一种快速提取丝状真菌染色体DNA的方法

介绍了一种适用于丝状真菌染色体DNA大片段的快速提取方法,该方法以(100mM Tris,100mM NaGl,50mM EDTA-Na2 2%SDS,pH值9.0)为提取液,经石英砂研磨破壁.应用该方法成功地提取了粗糙脉胞菌(Neurospora crassa)、米曲霉(Aspergillus oryzae)、产黄青霉(Penicillium chrysogenum)和头孢霉菌(Cep- halosporium sp.)等4种不同丝状真菌的染色体DNA大片段,且所提DNA片段均大于20kb,可直接用于限制性酶切、PCR等分子生物学研究.     

一种快速提取真菌染色体DNA的方法

介绍了一种适用于多种真菌染色体DNA的快速提取方法。该方法用石英砂振荡破壁 ,快速便捷地提取真菌染色体DNA ,提取时间仅用 1～ 2h。作者应用该方法成功地提取了粗糙脉胞菌 (Neurosporacrassa)、米曲霉 (Aspergillusoryzae)、羊肚菌 (Morchellaesculcnta)、酿酒酵母 (Saccharomycescervisae)等 4种不同真菌的染色体DNA ,所提DNA片段均大于2 0kb ,可直接用于限制性内     

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。     

细菌基因组DNA提取方法概述

细菌基因组DNA提取是分子生物学研究的基础,DNA提取的质量和效率可直接影响后续实验结果的准确性和精确性。综述并比较了近10年细菌DNA提取的几种方法,分析不同方法的提取效率,以期为分子生物学研究提供更可靠的基础。     

关于细菌人工染色体(BAC)文库载体DNA制备的研究

细菌人工染色体（BAC）文库在基因组研究中起着关键作用。构建BAG文库的一个关键步骤就是BAC载体DNA的制备,制备高质量的BAC载体DNA受到包括酶切,脱磷等诸多因素的影响。以BAC载体pECBAC1为材料,分别采用限制性内切酶BamHⅠ和HK脱磷酶对其进行酶切和脱磷,并结合凝胶回收缩化技术,制备了可用于进一步构建BAC文库的线性载体DNA。并在此基础上,确定了制备BAC载体DNA的适宜条件,其中包括确定适宜限制内切酶用量及酶切时间,脱磷酶种类及浓度和凝胶回收纯化线性载体DNA等关系步骤。     

细菌DNA的一种大量提取方法

本文介绍了一种大量提取细菌DNA的方法。该法是用60℃裂解菌体,以氯仿-苯酚去除蛋白,用Rnase去除RNA,用1倍体积95%乙醇沉淀DNA,省略了异丙醇沉淀步骤。而且该方法操作方便,对仪器要求不高。该法改进结果是:可稳定地得到50—60%的DNA回收率。可用于革兰氏阴性和阳性细菌的DNA提取。有些菌株用Marmur法和Zaslott法提取,DNA回收率在10%以下,采用本法仍能得到50%的回收率。每次提取的DNA量在毫克数量级。特别适合用于Tm法测定DNA G+C mol%含量所需DNA样品的制备。     

一种快速提取肠道微生物总DNA的方法

采集的兔肠道内容物及其粪便样品,通过分散浸泡、震荡洗涤、分级离心、滤器过滤、DNA提取试剂盒提取纯化,可以获得纯度很高的DNA样品。经0.8%琼脂糖凝胶电泳检测和紫外分光光度计测定,样品A260/A280的比值为1.72±0.02。分别以提取的DNA样品为模板,通过设计的细菌特异引物,对其16S rDNA基因进行PCR扩增,获得了1.6 kb大小特异性很好的预期条带。这为肠道微生物群落的分子生态学研究提供了一种简便、可靠的DNA提取方法。     

一种简单、快速的苏云金芽胞杆菌基因组DNA提取方法

从培养时间、裂解溶液、抽提和沉淀时间等方面对苏云金芽胞杆菌(Bacillus thuringiensis,Bt)基因组DNA提取技术进行改进.提取8株对蛴螬有杀虫活性的野生Bt菌株的基因组DNA,分析其纯度,并进行PCR分析与酶切分析.试验结果表明,新的提取方法耗时36 min,明显短于旧方法所用时间(97 min);两种方法提取的基因组DNA A260/A280均大于1.8,无明显区别;琼脂糖凝胶电泳结果显示,上样量相同时,新方法提取的基因组DNA浓度是旧方法的5倍以上;新方法提取的基因组DNA能作为模板灵敏地扩增出测试基因,所提取的DNA能被限制性内切酶完全酶切,证明DNA具有很高的纯度.本研究改进的基因组DNA提取方法耗时短、产量高,并能满足PCR扩增、酶切等分子生物学需要.     

蔓茎堇菜基因组DNA 3种提取方法的比较研究

以SDS作为解离剂,采用常规SDS法、改进的SDS法及SDS高盐低pH法分离提取蔓茎堇菜基因组DNA.比较3种方法的提取效果,结果表明：常规SDS法DNA得率高但完整性及纯度较差;改进的SDS法DNA质量好但得率相对较低;只有SDS高盐低pH法是最为理想的提取方法,不同组织提取的DNA相对分子质量均大于48 kb,260 nm/280 nm光密度比值在1.7～1.9之间,产率为479.0～543.9μg/g,所得DNA可直接用于限制性内切酶酶切,并适于进行RAPD分析.     

A method for obtaining DNA from compost

An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical–chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA were amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme HhaI.     

Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis

Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations > 0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n = 24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories.     

多环芳烃降解菌X20的鉴定及降解特性

从多环芳烃降解高效的混合菌群中分离筛选到1株多环芳烃降解菌X20,经形态观察和16SrRNA序列分析,属于假单胞菌(Pseudomonas sp.)。采用室内摇瓶培养的方法,研究了该菌在不同环境条件下对菲和芘的降解。结果表明:弱碱环境有利于菌株X20对菲和芘的降解,最适pH为8.0;葡萄糖对菲芘降解率的影响呈抛物线变化,当葡萄糖浓度为0.2%时,X20对菲和芘的降解达到最高;X20对菲和芘的降解率随其初始浓度的上升而降低,菲和芘在初始浓度为10、20和40mg.L-1时的7d降解率分别为56.3%、39.25%、29.75%和41.8%、29.55%、23.50%,芘对X20降解的抑制强度高于菲。本研究结果将为构建高效的多环芳烃降解菌群,提高多环芳烃原位污染土壤的生物修复效果奠定基础。     

中华绒螯蟹与合浦绒螯蟹两地理亚种的线粒体DNA序列变异

根据线粒体16SrDNA的PCR／RFLP鉴定和对Cyt b基因全序列的分析对中国大陆7水系绒螯蟹地理种群的遗传分化进行探讨。结果表明：中华绒螯蟹与合浦绒螯蟹两亚种在17条Cyt b全序列上以40个固定的碱基变异位点相区别。前者分布在瓯江及其以北的水系;后者主要分布在瓯江及其以南的水系。发现了4种在鸭绿江以南的北方水系中生活的合浦亚种单元型,对这些单元型的形成提出了3种可能的解释[动物学报51（5）：862—866,2005]。     

Chelex-100快速提取放线菌DNA作为PCR扩增模板

旨在建立有效扩增16S rRNA基因序列的放线菌DNA快速提取的方法。采用Chelex-100法提取放线菌DNA,使用PCR扩增16S rRNA基因序列评价提取核酸的质量。结果显示,Chelex-100法能够在10 min之内从放线菌中快速提取DNA,所提取的DNA可以直接用于PCR扩增反应,PCR扩增产物电泳条带清晰,符合理论预期结果。因此,Chelex-100法提取放线菌DNA可以作为16S rRNA基因序列PCR扩增的模板,该方法具有经济、简便、快速的特点,适合于放线菌菌株大规模地筛选和分类鉴定。     

内蒙古典型草原细菌群落结构的PCR-DGGE检测

用液氮冻融法和蛋白珠法对内蒙古典型草原土壤基因组DNA进行提取,用PCR—DGGE对细菌群落结构进行分析,并对主要的条带进行测序。发现蛋白珠法比液氮冻融法更能反应出实际的微生物群落结构和组成。内蒙古典型草原土壤细菌主要有5个分支：放线菌门（Actinobacteria）,变形菌门（Proteobacteria）的α、β及γ类群,拟杆纲门（Bacteriodetes）,芽单胞菌门（Gemmatimonadetes）和酸杆菌门（Acidobacteria）。与基因库中进行比较后发现有4个序列和已知的细菌种类相似达到了99％以上。     

PCR-DGGE对长江河口八种野生鱼类肠道菌群多样性的比较研究

目的分析长江河口捕获的8种野生鱼类的肠道菌群多样性的差异并观察这种差异与食性的联系。方法采用PCR-DGGE(denaturing gradient gel electrophoresis)技术,DGGE图谱用PCA(principal component analy-sis)方法进行分析。结果建立了长江口8种鱼野生条件下肠道菌群的DGGE指纹图谱,观察到它们在野生条件下的肠道菌群的差异。其中,营底栖生活的舌鰕虎鱼的肠道菌群和其他7种野生鱼有着明显的差异,其他7种鱼的肠道菌群多样性的差异与它们的食性差异相关。结论PCR-DGGE技术是一种能够快速有效地分析研究鱼类肠道菌群结构的技术。8种野生鱼的肠道菌群的结构有明显的差别,并且食性差异大的鱼类之间肠道菌群差异也     

Comparison of DNA extraction methods for human gut microbial community profiling

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome.     

快速评价肠道微生物的qPCR方法的建立

【背景】近些年,16S rRNA基因测序与宏基因组分析常用于肠道微生物病原体检测。【目的】为了使检测不受限于高成本与耗时长的问题,基于荧光探针的实时荧光定量PCR(real-time fluorescence quantitative PCR, qPCR),建立一种评估人类肠道微生物群组成的平台用于检测肠道微生物丰度。【方法】从公共数据库筛选10种肠道中普遍存在的微生物分类群,使用20个粪便样本验证为10种靶标所设计的特异性引物与探针,最后通过比较qPCR方法和16SrRNA基因测序技术的检测结果来评估该平台的有效性。【结果】10对引物及其探针对靶标分类群具有特异性并且在HITdb数据库中靶向菌种的覆盖率超过70%;样本检测结果的变异系数(coefficient of variation,CV)小于10%,证明了该方法具有很高的稳定性;qPCR方法检测样本中物种的相对丰度与16S rRNA基因序列生物信息学分析结果大部分具有显著相关性(P<0.05)。【结论】本研究根据HITdb数据库设计的靶向微生物群的引物和探针检测到的粪便样本中微生物的相对丰度结果与16S rRNA基因测序结...