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1.
线粒体是存在于绝大多数真核细胞内的一种基本的重要的细胞器,其具有相对独立的遗传系统。线粒体基因在真核生物具有高保守性,线粒体DNA(mtDNA)已被广泛应用于发病机理、临床诊断、遗传变异、生物进化等多方面的研究。1981年,Anderson用氯化铯密度梯度分离得到线粒体DNA(mtDNA),进行了全序列分析。此后,mtDNA的研究日益得到重视。已有的mtDNA提取方法概括起来可分为密度梯度离心法、酶消化法、柱层析法、氯化铯超速离心法、碱变性法和改进高盐沉淀法等,通过对以上方法的比较,发现改进高盐沉淀法具有简便、经济、易重复等优点。  相似文献   

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一种改进的动物线粒体DNA提取方法   总被引:80,自引:11,他引:80       下载免费PDF全文
王文  施立明 《动物学研究》1993,14(2):197-198
线粒体DNA(mtDNA)已被广泛用于动物群体遗传学和进化生物学的研究,并取得了许多有意义的结果。有效的mtDNA提取方法无疑是开展这方面研究的前提。关于动物mtDNA的提取方法,国内外已有不少报导。概括起来,可分为:1)氯化铯超速离心法,2)柱层析法,3)DNase法,4)碱变性法。本文报道了一种改进的碱变性提取法,与其它方法相比,具有应用范围广、简便、经济等优点。  相似文献   

4.
大白菜线粒体DNA有效、快速提取方法   总被引:6,自引:0,他引:6  
目的:建立一种有效、快捷的大白菜线粒体DNA(mtDNA)提取方法。方法:采用差速离心和蔗糖衬垫相结合的方法先从大白菜胞质雄性不育系及保持系叶片中分离出线粒体,然后用SDS—蛋白酶K裂解法提取mtDNA。结果:琼脂糖凝胶电泳表明,采用该方法提取的mtDNA片段大小在30~45kb之间,电泳条带清晰,无明显降解发生;以提取的mtDNA为模板进行RAPD反应,不育系和保持系均扩增出较多的条带,而且呈现出丰富的多态性,大小分布在500bp-3000bp之间,且条带清晰,没有脱尾现象。结论:该方法提取mtDNA,省时、省钱,对实验设备要求较低,而且分离出的mtDNA质量较高,能满足后续分子生物学研究工作需要。  相似文献   

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线粒体DNA提取方法的比较   总被引:6,自引:0,他引:6  
目的:将提取线粒体DNA的碱变性法、Triton法、改进高盐沉淀法加以比较,以得到最方便快速提取线粒体DNA的方法。方法:分离Wistar大鼠小肠上皮细胞,用3种方法提取线粒体DNA,紫外分光光度法定量。用琼脂糖凝胶电泳和线粒体ATPase 8亚基基因PCR扩增产物鉴定所提取的线粒体DNA。结果:改进高盐沉淀法线粒体DNA量最多,Triton法最少。OD260/OD280均在1.78-l.85间。将改进高盐沉淀法提取线粒体DNA用于PCR扩增,测定出了线粒体DNA ATPase 8亚基基因序列。结论:改进高盐沉淀法提取线粒体DNA具有操作简单,产量多的优点,该法所提取mtDNA可用于mtDNA测序。  相似文献   

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目的:对2种常用的线粒体DNA提取方法进行比较,分析提取物中核DNA的存在情况,同时建立新检测方法降低线粒体假基因干扰。方法:以仅在核DNA中存在的基因β-actin作为核DNA存在的标定基因,通过PCR方法扩增线粒体DNA上的一段基因MTND5-2,并以β-actin做参比,比较常用的2种提取线粒体DNA方法的优劣,即碱法Ⅰ(先提取完整线粒体后从中获得线粒体DNA)和碱法Ⅱ(根据线粒体DNA与核DNA的结构差异,从中获得双链环状的线粒体DNA)。结果:2种线粒体DNA提取方法并不能获得仅含线粒体DNA的纯提取物,碱法Ⅰ获得的线粒体DNA纯度相对较高;以碱法Ⅰ提取物为模板进行PCR,可获得更多较纯的线粒体目的基因。结论:碱法Ⅰ较碱法Ⅱ可获得更纯的线粒体来源的目的基因;新建方法可获得较纯的线粒体基因,且是一种简单、方便、经济的方法。  相似文献   

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应用随机扩增多态DNA(Random amplified polymorphic DNAs,RAPD)技术,对普通小麦细胞质雄性不育系(A)及其保持系(B)线粒体基因组(mitochondrial genome)的指纹图谱进行了分析。共使用引物41个,其中20个引物得到了扩增产物,部分引物扩增结果表现多态性。说明线粒体DNA(mitochondrial DAN,mt,DNA)序列在不育系和保持系之  相似文献   

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十字花科植物线粒体DNA的提取和纯化   总被引:8,自引:4,他引:8       下载免费PDF全文
以十字花科植物芸芥 Eruca sativa 、白菜型油菜 Brassica rapa 和甘蓝型油菜 Brassica napus :武39- 1、武4 3- 1、武5 0 - 2为试材,在蔗糖衬垫法分离纯化m t DNA方法优点的基础上,通过4组不同离心力配比,采用差速离心法、改变缓冲液A的成分以及增加缓冲液的用量,建立了一种简便提取十字花科植物mt DNA方法.研究结果表明:分离细胞破碎组织的离心力、分离线粒体的离心力以及纯化线粒体时的离心力分别为10 0 0 g、2 0 0 0 0 g、180 0 0 g时可分离到高产量的线粒体,且在缓冲液A 蔗糖0 .5 m ol·L- 1 ,Tris- Cl5 0 mm ol·L- 1 ,EDTA5 m mol·L- 1 ,BSA 0 .1% ,PVP 0 .5 % ,0 .1%β-巯基乙醇,p H=7.5 中加入0 .5 % PVP可提高mt DNA的产量,经DNA电泳检测、RAPD扩增证明此方法得到的mt DNA可完全达到RAPD标记和其它分子生物学研究的要求.  相似文献   

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一种改进的禽类线粒体DNA提取方法   总被引:6,自引:0,他引:6  
介绍一种碱变性法从禽类脏器中提取大量线粒体DNA,比较了从不同脏器提取mtDNA的难易程度和得率。结果表明:肝脏、脾脏的匀桨操作易于心脏,且肝脏的得率高于心脏,更高于脾脏;同时对影响mtDNA产量和质量的匀桨速度和次数,差速离心速度,溶液Ⅱ中SDS含量及溶液pH值等因素进行了初步分析和探讨。在借鉴前人研究基础上对禽类脏器mtDNA提取方法上进行了改进和优化,建立了一种从禽类脏器中有效制备mtDNA的方法。结果表明,这种方法得到的mtDNA可以满足限制性酶切实验的要求。  相似文献   

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一种棉花线粒体DNA的提取方法   总被引:2,自引:0,他引:2  
线粒体是重要的细胞器,它有自身的基因组。其基因组DNA与细胞核基因组DNA相比,含量较低。棉花当中富含棉酚、丹宁等物质,这对提取DNA有很大的影响。因此我们根据棉花自身的特点,找到了一种提取棉花线粒体DNA经济有效的方法,其质量可以满足限制性酶切、PCR、分子杂交等实验的要求。  相似文献   

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对新选育的甘蓝型油菜PL CMS不育系和Pol CMS及Ogu CMS不育系材料花器形态进行比较,并对3种不育系10份材料mtDNA进行RAPD分析.结果表明,新选育的PL CMS不育系与Pol CMS、Ogu CMS在花瓣长和宽、花冠直径、雄蕊大小等形态特征方面有极显著差异;mtDNA分析结果表明,新发现的细胞质雄性不育材料L04-02A、L04-05A、L04-01A为同一类型的细胞质雄性不育系,它们与Pol CMS及Ogu CMS不育材料有很大差异,由此推知PL CMS不育系是一种新型甘蓝型油菜不育系.  相似文献   

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 RAPD profiles were generated using mitochondrial DNA (mtDNA) isolated from two cytoplasmic male-sterile lines, two restorer lines and four maintainer lines of rice. Of the 40 primers tested, 25 generated consistent and easily scoreable patterns that were used for the computation of pairwise similarities as well as UPGMA analyses. The different lines of rice, including lines IR58025A and IR62829A that contained the same wild abortive (WA) cytoplasm, were distinguishable on the basis of RAPD profiles. These latter two lines were not distinguishable from each other by mtDNA RFLP analyses with as many as 16 mtDNA probes. The data illustrate the utility of the RAPD technique as a powerful tool for distinguishing different cytoplasms that by other techniques appear to be similar. To our knowledge, this is the first report wherein RAPD profiles obtained with isolated mtDNA templates enable the distinction between two or more types of cytoplasms in rice. Received: 1 April 1997 / Accepted: 2 June 1997  相似文献   

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小鼠基因组研究进展李善如1,2王冬平1陈永福2(1.军事医学科学院实验动物中心,北京100071)(2.中国农业大学生物学院,北京100094)TheDevelopmentofMouseGenomeResearchLIShanru1,2WANGDon...  相似文献   

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Genetic distances were lower between Upeneus moluccensis and Pseudopeneus prayensis than between those species and Mullus barbatus and Mullus surmuletus . However, for allozymes, the two Mullus species were found genetically more distinct from U. moluccensis than from P. prayensis , but RAPD and mtDNA analysis showed the opposite. RAPDs revealed less interspecific divergence compared with allozymes and the results they produced were more consistent with mtDNA analysis. Although RAPDs did not add any supplementary taxonomic information, they proved valuable tools for quick and reliable species discrimination compared with allozymes and mtDNA.  相似文献   

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玉米同核同质系在研究细胞质雄性不育中的应用   总被引:1,自引:0,他引:1  
以94个Operon公司的10碱基RAPD引物,对玉米同核同质系(I)中的野生型正常可育株478(F)和其突变的不育株478-cms的线粒体DNA(mtDNA)进行了RAPD分析。结果表明:仅OPZ-19、OPAA-15与OPS-01 3个引物在同核同质系(I)的野生型正常可育株和其突变的不育株之间扩增出多态产物OPZ-19420、OPAA-15600与OPS-1400。提示在同核同质系的野生型正常可育株和其突变的不育株的mtDNA之间同源性很高,多态性很低,同核同质系相当于在mtDNA水平上的近等基因关系,所找到的多态就在所研究的目标基因附近,因此它是研究mtDNA基因的良好体系。上述3个多态扩增结果在同核同质体系(I)的姐妹同核同质系(Ⅱ)中进行了验证, 结果表明仅2个引物OPZ-19、OPAA-15在姐妹同核同质系(Ⅱ)的野生型正常可育株苏478(F)和其突变的不育株苏478-cms之间获得了与同核同质系(Ⅰ)相同的多态性扩增产物OPZ-19420、OPAA-15600。即这两个多态性产物在两套同核同质系中同时存在,因而这两个多态性产物与育性直接相关的可能性很大。  相似文献   

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The pond loach Misgurnus dabryanus is a freshwater fish with a distribution range spanning the eastern part of the Asian continent, the Korean Peninsula, and Taiwan. The pond loach was transplanted to the Japanese archipelago through the co-inclusion with dojo loach (Misgurnus anguillicaudatus species complex) populations, which were imported live from China for food materials, and it is currently distributed widely across Japan. A previous mitochondrial DNA (mtDNA) analysis revealed that a pond loach population in Ehime Prefecture (Shikoku Island, Japan) included two highly diverged mtDNA groups (Groups I and II). To examine the origin of these two distinct forms of mtDNA within the Japanese pond loach population, we performed phylogenetic analyses using sequences based on the mtDNA of cytochrome oxidase b (cyt b) and the nuclear DNA recombination activating gene 1 (RAG-1). We also conducted a random amplified polymorphic DNA (RAPD) analysis to examine the establishment of reproductive isolation between sympatric pond loaches with two different mtDNA groups. Our mtDNA phylogenetic results indicated that the two diverged pond loach mtDNA sequences showed polyphyletic relationships among Misgurnus species and its related genus Cobitis. In contrast, there were no clear divergence in nuclear DNA among the pond loaches irrespective of their mtDNA groups, and they all formed monomorphic clades in the phylogenetic relationships among the species. The discrepancy between the mtDNA and nuclear DNA genes support that the existence of two diverged forms of DNA within the pond loach population could be attributed to past mtDNA introgressions from other species rather than convergent evolution. Previous mtDNA phylogenetic studies among Cobitidae revealed that the dojo loach also consisted of two genetically diverged polyphyletic clades: an original Misgurnus mtDNA and an introgressed mtDNA from Cobitis species. In our mtDNA result, the Group II haplotype of the pond loach was included in the mtDNA from the introgressed dojo loach. This suggested that the Group II haplotype was derived from introgressed dojo loach mtDNA. The close relationships between the introgressed dojo loach and the pond loach mtDNA indicated that this secondary introgression had recently occurred via hybridization in a recent artificial aquaculture or transportation process. Common RAG-1 alleles and RAPD bands were shared between the sympatric pond loaches with original and introgressed mtDNAs. This indicates that the introgressed mtDNA haplotype is included as one of the polymorphic genotypes within the pond loach populations, and does not represent existence of different cryptic species.  相似文献   

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姚鸿  王斌 《遗传学报》1998,25(1):67-73
msD2-CA8057是新育成的具有粗厚山羊草(Ae.crasa,6x)胞质的D2型小麦细胞质雄性不育系。采用RFLP和RAPD方法对该不育系及其具有普通小麦(T.aestivum)胞质的保持系CA8057和恢复系保-769-22-6的线粒体DNA进行分析和比较,发现该不育系的线粒体DNA组织结构明显不同于其保持系,也不同于其恢复系。Southern结果表明,该不育系线粒体基因组在atpA、atp9、cob和coxⅡ基因上或附近具有显著的组织结构差异。RAPD分析证实了这一点。相反,RFLP和RAPD结果都表明保持系与恢复系之间线粒体基因组结构非常相似。这支持了该不育系的胞质遗传特点来源于与普通小麦胞质差异较大的野生型胞质的事实。推测这种胞质差异与育性有关  相似文献   

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水稻线粒体DNA中与雄性不育有关特异片段的克隆及序列分析   总被引:11,自引:1,他引:11  
该项研究利用RAPD技术,对野败型,矮败型和BT型细胞质雄性不育系,保持系,基因组DNA进行PCR扩增,分离到6条不育系特有的扩增片段,并对野败型,矮败型不育系共有的片段进行了Southern分析,DNA序列分析和SCAR验证,该片段全长1879bp,包含6个开放阅读框架和8对重复序列,BLAST分析表明,该片段部分区域与Elytrigia elongata,小麦线粒体tRNA-Asp基因上游一段序列同源,并对该片段在线粒体DNA中的可能位置等进行了讨论。  相似文献   

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应用PCR和凝胶电泳等技术,对双孢蘑菇杂交菌株As2796及其亲本和子代作分子遗传标记跟踪分析,结果如下: 1) 总DNA的RAPD分析表明,随着遗传代数的增加,杂种子代和出发异核体亲本间的遗传差异逐渐增大; 2) mtDNA的酶切图谱表明,亲本8213及其杂交子代具有相同的基因型,表明双孢蘑菇的mtDNA呈单亲遗传; 3) Est同工酶的PAGE图谱表明, 结合了亲本02高产特征和8213优质特征的杂交子代具有两个亲本的标记带型,证明Est同工酶标记是双孢蘑菇新菌株特性预测或鉴定的有效指标。  相似文献   

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