小麦线粒体DNA的高效提取方法

以小麦黄化苗为材料, 通过简单差速离心、DNaseⅠ处理得到无核DNA杂质的线粒体, 用SDS和蛋白酶K裂解线粒体, 经酚/氯仿抽提除去蛋白, 并用RNase A消化而得到单纯线粒体DNA(mtDNA)。对所提取的mtDNA进行紫外吸收光度分析, A₂₆₀/A₂₈₀ 平均为1.92, A260/A230 平均为2.09, 平均每克黄化苗可提取mtDNA 26.85 mg; 并对mtDNA进行琼脂糖凝胶电泳和RAPD扩增, 均得到清晰的电泳图谱。结果表明: 此提取方法得到的mtDNA, 不但产率高、结构完整, 而且能有效去除核DNA、RNA和蛋白质等杂质, 获得高质量的mtDNA用于PCR反应和各种遗传学分析。研究还发现, 通过调整线粒体裂解温度(先50℃裂解1 h, 再37℃裂解1 h), 亦可大幅度提高mtDNA的产率。     

一种改进的动物线粒体DNA提取方法

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。     

大白菜线粒体DNA有效、快速提取方法

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。     

线粒体DNA提取方法的比较

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。     

提取线粒体DNA方法的比较及较纯线粒体基因获取方法的确立

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。     

一种改进的禽类线粒体DNA提取方法

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。     

一种制备酵母菌线粒体DNA的简便方法

ASimpleProcedureforPreparationmtDNAinYeastJinJianlingGaoDongSunZhongdong(MicrobiologyDepartment,ShandongUniversity,Jinan250100)目前,制备酵母mtDNA的常用方法大致有两类：一是先提取混合DNA（核DNA＋mtDNA）,再通过CsCI密度梯度离心或柱层析法分离纯化mtDNA（’,’,”’;二是先通过蔗糖不连续梯度超离心法分离纯化线粒体,再从线粒体中提取mtDNA”’。这些方法虽然能够得到纯度较高的mtDNA,但超离心法需要配套设备（超速离心机等）,柱层析法对样品的回收浓缩比较复杂。本文报道的制备mtDNA的方法,不需…     

一种棉花线粒体DNA的提取方法

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。     

线粒体DNA及其表达的研究进展

线粒体属于半自主性细胞器 ,含有环状ＤＮＡ ,能进行自我复制 (重组和修复机制也包括在内 )。但线粒体ＤＮＡ(ｍｔＤＮＡ)的复制仍受细胞核的控制 ,因为不仅构成线粒体的蛋白质几乎都受核基因编码、在细胞质中合成 ,而且与ｍｔＤＮＡ有关的特定蛋白质(如ＤＮＡ聚合酶、重组与修复所需的酶、ＲＮＡ聚合酶、ＲＮＡ加工酶 )也都是由核基因编码的。ｍｔＤＮＡ的复制、转录、翻译及蛋白质的输入有其特殊规律 ,阐明线粒体的分子遗传规律既有助于理解线粒体在凋亡或程序性细胞死亡中发挥的作用 ,因而可更深入地对发育生物学、癌症、老化及机体死亡…     

两种获取鱼类线粒体DNA的方法比较

直接从鱼类组织提取线粒体DNA和先提取基因组DNA再用相应引物PCR扩增所需mtDNA某一区域序列片段是获取鱼类mtDNA切实可行的两种方法。对这两种方法在使用过程中的注意事项、各自的优缺点及应用范围进行了较为细致的比较,为更好地利用mtDNA这一重要分子标记研究鱼类mtDNA的遗传多态提供参考。     

鱼类线粒体DNA多态性研究

介绍了鱼类线粒体ＤＮＡ多态性研究的方法及其在鱼类各学科领域中的广泛应用。     

Mitochondrial DNA Medicine

The small, maternally inherited mitochondrial DNA (mtDNA) has turned out to be a hotbed of pathogenic mutations: 15 years into the era of ‘mitochondrial medicine’, over 150 pathogenic point mutations and countless rearrangements have been associated with a variety of multisystemic or tissue-specific human diseases. MtDNA-related disorders can be divided into two major groups: those due to mutations in genes affecting mitochondrial protein synthesis in toto and those due to mutations in specific protein-coding genes. Here we review the mitochondrial genetics and the clinical features of the mtDNA-related diseases.     

猕猴桃属植物线粒体DNA片段PCR-RFLP研究初报

猕猴桃属 ( Actinidia)植物 ,全世界有 66个种 ,约 1 1 8个种下分类单位 (变种、变型 ) [1] 。近来通过对猕猴桃属植物的细胞质 DNA研究证实 ,该属植物细胞质 DNA为单亲遗传 ,其线粒体 DNA为严格的母性遗传 [2 ] ,这一单亲遗传特性为研究猕猴桃属植物分类及系统学提供了新的途径。在高等植物中 ,由于线粒体 DNA的高度保守 ,以及重排率高而突变率低 ,加之在植物组织中的含量较低 ,限制了它在系统学研究中的应用 ,不适合属以上高阶元间的比较研究[3 ] ,但线粒体 DNA序列进化速度快 ,在近缘种内或种间显示出了更大的变异性 ,并且线粒体 …     

太湖新银鱼线粒体DNA的制备

建立了适合太湖新银鱼等小型鱼类ｍｔＤＮＡ分离纯化的有效方法。改进匀浆条件,采用ＤＮａｓｅＩ和ＲＮａｓｅ纯化方法,可用常规方法由小量太湖新银鱼中制备出ｍｔＤＮＡ。采用改进的碱裂解法,能够在２小时内由单尾太湖新银鱼中快速分离纯化出适于限制性分析用的ｍｔＤＮＡ。     

Mapping Topoisomerase Sites in Mitochondrial DNA with a Poisonous Mitochondrial Topoisomerase I (Top1mt)

Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin O_H, in the ribosomal genes (12S and 16S) and at the light strand replication origin O_L. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.     

Mitochondrial DNA mutations and breast tumorigenesis

Breast cancer is a heterogeneous disease and genetic factors play an important role in its genesis. Although mutations in tumor suppressors and oncogenes encoded by the nuclear genome are known to play a critical role in breast tumorigenesis, the contribution of the mitochondrial genome to this process is unclear. Like the nuclear genome, the mitochondrial genome also encodes proteins critical for mitochondrion functions such as oxidative phosphorylation (OXPHOS), which is known to be defective in cancer including breast cancer. Mitochondrial DNA (mtDNA) is more susceptible to mutations due to limited repair mechanisms compared to nuclear DNA (nDNA). Thus changes in mitochondrial genes could also contribute to the development of breast cancer. In this review we discuss mtDNA mutations that affect OXPHOS. Continuous acquisition of mtDNA mutations and selection of advantageous mutations ultimately leads to generation of cells that propagate uncontrollably to form tumors. Since irreversible damage to OXPHOS leads to a shift in energy metabolism towards enhanced aerobic glycolysis in most cancers, mutations in mtDNA represent an early event during breast tumorigenesis, and thus may serve as potential biomarkers for early detection and prognosis of breast cancer. Because mtDNA mutations lead to defective OXPHOS, development of agents that target OXPHOS will provide specificity for preventative and therapeutic agents against breast cancer with minimal toxicity.     

线粒体基因突变与NIDDM发生的关系

采用PCR-SSCP、PCR-RFLP及PCR产物直接测序等技术对90例NIDDM(即非胰岛素依赖型糖尿病)及80例正常对照个体的血细胞线粒体DNA进行了突变分析。结果在2例患者中发现线粒体DNA(mitochondrial DNA,mtDNA) ND1 (NaDH Dehydrogenase subunitⅠ)基因上3316位点存在G→A的点突变,导致丙氨酸错义突变成苏氨酸,而在80例正常对照个体中均不存在此位点突变。国内外已证实的和1.5%NIDDM发生有关的mtDNA tRNA Leu^(UUR)|基因上3243位点A→G的突变在本实验中并未发现。由此推断,3316位点G→A的突变可能与NIDDM的发生在关,3243位点A→G的突变率确实很低,可见糖尿病的发生在线粒体遗传上具有广泛的异质性。 Abstract:Using PCR-SSCP,PCR-RFLP and PCR product direct sequencing techniques,we analysed the mitochondrial DNAs(mtDNAs)of 90 patients with NIDDM (Non Insulin-Dependent Diabetes Mellitus)and those of 80 normal controls.The results showed that a G to A mutation which leads alanine’s missence mutaton to threonine in the mitochondrial ND1(NaDH Dehydrogenase subunit I) gene at nucleotide pair 3316 occurred in the blood cells of 2 patients.We have not however,indentified with the A to G mutation at nucleotide pair 3243 of the mitochondrial tRNA Leu(UUR) gene,which has been reported to associate with NIDDM in about 1.5% of the diabetic population.We infer that the mutation at position 3316 is perhaps associated with the development of NIDDM,the occurance of the mutation at position 3243 is actually rare,and NIDDM has an intensive mitochondrial genetic heterogenous background.     

水稻线粒体DNA的提取与分析

为了研究水稻细胞质雄性不育的分子基础,我们比较了各种提取线粒体ＤＮＡ（ｍｔＤＮＡ）的方法,并提出了一些改进措施。以丛广４１Ａ、丛广４１Ｂ和杂种一代广优青为材料,对所提取的材料ｍｔＤ－ＮＡ进行了紫外扫描、ＯＤ值测定、电泳、酶切等分析,结果表明,以新鲜材料进行不连续蔗糖密度梯度超速离心对提取高纯度的线粒体ＤＮＡ效果较好。