Synthesis of novel porous magnetic silica microspheres as adsorbents for isolation of genomic DNA

An improved procedure is described for preparation of novel mesoporous microspheres consisting of magnetic nanoparticles homogeneously dispersed in a silica matrix. The method is based on a three-step process, involving (i) formation of hematite/silica composite microspheres by urea-formaldehyde polymerization, (ii) calcination of the composite particles to remove the organic constituents, and (iii) in situ transformation of the iron oxide in the composites by hydrogen reductive reaction. The as-synthesized magnetite/silica composite microspheres were nearly monodisperse, mesoporous, and magnetizable, with as typical values an average diameter of 3.5 microm, a surface area of 250 m(2)/g, a pore size of 6.03 nm, and a saturation magnetization of 9.82 emu/g. These magnetic particles were tested as adsorbents for isolation of genomic DNA from Saccharomyces cerevisiae cells and maize kernels. The results are quite encouraging as the magnetic particle based protocols lead to the extraction of genomic DNA with satisfactory integrity, yield, and purity. Being hydrophilic in nature, the porous magnetic silica microspheres are considered a good alternative to polystyrene-based magnetic particles for use in biomedical applications where nonspecific adsorption of biomolecules is to be minimized.     

Manipulation and extraction of genomic DNA from cell lysate by functionalized magnetic particles for lab on a chip applications

A novel approach for extracting living cells' genomic DNA materials utilizing functionalized magnetic particles (MPs) is reported in this investigation. This strategy is amenable to handle bio-samples in a miniaturized environment and it offers a possibility to separate and purify DNA from other cell lysate mixtures "on-chip", which is known to be a bottle-neck step in an integrated micro-total-analysis-system (muTAS). "Species-specific" genomic DNA of interest is captured by the MPs based on the hybridization interaction between the biotinylated probes modified MPs and a complementary region of the targeted genome. The genome DNA anchored on the particles can be separated from the rest of cellular mixtures by a simple buffer washing upon the exertion of external magnetic force. Surface modifications of MPs and hybridization conditions affecting the genome capturing efficiency are investigated. Extraction of genomic DNA from E. coli is demonstrated in a silicon/glass-based micro-reactor patterned with a platinum heater and sensors. On-chip extraction and manipulation of genomic DNAs illustrated in this study is a step forward toward a total integrated bioanalytical microsystem for crude cells/sample analysis.     

Chitosan functionalized magnetic particle-assisted detection of genetically modified soybeans based on polymerase chain reaction and capillary electrophoresis

The high quality of DNA template is one of the key factors to ensure the successful execution of polymerase chain reaction (PCR). Therefore, development of DNA extraction methods is very important. In this work, chitosan modified magnetic particles (MPs) were synthesized and employed for extraction of genomic DNA from genetically modified (GM) soybeans. The extraction protocol used aqueous buffers for DNA binding to and releasing from the surface of the MPs based on the pH inducing the charge switch of amino groups in chitosan modified MPs. The extracted DNA was pure enough (A(260)/A(280)=1.85) to be directly used as templates for PCR amplification. In addition, the PCR products were separated by capillary electrophoresis for screening of GM organisms. The developed DNA extraction method using chitosan modified MPs was capable of preparation of DNA templates, which were PCR inhibitor free and ready for downstream analysis. The whole process for DNA extraction and detection was preferable to conventional methods (phenol-chloroform extraction, PCR, and gel electrophoresis) due to its simplicity and rapidity as well as its avoiding the use of toxic reagents and PCR inhibitors.     

Application of gelatin-coated magnetic particles for isolation of genomic DNA from bacterial cells

Gelatin-coated magnetic particles were implemented for bacterial genomic DNA isolation in this study. Based on structural differences in the cell wall, the standard strains Staphylococcus aureus and Escherichia coli were selected. The quantity, quality, and timing process for DNA extraction using gelatin-coated magnetite were compared to reference phenol-chloroform extraction and a commercially available kit. Approximately twice as much DNA was recovered with the use of coated magnetite, providing greater yields than other DNA extraction methods. In addition, the DNA quality was determined using 16S ribosomal DNA (rDNA) gene amplification by polymerase chain reaction (PCR). The described technique is rapid, simple, and a well-suited method to use with PCR for diagnosis of bacterial infections.     

一种快速提取基因组DNA的方法

采采用氧化硅超顺磁性纳米磁珠和自己设计的试剂体系及提取流程,建立了一种基因组DNA的快速提取方法,该方法以氧化硅磁珠为固相吸附载体,盐酸胍、 -巯基乙醇和SDS为主要裂解吸附试剂。以全血或培养细胞为实验材料进行基因组DNA的提取结果显示用本文建立的方法提取100 L小鼠抗凝血,可得2～3 g基因组DNA, OD260/OD280为1.8 ± 0.05,其纯度可满足后续的酶切和PCR生物操作要求。该方法整个提取过程只需12分钟,不需特殊实验条件同时可省略蛋白酶K的消化过程和离心操作,适用于一般实验室的需求,是一种操作简便、快速高效的提取方法。     

石斛干品基因组DNA的提取与RAPD分析

市场中药干品的药性差异一直是影响中药标准化的瓶颈,而检测技术相对落后是导致这一现象的主要原因。DNA分子水平检测的困难是药材干品的基因组DNA难以提取。本文以铁皮石斛(Dendrobium candidum)干茎为材料,采用了四种方法从干品石斛中提取基因组DNA。结果表明,采用改良的CTAB法可从石斛干品尤其是干茎皮中提取质量较高的基因组DNA,其分子量大于23kb,以此DNA为模板进行不同引物的PCR扩增可获得清晰的RAPD条带。该研究初步建立了石斛干品合适的RAPD技术体系。     

一种简便高效的酵母基因组提取方法

提取基因组进行检测是酵母研究过程中的必要步骤之一。以毕赤酵母菌株GS115作为研究对象,主要成分为0.2 mol/L醋酸锂和1% SDS的酵母裂解液能高效的裂解酵母细胞壁。与两种酵母基因组提取试剂盒相比,该方法从相同体积的酵母培养液中获得的基因组的量高5倍以上,并且操作简便、快速,能在2 h内完成一次提取过程,极大地缩短了时间。以GS115中的内源AOX基因为目的基因,对提取的基因组进行PCR检测和Southern杂交检测,进一步验证了基因组的质量。因此,本文建立了一种简便、快速、经济而高效的酵母基因提取方法。     

Does extraction of DNA and RNA by magnetic fishing work for diverse plant species?

An automated nucleic acid extraction procedure with magnetic particles originally designed for isolation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from animal tissues was tested for plant material. We isolated genomic DNA and total RNA from taxonomically diverse plant species representing conifers (Scots pine), broad-leaved trees (silver birch and hybrid aspen), dwarf shrubs (bilberry), and both monocotyledonous (regal lily) and dicotyledonous (Saint John's wort, round-leaved sundew, and tobacco) herbaceous plants. Buffers developed for DNA extraction were successfully used in addition to manufacturer's extraction kits. The quality of RNA was appropriate for many applications, but the quality of DNA was not always sufficient for polymerase chain reaction (PCR) amplification. However, we could strikingly improve the quality by eliminating the adherent compounds during the extraction or later in the PCR phase. Our results show that the use of the procedure could be extended to diverse plant species. This procedure is especially suitable for small sample sizes and for simultaneous processing of many samples enabling large-scale plant applications in population genetics, or in the screening of putative transgenic plants.     

A simplified universal genomic DNA extraction protocol suitable for PCR

Conventional genomic DNA extraction protocols need expensive and hazardous reagents for decontamination of phenolic compounds from the extracts and are only suited for certain types of tissue. We developed a simple, time-saving and cost-efficient method for genomic DNA extraction from various types of organisms, using relatively innocuous reagents. The protocol employs a single purification step to remove contaminating compounds, using a silica column and a non-hazardous buffer, and a chaotropic-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from cell lysates. We used this system to extract genomic DNA from different tissues of various organisms, including algae (Dunaliella salina), human peripheral blood, mouse liver, Escherichia coli, and Chinese hamster ovary cells. Mean DNA yields were 20-30 μg/cm(3) from fresh tissues (comparable to yields given by commercial extraction kits), and the 260/280 nm absorbance ratio was 1.8-2.0, demonstrating a good degree of purity. The extracted DNA was successfully used in PCR, restriction enzyme digestion and for recombinant selection studies.     

煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较

目的:在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法:分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果:相同处理量(108个)的革兰氏阳性菌(1株)、革兰氏阴性菌(4株)、古菌(1株)经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260/OD280的值更接近1.8-2.0(纯DNA的OD260/OD280在1.8-2.0之间),前者所提DNA回收率最大可达后者的16.7倍;煮沸裂解法只需较少菌(102个)便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论:两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随菌浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。     

An improved protocol and a new grinding device for extraction of genomic DNA from microorganisms by a two-step extraction procedure

Current protocols to extract genomic DNA from microorganisms are still laborious, tedious and costly, especially for the species with thick cell walls. In order to improve the effectiveness of extracting DNA from microbial samples, a novel protocol, defined as two-step extraction method, along with an improved tissue-grinding device, was developed. The protocol included two steps, disruption of microbial cells or spores by grinding the sample together with silica sand in a new device and extraction of DNA with an effective buffer containing cell lysis chemicals. The device was prepared by using a commercial electric mini-grinder, adapted with a grinding stone, and a sample cup processed by lathing from a polytetrafluoroethylene rod. We tested the method with vegetative cells of four microbial species and two microbial spores that have thick cell walls and are therefore hard to process; these included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S. cerevisiae and T. viride, respectively, representing Gram-positive bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this new method and device extracted usable quantities of genomic DNA from the samples. The DNA fragments that were extracted exceeded 23 kb. The target sequences up to about 5 kb were successfully and exclusively amplified by PCR using extracted DNA as the template. In addition, the DNA extraction was finalized within 1.5 h. Thus, we conclude that this two-step extraction method is an effective and improved protocol for extraction of genomic DNA from microbial samples.     

Methods for DNA extraction from Candida albicans

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.     

革兰氏阳性细菌基因组DNA提取方法的比较及优化

【目的】基因组DNA提取效率和质量对分子生物学相关研究起着关键的作用,革兰氏阳性细菌由于细胞壁较厚、难破裂使其基因组DNA提取的难度增大,本文旨在寻找一种高效稳定的DNA提取方法。【方法】以Clostridium thermocellum和Thermoanaerobacterium thermosaccharolyticum为实验菌株,使用6种DNA提取方法对C. thermocellum基因组DNA进行提取,对比其提取效果和产率。【结果】改良的SDS-碱裂解法提取得到的DNA浓度较高(400 mg/l左右),且平行样间浓度和纯度稳定。【结论】为革兰氏阳性细菌基因组DNA提取提供参考。     

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.     

煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较

目的：在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法：分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果：相同处理量（108个）的革兰氏阳性菌（1株）、革兰氏阴性菌（4株）、古菌（1株）经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260／OD280的值更接近1．8-2．0（纯DNA的OD260／OD280在1．8—2．0之间）,前者所提DNA回收率最大可达后者的16．7倍;煮沸裂解法只需较少菌（102个）便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论：两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随茵浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。     

Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

马蹄金基因组DNA的提取方法比较

对马蹄金基因组DNA的提取方法———快速提取法、小量提取法和大量提取法进行了对比分析,结果表明,利用快速提取法可在20 min内快速可靠地从马蹄金(Dichondra repensForst.)组织中提取DNA,与小量提取法和大量提取法获得的DNA用于PCR检测结果一致,可为转基因植物的快速检测提供方法。     

An improved method to extract DNA from mango Mangifera indica

High quality genomic DNA is the first step in the development of DNA-based markers for fingerprinting and genetic diversity of crops, including mango (Mangifera indica L.), a woody perennial. Poor quality genomic DNA hinders the successful application of analytical DNA-based tools. Standard protocols for DNA extraction are not suitable for mango since the extracted genomic DNA often contains secondary metabolites that interfere with analytical applications. In this study, we employed an additional step to remove polysaccharides, polyphenols and secondary metabolites from genomic DNA extracted from young or mature leaf tissue; then a modified traditional cetyl trimethyl ammonium bromide (CTAB) method was applied. The use of 0.4 M glucose improved DNA quality and avoided contamination and browning by polyphenolics, relative to the traditional CTAB method. This is an easy and efficient method for genomic DNA extraction from both young and mature leaves of mango. The isolated DNA was free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by its clear colour, its viscosity, A²⁶⁰/A²⁸⁰ ratio and suitability for PCR-based reactions. This modified protocol was also used to extract high quality genomic DNA from other woody perennials, including walnut, guava, lychee, pear, grape and sugarcane.     

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.     

小鼠胚胎胃肠道细菌基因组DNA提取方法比较

目的:筛选能均衡地提取小鼠胚胎胃肠道微生物区系各种细菌总DNA的方法.方法:分别采用反复冻融法、CTAB -SDS法、柱式基因组DNA提取试剂盒法提取小鼠胚胎胃肠道细菌基因组DNA,对其进行琼脂糖凝胶电泳、紫外分光光度计测定、PCR扩增等质量检测.结果:CTAB -SDS方法提取的基因组DNA纯度较高,OD260/OD280平均值最高,为1.845,电泳条带清晰,能满足下游的PCR扩增等分子操作.结论:确定CTAB -SDS方法为提取小鼠胚胎胃肠道细菌基因组DNA的最佳方法,为研究不同种动物胚胎的肠道菌群的结构和多样性奠定了基础.