛋白基因的克隆和生物信息学分析

为探讨人高亲和力钠离子依赖性二羧酸转运蛋白 (humanhigh affinitysodium dependentdicar boxylatetransporter,hSDCT2orhNaDC3 )基因在人体内的生理功能及其与疾病的关系 ,借助生物信息学成功地从人肾中克隆了hSDCT2基因 (GenBank接收号 :AY0 72 810 ) .首先将大鼠SDCT2cDNA与人EST数据库进行同源性比较 ,获得具有高度同源性EST片段并用DNAstar软件将它们拼接成EST重叠群 .在重叠群上设计PCR引物从人肾总RNA中用RT PCR扩增出hSDCT2基因并测序 ,然后用软件对其结构特性、组织分布及基因定位进行分析 .序列测定结果显示 ,hSDCT2开放阅读框为180 9bp ,共编码 6 0 2个氨基酸 .蛋白同源性分析表明 ,其氨基酸序列与大鼠及小鼠SDCT2分别有85 %和 87%相同 .二级结构分析显示 ,该蛋白有 12个跨膜螺旋区 .Northern分析显示 ,该基因可在肾、肝、脑、胎盘等多种组织中表达 ,并定位于 2 0号染色体的q12～q13 1     

耐辐射球菌转录因子DdrO 的基因克隆与生物信息学分析

目的:克隆耐辐射球菌ddrO基因,并对其进行生物信息学分析,预测其功能。方法:根据耐辐射球菌ddrO基因序列,由Primer Premier 5设计一对引物,以提取的耐辐射球菌基因组为模板,PCR扩增获得耐辐射球菌ddrO基因,序列测定并利用生物信息学软件对ddrO基因的理化性质、高级结构及生物学功能等进行分析与预测。结果:成功获得了ddrO基因。生物信息学分析发现,ddrO基因核苷酸序列长度为396bp,编码一个131aa组成的相对分子质量为14.993kD的预测的DdrO转录因子。核酸同源性搜索及比较分析仅在与耐辐射球菌同属的Deinococcus geothermalis和Deinococcus deserti中发现高度相似的序列;蛋白同源性搜索发现一些与DdrO显著同源的蛋白,如Deide₂0570（95%）,Dgeo₀336（90%）,Deide₃p02170（82%）等;结构域分析发现DdrO含有HTH（helix-turn-helix）DNA结合结构域。结论:根据生物信息学结果预测DdrO蛋白可能具有转录调控作用,参与DNA修复和复制,在耐辐射球菌的DNA损伤修复过程中发挥一定作用。     

EasyExonPrimer

EasyExonPrimer is a web-based software that automates the design of PCR primers to amplify exon sequences from genomic DNA. EasyExonPrimer is written in Perl and uses Primer3 to design PCR primers based on the genome builds and annotation databases available at the University of California, Santa Cruz (UCSC) Genome Browser database (http://genome.ucsc.edu/). It masks repeats and known single nucleotide polymorphism (SNP) sites in the genome and designs standardised primers using optimised conditions. Users can input genes by RefSeq mRNA ID, gene name or keyword. The primer design is optimised for large-scale resequencing of exons. For exons larger than 1 kb, the user has the option of breaking the exon sequence down into overlapping smaller fragments. All primer pairs are then verified using the In-Silico PCR software to test for uniqueness in the genome. We have designed >1000 pairs of primers for 90 genes; 95% of the primer pairs successfully amplified exon sequences under standard PCR conditions without requiring further optimisation. AVAILABILITY: EasyExonPrimer is available from http://129.43.22.27/~primer/. The source code is also available upon request. CONTACT: Xiaolin Wu (forestwu@mail.nih.gov).     

MethPrimer: designing primers for methylation PCRs

MOTIVATION: DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications. RESULTS: MethPrimer, based on Primer 3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.     

弓形虫RH株SAG1基因序列体外扩增及生物信息学分析

根据sAG1基因序列,自行设计一对寡核苷酸引物,利用PCR技术从弓形虫RH株基因组DNA中成功扩增出编码SAG1抗原的基因片段,扩增出的基因片段大小与预期长度（1006bp）相符,结果经测序验证,并利用生物信息学方法对SAG1蛋白理化性质、结构和功能进行了预测．     

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

恶性疟原虫红内期Pf332抗原基因未知序列的测定及分析

测定恶性疟原虫红内期Pf332抗原 (Ag332 )基因的未知序列 ,并进行序列分析 .根据非洲恶性疟原虫Palo alto株Pf332基因的G1片段序列 ,设计 1对引物 ,从中国恶性疟原虫海南株 (FCC1 HN)基因组DNA中扩增出P332 1片段 .Pf332基因中经常出现SVTEEI短肽的编码序列 ,据此分别设计非特异的正、反义寡核苷酸引物 (NSP1、NSP2 ) ,应用低严谨PCR(LSPCR)分别扩增出P332 1邻近的未知序列片段P332 up1和P332 dow1.根据恶性疟原虫Palo alto株Pf332基因G1片段上、下游的G9和C1片段序列以及测定的P332 up1和P332 dow1序列 ,分别设计 2对特异引物继续扩增邻近的未知序列片段P332 up2和P332 dow2 .根据P332 dow2片段的 3'端序列 ,设计 2条特异引物分别与非特异引物NSP2行LSPCR和巢式PCR ,扩增出P332 dow2邻近的未知序列片段P332 dow3.对获得的Pf332基因片段进行序列测定 ,并用分子生物学软件辅助进行序列分析 .序列测定和拼接结果显示 ,共获得了连续 6 14 4bp的恶性疟原虫FCC1 HN株Pf332基因序列 .序列分析表明 ,所获得的 614 4bp序列位于Pf332基因的编码区内 ,不含内含子 ,编码 2 0 4 8个氨基酸残基 ,包含 5个氨基酸残基重复区 .对恶性疟原虫FCC1 HN株Pf332基因 6 14 4bp序列的测定和分析 ,为获得Pf332全基因     

Inter-simple-sequence-repeat (ISSR) polymorphisms are useful for finding markers associated with disease resistance gene clusters

 We describe a simple and new approach, based on inter-simple sequence repeats (ISSRs), for finding markers linked to clusters of disease resistance genes. In this approach, simple sequence repeats (SSR) are used directly in PCR reactions, and markers found to be linked to disease resistance genes provide important information for the selection of other sequences which can be used with PCR to find other linked markers. Based on an ISSR marker linked to a gene of interest, many new markers can be identified in the same region. We previously demonstrated that ISSR markers are useful in gene tagging and identified a marker, UBC-855₅₀₀, linked to the gene for resistance to fusarium wilt race 4 in chickpea. This ISSR marker provided the information used in the present study for selecting other primers which amplified a region linked to the gene for resistance to fusarium wilt race 4. The primers were based on homology with the (AC)_n sequence and were used for PCR amplifications. Changes in the sequence were at the anchor region of the primers. The repeat (AC)₈T amplified a marker, UBC-825₁₂₀₀, which was located 5.0 cM from the gene for resistance to fusarium wilt race 4 and was closer than other markers. These results indicated that ISSR markers can provide important information for the design of other primers and that by making changes at the 3′ and 5′ anchors close linkage to the desired gene can be found. The approach allows rapid scanning of the targeted region and may provide important information for genome analysis of plant species. Received: 20 January 1998 / Accepted: 19 March 1998     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.