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1.
赵珺 《内蒙古科技与经济》2008,(6)
从阿尔巴斯绒山羊及蒙古绵羊血中提取基因组DNA,根据已报道的BMP2基因的保守序列设计上下游引物,扩增山羊及蒙古绵羊的BMP2基因部分序列,将此片段克隆到pMD18-T载体中,筛选阳性克隆,提取质粒DNA,并进行序列测定,并将两者序列进行比对.结果山羊该核苷酸片段与蒙古绵羊的同源性达99%,另与GenBank中人、小鼠、牛、绵羊的同源性分别为87%,85%,98%和99%.说明该基因在不同物种间具有较高的保守性,尤其是山羊与绵羊之间具有更高的保守性. 相似文献
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萤火虫荧光素酶基因cDNA真核表达载体pGL2可在原核细胞中高效表达。该载体中含有SV40早期启动子及荧光素酶基因cDNA5'端总长共758bp的DNA碱基序列，经计算机分析发现，在SV40早期启动子中含有SV40基因组HindⅢB片断中一段长为49bp的DNA序列，且荧光素酶基因cDNA编码区的5'端含有一个原核细胞基因表达调控区相似序列。将pGL2中的荧光素酶基因cDNA（Luc）克隆入真核表达载体pED中，构建成pED-Luc。该载体经表达测定后，只在真核细胞中表达，在原核细胞不中表达。这说明荧光素酶基因cDNA编码区的5'端的原核细胞基因表达调控区相似序列不具表达有活性的荧光 素酶的功能。经DNA序列对比研究，认为pGL2含有的SV40基因组Hind ⅢB片段5123bp-5171bp长49bp的DNA序列可能就是Hind ⅢB片段的原核增强子功能的决定序列。 相似文献
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由于大豆种子具有高油高蛋白,且可与土壤微生物共生进行生物固氮的特性,大豆已成为最重要的农作物品种之一。大规模的收集全长c DNA对于基因组序列的准确注释和基因及其产物的功能分析是十分必要的。我们用不同发育阶段和环境条件下的大豆构建了全长c DNA文库,并从中获得了总共39,936个大豆c DNA克隆。分别从每个克隆的5’端和3’端进行测序后,共得到68,661个表达序列标签(Expressed Sequence Tag,EST)。这些EST序列被聚类成包含2580个全长序列的22674个长片段。此外,我们对4712个全长c DNA进行了测序。去除重叠后,我们目前共得到6570条新的大豆c DNA序列。我们的数据表明87.7%的大豆c DNA克隆包含除5’-UTR和3’-UTR的完整编码区序列。已有数据证明我们测得的全长c DNA覆盖了大量不同种类的基因。通过将大豆序列与拟南芥、水稻和其他豆科植物数据进行对比分析后发现,我们的结果中包含一些特异基因,并且其中很大一部分经注释后发现存在未知功能。本研究报道的这组大豆全长c DNA克隆,将为大豆基因发掘提供有用的资源,并有助于大豆基因组的精确注释。 相似文献
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根据鸡白细胞介素18（IL-18）cDNA基因序列设计了1对特异性引物，应用RT-PCR技术，从脂多糖（lipopolysaccharide，LPS）刺激的我国地方品种萧山鸡原代鸡脾细胞中扩增并克隆鸡IL-18全长基因（Genbank accession，AY628648）。扩增片段全长591bp，共编码197个氨基酸的前体蛋白，其中含有表达完整功能蛋白所必需的起始密码子和终止密码子。该序列与国外报道的鸡IL-18全长基因核苷酸序列及推导的氨基酸序列的同源性为98．99％。只在引导序列中出现两个氨基酸残基的缺失，分子进化分析表明萧山鸡IL-18基因与火鸡以及家鸭基因形成一个独立的分支。将萧山鸡IL-18成熟蛋白序列插入pGEX-4T-2载体并在Ecoli中得到表达，获得45kD的GST-IL-18融合蛋白。经纯化后用于制备多克隆抗体。本研究对萧山鸡白细胞介素18的全长基因进行了克隆及表达，并对其分子进化进行了分析。为深入研究其功能奠定了基础。 相似文献
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目的克隆小鼠内皮抑素（Endostatin）编码区cDNA序列及测序鉴定并构建pEgr-IFNγ-EndostatinA基因表达载体。方法从小鼠肝细胞提取总RNA，经逆转录一聚合酶链式反应（RT-PCR）扩增获得全长Endostatin基因，测序并利用基因重组技术构建pEgr-IFNγ-EndostatinYT．基因共表达质粒。结果分光光度计测定50倍稀释的总RNA抽提产物，A260=0．834，A280=0．407，A260：A280=2．049，总RNA浓度为1．668g／L。EndostatincDNA的RT-PCR产物与载体pMD18T连接并经测序证实Endostatin序列与文献报道完全一致，并构建了含Egr-1启动子的IFNγ和DEndostain双基因共表达质粒pEgY-IFNγ-Endostatin。结论成功克隆小鼠Endostatin基因，构建了pEgr-IFNγ-Endostatin双基因共表达质粒。 相似文献
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一株嗜热梭菌β-葡聚糖酶基因的克隆和表达 总被引：1，自引：0，他引：1
提取嗜热梭菌(Clostridium.sp)基因组DNA,首次通过PCR克隆了该菌的β-葡聚糖酶基因全长,结果表明:该基因全长1040bp,ORF为1003bp,编码334个氨基酸,计算分子量为37.8kD,等电点为7.67。经Blast分析,该序列与热纤梭菌同源性最高(99%),而与基因库中嗜热梭菌的同源性为94%,该基因已被GenBank接受(AY225318)。用BamHⅠ和XhoⅠ双酶切目的片段和表达载体pET鄄30a( )后相连接,构建重组表达载体pET鄄clo,并导入BL21细菌中表达.酶学特性表明:SDS鄄PAGE电泳在37kD左右有表达蛋白带,该工程菌最适酶活29.4U/mL,是出发菌的15倍,最适温度在80℃左右,最适pH在9左右。该工程菌可构建耐热性好、酶活高的杂合基因工程菌。 相似文献
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目的:建立简便、快捷、稳定可靠的鉴定小鼠基因型方法,维护动物伦理福利。方法:基因敲除小鼠后代鉴定时,分别从鼠尾,鼠耳,趾甲三个部位提取DNA,通过优化DNA提取技术,经分光光度计检测,PCR扩增等技术,比较分析DNA纯度、提取时间,及基因型鉴定结果。结果:取组织提取DNA,经分光光度计检测,发现DNA浓度尾尖最高,耳尖次之,趾甲浓度最低,但是纯度最好。经PCR扩增技术发现不同基因型的不同部位提取DNA鉴定结果一致。结论:剪趾甲提取DNA操作方法相对于其他常规方法,操作简单,耗时最短,基因型鉴定结果可靠,对小鼠本身伤害更小,可用于规模化的小鼠基因型鉴定实验中。 相似文献
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目的克隆小鼠内皮抑素(Endostatin)编码区cDNA序列及测序鉴定并构建pEgr-IFN(Y)-Endostatin双基因表达载体.方法从小鼠肝细胞提取总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增获得全长Endostatin基因,测序并利用基因重组技术构建pEgr-IFN(Y)-Endostatin双基因共表达质粒.结果分光光度计测定50倍稀释的总RNA抽提产物,A260=0.834,A280=0.407,A260A280=2.049,总RNA浓度为1.668g/L.EndostatincDNA的RT-PCR产物与载体pMD18T连接并经测序证实Endostatin序列与文献报道完全一致,并构建了含Egr-1启动子的IFNY和Endostain双基因共表达质粒pEgY-IFN(Y)-Endostatin.结论成功克隆小鼠Endostatin基因,构建了pEgr-IFN(Y)-Endostatin双基因共表达质粒. 相似文献
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文章以樱草杜鹃、大花黄牡丹、长鞭红景天、露蕊乌头、苞叶雪莲、藏橐吾、独一味、砂生槐、拉萨蒲公英、拉萨小檗10种藏药材为研究对象,提取基因组DNA,对其线粒体细胞色素c氧化酶第Ⅲ亚基(cox3)的部分片段进行了PCR扩增、测序及序列分析,用Bioedit软件与Gen Bank数据库中相关植物的序列进行序列比对,并用MEGA6.0软件做系统进化树(NJ),运用Codon W软件计算密码子的RSCU值。通过PCR扩增获得了10种藏药材的长度为750bp的目的片段,通过序列比对发现cox3基因在不同植物中变异位点较少,序列比较保守;基于cox3部分基因序列构建的NJ树反映了物种的进化关系,可以作为物种系统发育分析的补充;密码子偏好性分析发现,cox3基因偏好使用以AU结尾的密码子。樱草杜鹃等10种藏药材的COXⅢ基因部分片段的扩增与序列分析从序列比对分析、构建系统进化树、密码子偏好性分析水平上分析了cox3基因部分序列在樱草杜鹃等10种藏药材的差异,为植物cox3基因的功能、表达研究奠定了基础。 相似文献
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为给不结球白菜的品种配置和遗传育种提供理论基础,利用特异性PCR扩增、DNA测序和生物信息学方法鉴定了7个自交系的S基因型,PCR扩增结果表明,51-14、375-124、711-114、884-214等4个自交系的基因型为Class-Ⅰ SLG和SRK,表现为自交强不亲和.523-112和544-312的基因型为Class-ⅡSLG类型.表现为自交若不亲和,和田间试验结果一致.测序结果blast分析表明844-214 Class-Ⅰ型和Brassica rapa SLG56的相似性为99%,51-14的SRK PCR与SRK56的同源性达到100%. 相似文献
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Uwe Tangen Gabriel Antonio S. Minero Abhishek Sharma Patrick F. Wagler Rafael Cohen Ofir Raz Tzipy Marx Tuval Ben-Yehezkel John S. McCaskill 《Biomicrofluidics》2015,9(4)
Nanoscale synthetic biology can benefit from programmable nanoliter-scale processing of DNA in microfluidic chips if they are interfaced effectively to biochemical arrays such as microwell plates. Whereas active microvalve chips require complex fabrication and operation, we show here how a passive and readily fabricated microchip can be employed for customizable nanoliter scale pipetting and reaction control involving DNA. This recently developed passive microfluidic device, supporting nanoliter scale combinatorial droplet generation and mixing, is here used to generate a DNA test library with one member per droplet exported to addressed locations on microwell plates. Standard DNA assembly techniques, such as Gibson assembly, compatible with isothermal on-chip operation, are employed and checked using off-chip PCR and assembly PCR. The control of output droplet sequences and mixing performance was verified using dyes and fluorescently labeled DNA solutions, both on-chip and in external capillary channels. Gel electrophoresis of products and DNA sequencing were employed to further verify controlled combination and functional enzymatic assembly. The scalability of the results to larger DNA libraries is also addressed by combinatorial input expansion using sequential injection plugs from a multiwell plate. Hence, the paper establishes a proof of principle of the production of functional combinatorial mixtures at the nanoliter scale for one sequence per well DNA libraries. 相似文献
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Two simple gold nanoparticle (GNP)-based DNA analysis methods using a microfluidic device are presented. In the first method, probe DNA molecules are immobilized on the surface of a self-assembled submonolayer of GNPs. The hybridization efficiency of the target oligonulceotides was improved due to nanoscale spacing between probe molecules. In the second method, target DNA molecules, oligonulceotides or polymerase chain reaction (PCR) amplicons, are first bound to GNPs and then hybridized to the immobilized probe DNA on a glass slide. With the aid of GNPs, we have successfully discriminated, at room temperature, between two PCR amplicons (derived from closely related fungal pathogens, Botrytis cinerea and Botrytis squamosa) with one base-pair difference. DNA analysis on the microfluidic chip avoids the use of large sample volumes, and only a small amount of oligonucelotides (8 fmol) or PCR products (3 ng), was needed in the experiment. The whole procedure was accomplished at room temperature in 1 h, and apparatus for high temperature stringency was not required. 相似文献
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Anubha Paliwal Nivedita Bir Sowmya Raghuraman T. L. P. Reddy P. Usha Sarma 《Indian journal of clinical biochemistry : IJCB》1999,14(2):129-134
Oligonucleotide primers were synthesised based on the gene sequence of an 18 kDa allergen/antigen ofA. fumigatus isolated from a pathogenic strain. Polymerase chain reaction (PCR) was carried out using the forward and reverse primers
and genomic DNA ofA. fumigatus, A. flavus andA. niger as template. This resulted in a PCR product of 480 bp with onlyA. fumigatus. The absence of PCR product inA. flavus andA. niger with the primers of Asp fl facilitated use of these primers for detection ofA. fumigatus in clinical specimens of patients. The results were compared with microscopy, culture and serology. Application of PCR test
to clinical samples of aspergillosis patients is discussed. 相似文献
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Sheshadri Narayanan 《Indian journal of clinical biochemistry : IJCB》1999,14(1):40-48
The apo E gene located on chromosome 19 in humans is polymorphic. The three apo E isoforms E2, E3, and E4 are coded by three
common alleles of the gene. The amyloid plaques in brains of Alzheimer disease (AD) patients are known to contain apo E. There
is an increased prevalence of E4 allele in AD patients. apo E exhibits increased binding to a peptide Aβ deriving from amylold
precursor protein. apo E, the risk factor for late AD disease is unable to prevent formation of paired helical filaments which
in turn destabilizes neuronal microtubules.
A variety of molecular techniques are available for apo E genotyping using DNA amplified by the polymerase chain reaction
(PCR). The high guanine to cytosine content of apo E is problematic to the extent that the yield of PCR product and hybridization
stringency can be compromised. The specificity of diagnosis of late-onset AD can be improved when results of apo E genotyping
are evaluated together with clinical criteria. 相似文献
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Digital microfluidics is an elegant technique based on single droplets for the design, composition, and manipulation of microfluidic systems. In digital microfluidics, especially in the electrowetting on dielectric (EWOD) system, each droplet acts as an independent reactor, which enables a wide range of multiple parallel biological and chemical reactions at the microscale. EWOD digital microfluidics reduces reagent and energy consumption, accelerates analysis, enables point-of-care diagnostic, simplifies integration with sensors, etc. Such a digital microfluidic system is especially relevant for droplet digital PCR (ddPCR), thanks to its nanoliter droplets and well-controlled volume distribution. At low DNA concentration, these small volumes allow less than one DNA strand per droplet on average (limited dilution) so that after a fixed number of PCR cycles (endpoint PCR), only the DNA in droplets containing the sequence of interest has been amplified and can be detected by fluorescence to yield an accurate count of the sequences of interest using statistical models. Focusing on ddPCR, this article summarizes the latest development and research on EWOD technology for droplet PCR over the last decade. 相似文献
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The white spot syndrome virus (WSSV) is one of the deadly pathogens of penaeid shrimps and other crustaceans. The WSSV virion
consists of an enveloped rod-shaped nucleocapsid enclosing a large circular double stranded DNA genome of 305 Kb with 181
open reading frames. The two major structural genes, VP19 and VP28 were amplified from the genomic DNA of Chinese isolate
of WSSV and cloned in pUCm-T vector and sub cloned in pET-30a (+) vector. The expressions of genes inE. coli (BL21) were confirmed by SDS-PAGE analysis. The clones were sequenced, submitted to the gene bank and the Xiang Shan strain
of WSSV were compared with the previous reported sequence of WSSV of various regions which revealed that VP19 and VP28 gene
sequences had certain differences from the sequences of similar genes of the isolate already reported. The recombinant proteins
expressed, purified and characterized. 相似文献