用于微生物多样性分析的棉田土壤总DNA的提取

目的：探讨适用于微生物多样性研究的棉田土壤微生物总DNA提取方法。方法：采用4种方法提取不同连作和轮作处理的棉田土壤微生物总DNA,比较其纯度、产率、片段大小,并应用ARDRA技术验证其质量。结果：其中3种方法均可获得23kb的DNA片段,但不同方法提取的DNA的产率和纯度上有明显差异。改良CTAB-SDS法提取的DNA完整性好,得率为24.20μg.g-1干土,纯化后A260/A280和A260/A230为分别为1.80和1.70,纯化回收率可达70.1%,完全适用于后续的PCR分析。结论：采用该法提取棉田土壤总DNA简便而高效。对该法提取获得的棉田土壤微生物总DNA进行ARDRA和DGGE分析,所得图谱能较全面地反映不同处理间微生物多样性及群落结构的差别,为不同栽培体系下棉田土壤微生物的分子生态学研究提供了基础。     

红豆杉属植物三种不同总DNA提取方法的分析比较

红豆杉属植物均为濒危物种,也是国家一级保护植物.以红豆杉属植物叶片为材料,利用三种不同的DNA提取方法提取总DNA,用分光光度计和琼脂糖凝胶电泳方法检测所得总DNA的得率和纯度,用PCR扩增的方法检测所得总DNA的质量,并对三种不同提取方法的结果进行了比较分析.结果表明:CTAB法提取的DNA纯度和得率均较高,可直接用...     

两株假单胞菌菌株的RAPD分析及分子生物学鉴定

根据DNA随机扩增多态性(RandomAmplifiedPolymorphicDNA,简RAPD)分子标记技术设计鉴别引物,建立一种快速、准确检测病人体内新发现的假单胞菌菌株的分子生物学方法.采用RAPD分析方法对该菌种的对照菌株AcinetobactercalcoaceticusKHW14(简称A.calcoaceticusKHW14)和新分离的菌株Acinetobactercalcoaceticus(简称A.calcoaceticus)进行指纹分析,依据两菌株的差异序列设计两对引物,并建立最佳的PCR扩增体系,产物经1.2%琼脂糖凝胶电泳得菌株特异性电泳图谱.此图谱可作为鉴定两菌株的标准图谱,RAPD分析方法具有良好的重复性,同时也进一步验证了两菌株的同源性.     

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。     

Comparison of rapid DNA extraction methods applied to PCR identification of medicinal mushroom Ganoderma spp

Four different DNA extraction methods were used to extract genomic DNA of the medicinal mushroom Lingzhi from its developing stage materials, such as mycelium, dry fruiting body, or sliced and spore powder or sporoderm-broken spore powder. The DNA samples were analyzed using agarose gel electrophoresis, UV spectrophotometer, and PCR amplification. According to the average yields and purity of DNA, high salt concentrations and low pH methods were the best for DNA extraction. The mycelia and sporoderm-broken spore powder yielded higher and purer DNA. The method developed could effectively eliminate the influence of the secondary metabolites to DNA extraction. The DNA samples extracted from the developed method could be successfully used for PCR applications.     

Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes

A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.     

Comparison of Rapid DNA Extraction Methods Applied to PCR Identification of Medicinal Mushroom Ganoderma spp.

Abstract

Four different DNA extraction methods were used to extract genomic DNA of the medicinal mushroom Lingzhi from its developing stage materials, such as mycelium, dry fruiting body, or sliced and spore powder or sporoderm‐broken spore powder. The DNA samples were analyzed using agarose gel electrophoresis, UV spectrophotometer, and PCR amplification. According to the average yields and purity of DNA, high salt concentrations and low pH methods were the best for DNA extraction. The mycelia and sporoderm‐broken spore powder yielded higher and purer DNA. The method developed could effectively eliminate the influence of the secondary metabolites to DNA extraction. The DNA samples extracted from the developed method could be successfully used for PCR applications.     

基于矿石纳米材料的DNA转化的机理初探及方法改进

一种价格便宜、资源丰富和对人体无害的矿石纳米材料——海泡石,用于微生物DNA转化。该法简便、快速、对人体健康无害,但对这种转化方法机制的理解还有很多问题。通过小片段RNA竞争试验,发现了与先前报道不同的转化机制。同时,对该法进行了优化,结果可以实现对冷藏1个月的大肠杆菌EscherichiacoliDH5α单菌落直接转化,无需感受态制备和处理后的温育过程,可得到比钙转高的转化率。由于可优化的参数很多,所以这种转化方法可以提升的空间很大。同时,该方法可用于探索其他用钙转和电转未成功的微生物。     

一种快速提取细菌总DNA的方法研究

随着分子生物学技术应用于环境微生物研究的深入开展,占自然界微生物物种总数的90%以上的不能人工培养或培养困难的微生物已经可以借助分子生物学技术进行功能基因的开发和利用。而快速得到纯度较高,结构完整的细菌染色体DNA成为这一技术得以实现的前提。本文报道了利用高温处理和SDS的裂解作用相结合而建立的一种快速、简便的提取细菌染色体DNA的方法。经过脉冲电泳实验证明,利用本方法提取得到的几种革兰氏阳性和革兰氏阴性菌株的基因组DNA结构完整,并且无明显降解,无须经过纯化,可以直接进行PCR扩增和酶切等分子生物学操作,将此方法进一步应用于土壤环境DNA的提取方面,同样达到了快速得到大片段、高质量的环境微生物基因组的目的,为研究未培养的环境微生物多样性打下了坚实的基础,同时为环境基因组的提取提供了一个新的途径。     

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。     

改良CTAB法提取番石榴叶片总DNA

目的:从番石榴叶片中快速提取高质量的总DNA。方法:改良CTAB法。主要改进之处在于不用液氮,而是直接研磨硅胶干燥样品;用高浓度CTAB、低浓度乙醇与NaCl盐析相结合等方法去除多糖。结果:应用改良后的方法可以快速提取番石榴叶片总DNA,有效去除组织中的多糖、蛋白质,抑制提取过程中的组织褐变。提取的DNA可用于限制性内切酶酶切和PCR扩增。结论:传统CTAB法经过改良,可用于快速提取番石榴高质量DNA。     

红树林土壤总DNA不同提取方法比较研究

获得高浓度、大片段、无偏好的土壤微生物总DNA是土壤微生物分子生态学研究和宏基因组文库构建的基础。本研究采用了5种方法从红树林土壤中提取DNA,并对5种方法提取出的DNA的质量和产量进行比较评价。结果表明,5种方法均可从土壤中提取到DNA,但不同方法提取到DNA的产量和质量存在明显差异。Bio101FastPrep?SPINKit(forSoil)抽提到的DNA得率最高,适合分子生态学研究;SDS-GITC-PEG法提取的DNA纯度最高,所得到的DNA片段较大(>48kb),有利于构建宏基因组文库。     

High yield DNA extraction from the snake cast-off skin or bird feathers using collagenase

A simple method to extract DNA in high yield from the snake cast-off skin or bird feathers was developed. The molecular weight of the extracted DNA was higher by this than the conventional method and the yield of DNA was increased by more than one hundred fold. The DNA extracted by this method could be used for PCR and other analyses. This method could be applied to various samples, for instance, extracting DNA from bird feather in general.     

Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies.     

An effective protocol to solve the problem in genomic DNA isolation of tung tree

Tung tree (Vernicia fordii) is a native and oil-producing woody plant in China. The oil is industrially important and promising biodiesel raw material. However, until recently the lack of effective protocols for the extraction of genomic DNA had made DNA-based molecular studies of tung tree difficult. Here, four conventional protocols and one novel protocol were compared for their capacity in isolating DNA from tung tree leaves of different age. Our results showed that all the four conventional protocols could isolate DNA from old leaves, two from matured leaves, but none from young leaves. However, the detectable DNA samples contained many contaminations, leading to overestimation of DNA concentration measured by ultraviolet spectrophotometer, also interfering with the downstream PCR reaction. The novel protocol could produce high-yield and good-quality DNA from tung tree leaves regardless of leaf age. Its key steps were that a single leaf tissue sample could be recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations (termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively) were conducted. All the four DNA samples of a single tissue were good template for PCR reaction. The novel protocol is an effective method for genomic DNA isolation of tung tree.     

东方白鹳粪便总DNA五种提取方法的比较

本研究比较东方白鹳(Ciconia boyciana)粪便总DNA的5种提取方法,旨在为后续物种性别鉴定和DNA条形码鉴定提供合适的方法.采用十六烷基三甲基溴化铵法(CTAB法)、十二烷基磺酸钠裂解法(SDS法)、Tiangen试剂盒法、Qiagen试剂盒法和异硫氰酸胍法(GuSCN法)对取自天津动物园的东方白鹳新鲜粪...     

高温环境样品总DNA直接和间接提取方法的比较

分别采用两种环境总DNA直接提取法和一种间接提取法从6种温泉菌席样品中提取总DNA,以DNA粗产物的纯度、能否用于后续PCR扩增及PCR-DGGE(变性梯度凝胶电泳)所反映的微生物多样性为评价指标对两类方法进行比较和评价。研究发现,虽然间接提取法效率低下,但对于高温极端环境中生物量较小的样品,间接法能得到有研究价值的、纯度较高的环境样品总DNA,而直接法得到的DNA量小且不适于PCR扩增操作。在使用这2类方法都能得到可用于研究操作的DNA的情况下,间接提取法能更好的体现环境样品中微生物的多样性。     

Detection of single nucleotide polymorphisms by PCR conformation-difference gel electrophoresis

The most common genetic variations in the human genome, single nucleotide polymorphisms (SNPs), are ideal biomarkers and are used extensively in disease research. Here we introduce a novel method of PCR-conformation-difference gel electrophoresis (PCR-CDGE) used for detecting SNPs. The principle of PCR-CDGE relies PCR products from different homozygous DNA samples showing dissimilar migration patterns upon PAGE due to their conformational differences. PCR products from heterozygous DNA samples may exhibit two or more bands in PAGE because of the existence of DNA homoduplexes and heteroduplexes. In this study, analysis of two SNPs showed that PCR-CDGE is an accurate, simple, rapid, low-cost, and high-throughput genotyping method that could be used in most laboratories.     

Detection of varicella-zoster virus DNA by field-inversion gel electrophoresis

A new method for detection of varicella-zoster virus (VZV) DNA using field-inversion gel electrophoresis (FIGE) was devised. VZV-genomic DNA could be differentiated from the host cell DNA of human embryonic lung (HEL) fibroblasts infected with VZV under electrophoretic conditions allowing resolution of linear and double-stranded DNAs in the 49-230 kilobase pairs (Kb) range. The detection of VZV-genomic DNA from infected HEL cells was successful regardless of whether the VZV was a laboratory strain, live vaccine strain, or fresh isolate. Under the same electrophoretic conditions, DNA of VZV-infected HEL cells could be clearly differentiated from DNA obtained from HEL cells infected with herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), or human cytomegalovirus (HCMV). Furthermore, VZV genomic DNA could be detected from as small a sample as 1.9 x 10(4) VZV-infected HEL cells. Finally, we could detect VZV genomic DNA from 10 samples of vesicle tissue (blister lids, each about 1-4 mm2) and one sample of vesicle fluid (about 5 microliters) obtained from patients diagnosed as having herpes-zoster. The results of this study indicate that FIGE is a simple and promising method for the detection of VZV from clinical materials as well as infected in vitro cultured cells.     

Transfer of plasmid DNA to Brevibacterium lactofermentum by electrotransformation

The Escherichia coli-Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed. Optimal conditions gave an efficiency of 10(6) transformants per microgram of DNA. Two recalcitrant strains could be electrotransformed when an ampicillin pretreatment step was used. Electrotransformation experiments using DNAase or different structural forms of plasmid DNA showed that the electrotransformation process is quite different from natural transformation involving competence development. Restriction-modification-proficient B. lactofermentum could be efficiently electrotransformed with pBLA DNA isolated from E. coli. This restriction-modification system therefore seems to be overcome by electrotransformation. Thus electrotransformation may efficiently replace the protoplast bacterial transformation method.