对中国黄牛毛囊DNA六种提取方法的研究

本研究的目的是寻求一种简便、高效和对黄牛没有创伤的DNA提取方法,从而为黄牛基因组DNA的制备提供最优可行方案。本研究以中国黄牛毛囊作为DNA的提取材料,设计了离心柱法、磁珠法、SDS法、CTAB法、碱裂解法和煮沸法等6种DNA提取方法,然后分别对提取的DNA样本进行了分光光度计鉴定、PCR扩增和琼脂糖凝胶电泳。发现在这6种方法中,耗时最短且成本最低的是煮沸法;DNA纯度、浓度和完整度最理想的是CTAB法;而磁珠法可实现大规模高通量提取,能减少实验者的手动工作量,并且适用于基因测序各种常规操作。以此得出,适用于基因测序的6种黄牛毛囊DNA提取方法中最优的是磁珠法。本研究对中国黄牛毛囊DNA的6种提取方法进行了总结和分析,为今后提取毛囊DNA的研究提供了借鉴和参考。     

木棉基因组DNA提取方法的比较研究

木棉组织细胞含有多酚类、糖类、萜类化合物和其他次生代谢物质,很难得到高质量的基因组DNA。为了获得高质量的木棉基因组DNA,本实验以两个不同地方采集的木棉成熟叶片为材料,采用CTAB法、SDS法、普通试剂盒、磁珠法试剂盒以及对以上4种方法进行改良后的4种方法,提取总的木棉DNA。通过微量紫外-分光光度计、琼脂糖电泳对所提取DNA的纯度、得率,毒害及耗时等方面进行了比较。结果表明:经改良后提取的DNA质量都有所提高,且改良的磁珠法试剂盒效果最佳,可直接用于下游分子生物学实验。     

木棉基因组DNA提取方法的比较研究

木棉组织细胞含有多酚类、糖类、萜类化合物和其他次生代谢物质,很难得到高质量的基因组DNA。为了获得高质量的木棉基因组DNA,本实验以两个不同地方采集的木棉成熟叶片为材料,采用CTAB法、SDS法、普通试剂盒、磁珠法试剂盒以及对以上4种方法进行改良后的4种方法,提取总的木棉DNA。通过微量紫外-分光光度计、琼脂糖电泳对所提取DNA的纯度、得率,毒害及耗时等方面进行了比较。结果表明:经改良后提取的DNA质量都有所提高,且改良的磁珠法试剂盒效果最佳,可直接用于下游分子生物学实验。     

人snail真核表达载体构建与鉴定

目的:构建人snail基因真核表达载体并鉴定。方法:使用RT-PCR法获取人snail基因全长c DNA,经Bam H I、Eco R I双酶切、连接,插入pc DNA3.1(+)真核表达载体,转化TOP10感受态细胞,用含氨苄青霉素的LB培养基筛选阳性克隆,提取质粒双酶切电泳及测序鉴定,瞬时转染siha细胞Western-blot从蛋白水平鉴定重组质粒在真核细胞内的表达。结果:pc DNA3.1-snail重组质粒经酶切电泳符合预期片段,测序鉴定插入片段与NCBI Gen Bank文库中人snail序列一致,重组质粒瞬时转染后snail蛋白表达量明显增高。结论:成功构建pc DNA3.1-snail重组质粒载体,为进一步探讨snail基因生物学功能奠定了基础。     

磁珠法快速提取基因组DNA的实验研究

针对临床标本基因组DNA的提取方法缺乏广泛适用性,提取步骤繁琐,需要进行离心操作,并且提取过程中会用到苯酚、氯仿等有机试剂,会对操作人员有一定的危害性等不足,本研究拟建立一种适用于临床标本的基因组DNA快速提取方法。在传统的基因组DNA提取试剂和方法的基础上,本研究采用二氧化硅修饰的超顺磁珠设计了一种基因组DNA快速提取方法;探讨磁珠用量、裂解液p H、盐酸胍浓度等因素对基因组DNA提取效率的影响并采用凝胶电泳实验进行验证。当磁珠用量在50~100μg/100 mg样品,裂解液p H约为6,盐酸胍的浓度为6 mol/L时基因组DNA提取效果好、效率高,且磁珠的用量与吸附表面积成正比,但达到一定用量后不会增加提取量,对于100 mg肺组织,适宜的磁珠用量为80μg。此基因组DNA提取方法高效省时,简便快捷,性价比高,适用临床大量样本基因组DNA提取。     

骨皮质核DNA分析在法医学中的应用研究

目的:为探讨在白骨化案件的骨皮质中提取到一定质和量的可供核DNA分析的DNA模板,本文从受环境因素和生物因素影响较小的骨皮质中有效地提取到了核DNA,并成功地进行DNA分析,进行个人识别和亲子鉴定.方法:采集10根无关个体胫骨骨皮质,分别用有机法、Chelex-100法、有机法结合Chelex-100法、有机法结合磁珠纯化柱法4种方法提取骨皮质核DNA,用常规荧光标记复合STR基因分型法成功分析骨皮质核DNA,获得满意STR基因座分型.结果:有机法能提取到骨皮质核DNA,进行STR分型时部分样本图谱峰值不均衡;仅用Chelex-100法提取的核DNA得不到STR分型结果或出现较多的等位基因缺失;有机法结合Chelex-100法、有机法结合磁珠纯化柱法提取的核DNA均能成功进行STR分型,没有等位基因的缺失,其中有机法结合磁珠纯化柱法提取的核DNA检测成功率最高.结论:骨皮质中能提取到核DNA,可以成功地进行DNA分析.     

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。     

磁珠法快速提取鉴定DNA的实验研究

探索用磁珠法进行动植物DNA的快速提取与电泳鉴定实验。操作简捷直观、降低了实验成本,提升了实验水平,使得高纯度DNA的提取实验今后可以在大学生、中学生的生物学、遗传学实验室进行,给更多的学生和教师、科研人员参与生命科学的研究提供了机会。     

一种经济高效的用Silica提取纯化质粒DNA的方法

目的：为了达到批量提取质粒DNA的目的,在多次实验的基础上,建立一种经济、高效的质粒提取方法。方法：以pUC18、pET28b、pCAMBIA1304等3种质粒为材料,分别采用silica法和碱裂解法提取质粒DNA,通过质粒DNA浓度的紫外分光光度法定量测定、电泳分析和HindⅢ酶切鉴定,对两种质粒提取方法的效果进行了比较与评价;对silica法进行了改进和优化,进行大批量重组子的提取和验证。结果：silica法和碱裂解法提取质粒DNA效果相当,都可进行后续实验,但silica法具有经济、高效、无毒的优势。结论：silica法是一种简单、经济、高效的质粒提取方法,可用于批量质粒DNA提取。     

玉米基因组DNA提取及浓度测定方法评价

以非转基因玉米种子为材料,比较了常用的3种植物基因组DNA提取试剂盒及改良的CTAB法,通过琼脂糖凝胶电泳、紫外分光光度及实时荧光PCR扩增检测,对提取得到的基因组DNA的纯度、得率及4种提取方法的重复性、提取时间进行分析;比较紫外分光光度法、Qubit荧光法、Pico Green荧光分光光度法,以实时荧光定量PCR检测结果为参照,对3种DNA浓度测定方法的准确性进行分析.结果显示,磁珠法(Promega)最适合应用于快速、简便、高效检测中的植物基因组DNA提取,能有效获得纯度高、完整性好的基因组DNA,并且磁珠法提取效率高,重复性好,提取时间短;在基因组DNA浓度测定中,紫外分光光度法、Qubit荧光法、Pico Green荧光分光光度法的相对误差分别为99.8％、49.8％和28.9％,表明Pico Green荧光分光光度法测定DNA浓度的准确度最高.     

Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes

To meet the new challenge of generating the draft sequences of mammalian genomes, we describe the development of a novel high throughput 96-well method for the purification of plasmid DNA template using size-fractionated, acid-washed glass beads. Unlike most previously described approaches, the current method has been designed and optimized to facilitate the direct binding of alcohol-precipitated plasmid DNA to glass beads from alkaline lysed bacterial cells containing the insoluble cellular aggregate material. Eliminating the tedious step of separating the cleared lysate significantly simplifies the method and improves throughput and reliability. During a 4 month period of 96-capillary DNA sequencing of the Rattus norvegicus genome at the Baylor College of Medicine Human Genome Sequencing Center, the average success rate and read length derived from >1 800 000 plasmid DNA templates prepared by the direct lysis/glass bead method were 82.2% and 516 bases, respectively. The cost of this direct lysis/glass bead method in September 2001 was ~10 cents per clone, which is a significant cost saving in high throughput genomic sequencing efforts.     

大肠杆菌质粒DNA提取方法的优化

目的:简化操作流程,缩短提取时间,降低试验对操作者的危害。方法:该方法去除酚、氯仿等有害试剂,采用LiCl沉淀去除质粒制备物中小片段核酸(包括DNA和RNA);工程菌生长至对数期时通过加入氯霉素后不仅方便质粒DNA的提取过程中蛋白质的去除,而且可使质粒的拷贝数进一步增加,提高质粒DNA产量的目的。结果:实验改进方法所提取的质粒DNA产量高于常规方法,达到20μg/mL。结论:改进方法提取的质粒DNA,其下游的内切酶消化,PCR、重组质粒鉴定、转化大肠杆菌等实验的结果和重复性都令人满意,完全可用于一般的分子生物学研究。     

Comparing the performance of three ancient DNA extraction methods for high‐throughput sequencing

The DNA molecules that can be extracted from archaeological and palaeontological remains are often degraded and massively contaminated with environmental microbial material. This reduces the efficacy of shotgun approaches for sequencing ancient genomes, despite the decreasing sequencing costs of high‐throughput sequencing (HTS). Improving the recovery of endogenous molecules from the DNA extraction and purification steps could, thus, help advance the characterization of ancient genomes. Here, we apply the three most commonly used DNA extraction methods to five ancient bone samples spanning a ~30 thousand year temporal range and originating from a diversity of environments, from South America to Alaska. We show that methods based on the purification of DNA fragments using silica columns are more advantageous than in solution methods and increase not only the total amount of DNA molecules retrieved but also the relative importance of endogenous DNA fragments and their molecular diversity. Therefore, these methods provide a cost‐effective solution for downstream applications, including DNA sequencing on HTS platforms.     

Modified low‐salt CTAB extraction of high‐quality DNA from contaminant‐rich tissues

The increasing use of high‐throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low‐salt CTAB (MoLSC) extraction protocol to accommodate contaminant‐rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle. Comparisons of purity and molecular integrity showed that our in‐house protocol yielded genomic DNA generally free of contaminants and shearing, whereas the traditional CTAB method and some of the commercial kits yielded DNA unsuitable for some applications of massively parallel sequencing. Our open‐source MoLSC protocol provides a cost‐effective, scalable, alternative DNA extraction method that can be easily optimized and adapted for sequencing applications in other contaminant‐rich samples.     

Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples

Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next‐generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high‐quality DNA extraction procedures for obtaining the minute quantities of short‐fragmented food DNA. Automatic high‐throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole‐body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high‐throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor‐intensive, low‐throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost‐efficient and innovative methodology at low contamination risk also in trophic ecology.     

A comparison of DNA extraction methods for high‐throughput DNA analyses

The inclusion of next‐generation sequencing technologies in population genetic and phylogenetic studies has elevated the need to balance time and cost of DNA extraction without compromising DNA quality. We tested eight extraction methods – ranging from low‐ to high‐throughput techniques – and eight phyla: Annelida, Arthropoda, Cnidaria, Chordata, Echinodermata, Mollusca, Ochrophyta and Porifera. We assessed DNA yield, purity, efficacy and cost of each method. Extraction efficacy was quantified using the proportion of successful polymerase chain reaction (PCR) amplification of two molecular markers for metazoans (mitochondrial COI and nuclear histone 3) and one for Ochrophyta (mitochondrial nad6) at four time points – 0.5, 1, 2 and 3 years following extraction. DNA yield and purity were quantified using NanoDrop absorbance ratios. Cost was estimated in terms of time and material expense. Results show differences in DNA yield, purity and PCR success between extraction methods and that performance also varied by taxon. The traditional time‐intensive, low‐throughput CTAB phenol–chloroform extraction performed well across taxa, but other methods also performed well and provide the opportunity to reduce time spent at the bench and increase throughput.     

一种快速的胚胎组织总RNA的提取方法

简要介绍一种改进的提取人体器官组织总RNA的方法,该方法具有费用低,快速简便,重复性好的优点,提取的RNA无DNA等污染物,完全能满足基因表达研究的需要。     

TempliPhi: A Sequencing Template Preparation Procedure That Eliminates Overnight Cultures and DNA Purification

Preparing plasmid templates for DNA sequencing is the most time-consuming step in the sequencing process. Current template preparation methods rely on a labor-intensive, multistep procedure that takes up to 24 h and produces templates of varying quality and quantity. The TempliPhi™ DNA Sequencing Template Amplification Kit eliminates the requirement for extended bacterial growth prior to sequencing and saves laboratory personnel hands-on time by eliminating the centrifugation and transfer steps currently required by older preparatory methods. In addition, costly purification filters and columns are not necessary, as amplified product can be added directly to a sequencing reaction. Starting material can be any circular template from a colony, culture, glycerol stock, or plaque. Based on rolling circle amplification and employing bacteriophage Phi29 DNA polymerase, the method can produce 3–5 μg of template directly from a single bacterial colony in as little as 4 h. Implementation of these procedures in a laboratory or core sequencing facility can decrease cost on tips, plates, and other plasticware, while at the same time increase throughput.     

High-throughput plasmid DNA purification for 3 cents per sample

To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical difficulty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week.