黑麂粪便DNA提取及其PCR检测

采集了黑麂(Muntiacuscrinifrons)的新鲜粪便以及在野外自然条件下保存较长时间的粪便样品,晾干后带回实验室,提取其DNA;同时提取黑麂肌肉、皮张样品的DNA,用以对比粪便样品的提取效果。电泳检测结果显示,此方法使用实验室中常用的分子生物学试剂,可以从黑麂粪便样品中抽提到高质量的粪便DNA并克服分子粪便学研究中常见的PCR反应抑制物的影响。为其它濒危鹿科动物的非损伤性取样提供了的新途径,为其遗传结构、遗传多样性现状等研究提供了更加广阔的取材空间。     

野生动物分子水平研究中的取样方法进展

对野生动物进行分子水平研究时 ,在取材上经常存在野外找不到动物或得到的材料提取不到足够的DNA等困难。但随着PCR技术的产生和DNA提取技术的进步 ,研究材料也不再局限于新鲜的组织样本 ,从而取样的范围扩大到微量的血液、单根的毛发、羽毛、指甲、唾液、粪便、骨骼 ,甚至古代化石等样品 ,研究范围随之也不断扩大。     

野生动物分子水平研究中的抽样方法进展

对野生动物进行分子水平研究时,在取材上经常存在野外找不到动物或得到的材料提取不到足够的DNA等困难。但随着PCR技术的产生和DNA提取技术的进步,研究材料也不再局限于新鲜的组织样本,从而取样的范围扩大到微量的血液、单根的毛发、羽毛、指甲、唾液、粪便、骨骼,甚至古代化石等样品,研究范围随之也不断扩大。     

一种提取动物基因组总DNA的野外样品保存方法

为了确定一种方便的野外动物样品保存方法,以新鲜材料作对照,从-20℃冰箱、70%乙醇、含50mmol/L EDTA的70%乙醇、95%乙醇、液氮处理的高原鼠肌肉和肝脏组织中提取基因组总DNA。通过琼脂糖凝胶电泳和紫外分光光度计对提取的基因组总DNA质量进行检测。结果显示:相同处理的肝脏DNA产量大,肌肉组织提取的DNA质量好;各种保存方法提取的DNA降解程度依次为,-20℃冰箱、70%乙醇>含50mmol/L EDTA的70%乙醇、95%乙醇>液氮>新鲜。选择新鲜肌肉和95%酒精处理的肌肉样品提取的总DNA作模板,进行微卫星PCR扩增,均可获得清晰的电泳带。将该方法用于高原鼢鼠,进行线粒体12S rRNA、Cytb和D-loop区测序,结果显示该方法保存的样品与新鲜样品没有差别。因此,在野外用95%乙醇固定肌肉样品是一种可行的样品保存方法。     

陈旧皮张中DNA提取的新方法

对传统的馆藏陈旧皮张标本DNA提取方法进行了改进,所提DNA分子量可达1kb,而且具有样品用量少（约0.01g),消化时间短（约14h)和操作步骤简单等优点,利用所提DNA,对小熊猫等珍稀动物线粒体DNA的细胞色素b和控制区序列的部分片段进行了PCR扩增,序列测定和比较分析,证实所提DNA合格而无污染,完全可以用于珍稀动物保护遗传学研究。     

一种从毛发中提取DNA 的简易方法

用PCR 缓冲液及蛋白酶K 在PCR仪上对单支毛囊进行消化,　可获得用于PCR反应的足量的DNA。还对几种从毛发中提取DNA的不同方法进行了比较,并对从毛干中提取线粒体DNA进行了讨论。     

毛干DNA提取方法概述

毛干作为最容易取得的一种无创、运输和储存方便的生物样本,对从核酸分子水平上进行各方面研究有着十分重要的意义。但毛干中DNA含量低,不易提取,而且存在大量角蛋白和色素,纯化不净会对下游PCR扩增等反应产生抑制作用。基于毛干DNA提取现状,综述并比较了近三十年动物及人类毛发的毛干DNA提取、纯化等相关方法,拟为毛干DNA提取在分子生物学各领域的推广应用提供充分的文献支持和参考。     

香鱼基因组DNA提取方法的优化

为获得高质量的基因组DNA,分别采用传统酚-氯仿法、高盐法、试剂盒法和改进酚氯仿法提取香鱼肌肉基因组DNA。琼脂糖凝胶电泳检测结果表明,改进酚氯仿法提取的基因组DNA电泳条带整齐明亮且无降解。紫外分光度计测定DNA浓度和纯度,结果表明,改进酚氯仿法提取的鱼类基因组DNA浓度约为300μg/mL,A260/A280为1.80-1.86。用改进的酚氯仿法提取的DNA进行AFLP分析,扩增结果稳定,电泳条带清晰。综上所述,改进酚氯仿法能够获得高质量DNA,且可以用于进一步的分子生物学研究。     

虎物种特异性鉴定的PCR方法研究

为了建立可对虎DNA进行特异性检测的PCR方法,应用在野外调查中获得的虎疑似样品和保护执法工作中难以检查辨认的虎产品进行物种鉴定,从Genbank数据库下载5个虎亚种及其它6种猫科动物和6种鹿科动物的mtDNA细胞色素b基因序列,并用Wdnasis(V2．5)软件对上述不同动物的该基因碱基序列进行比较。在此基础上,综合考虑了设计引物的基本原则,选择了虎与其它动物碱基序列上差异位点较多的两个片段,设计出PCR引物(引物1、2)。用该对引物分别对从东北虎、华南虎及8种猫科动物和6种非猫科动物的肌肉、脏器组织、皮或毛发中提取的DNA进行：PCR(聚合酶链反应)扩增。结果表明,所设计的引物对虎DNA具有特异性,从而达到了对该物种进行特异性检测鉴定的目的。     

海南产桶形芋螺线粒体基因组DNA提取条件的优化

提取海南产桶形芋螺线粒体基因组完整DNA (mtDNA),并对提取条件进行优化。以桶形芋螺腹足肌肉、毒腺和肝胰脏三个不同组织为材料,分别采用改进高盐沉淀法、细胞器/磁珠法和试剂盒提取三种方法,提取桶形芋螺mtDNA,并利用琼脂糖凝胶电泳和紫外分光光度计对提取mtDNA的纯度和浓度进行测定。以coxⅠ-rRNA小亚基基因和α-芋螺毒素基因设计引物,通过PCR反应来确证所提取的DNA确实是mtDNA。试剂盒法提取肝胰脏、高盐沉淀法提取肝胰脏和腹足肌肉组织这三种方法的产率很高,分别为44.4μg/mg、43.3μg/mg和32.6μg/mg。A260/280比值表明,改进高盐沉淀法提取毒腺和腹足肌肉组织,细胞器磁珠法提取腹足肌肉组织的mtDNA纯度很高。综合比较,采用改进高盐沉淀法,利用桶形芋螺腹足肌肉组织所提取的mtDNA产率高、质量好、纯度高。高质量芋螺mtDNA的获取为利用分子生物学方法对芋螺进行遗传进化分析和系统分类提供了基础。     

Simple methods of enzymatic amplification of DNA for clinical practice]

The methods for enzymatic DNA amplification in vitro that allow to avoid the step of preliminary DNA extraction and purification are proposed. Lysates of blood cells in the solution or immobilized on the nylon membrane filters and dried blood spots on the filter paper blotters were used directly in amplification permitting one to solve the problems of adapting the method of polymerase chain reaction in clinical practice, for instance, in massive screening of genome mutations, viral infections etc.     

Ancient DNA extraction from bones and teeth

This method is designed to maximize recovery of PCR-amplifiable DNA from ancient bone and teeth specimens and at the same time to minimize co-extraction of substances that inhibit PCR. This is achieved by a combination of DNA extraction from bone powder using a buffer consisting solely of EDTA and proteinase K, and purification of the DNA by binding to silica in the presence of high concentrations of guanidinium thiocyanate. All steps are performed at room temperature (20-23 degrees C), thereby reducing further degradation of the already damaged and fragile ancient DNA and providing an optimal trade-off between DNA release and degradation. Furthermore, the purification step removes most of the various types of PCR inhibitors present in ancient bone samples, thereby optimizing the amount of ancient DNA available for subsequent enzymatic manipulation, such as PCR amplification. The protocol presented here allows DNA extraction from ancient bone and teeth with a minimum of working steps and equipment and yields DNA extracts within 2 working days.     

Comparative analysis of three methods from dried blood spots for expeditious DNA extraction from mosquitoes; suitable for PCR based techniques

The objective of this work was to compare the quality, purity and quantity of DNA isolated from dried blood spots (DBS) by three methods (Chelex-100, QIAamp DNA mini kit, and TE (Tris EDTA)-Buffer). Sample collection was performed in six districts in Odisha, India and screened for cases of clinical malaria and dengue and vector density. Mosquito abdomens were spotted on Whatman 3MM (MERCK) Filter paper and dried for 10 min at room temperature. DNA was isolated from DBS using three methods (Chelex-100, QIAamp DNA mini kit, and TE-Buffer), and PCR was used to determine the feeding behaviours of vector mosquitoes. DNA was quantified using a UV-spectrophotometer, and q-PCR was used to determine the target gene copy number to compare the methods. The QIAamp DNA mini kit method was used as the reference method. The yield and purity of DNA extracted with Chelex-100 and TE were 14–72 ng/µl and 1.51–1.85 and 9–50 ng/µl and 1.68–2.1, respectively. DNA extracted using the Chelex-100 method was stored for over 1 month at ? 20 °C and was suitable for later use. The Chelex-100 method had a sensitivity of 99.5% and specificity of 78%. A Bland–Altman plot suggested that the Chelex-100 method was similar to the QIAamp DNA mini kit method for determining the feeding behaviours of vector mosquitoes. The Chelex-100 method is simple, cost-effective, and safe and requires minimal time for DNA extraction from dried blood spots. In malaria and dengue research, detecting the feeding behaviours from mosquito DNA from dried blood spots on filter paper by PCR is an easy, minimally invasive and inexpensive molecular technique that can be performed in remote areas.

     

Increased sensitivity of malaria detection by nested polymerase chain reaction using simple sampling and DNA extraction

Malaria remains a disease of underdeveloped and remote regions of the world. The application of polymerase chain reaction (PCR) technology to malaria epidemiology has the potential for increasing our knowledge and understanding of this disease. In order to study malaria in all geographical locations it is important that specimen collection and DNA extraction for PCR be kept simple. Here we report a method for extracting DNA from dried blood spots on filter paper which is capable of detecting one Plasmodium falciparum and two Plasmodium vivax parasites/μl of whole blood by nested PCR without compromising the simplicity of specimen collection or DNA extraction.     

Comparison of DNA extraction methods for multiplex polymerase chain reaction

We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A_260/280), PCR inhibition ratio, and mitochondrial DNA/genomic DNA ratio were measured to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity, but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single-base extension on the arrayed primer extension 2 (APEX-2).     

Archived Guthrie blood spots as a novel source for quantitative DNA methylation analysis

Sodium bisulfite treatment followed by PCR and DNA sequencing is widely considered the gold standard for the analysis of DNA methylation patterns. However, this technique generally requires substantial quantities of genomic DNA as starting material and is often associated with degradation of DNA. Here, we assess the feasibility of performing bisulfite sequencing on DNA isolated from 3-mm diameter punches of dried blood Guthrie spots. We demonstrate that it is possible to perform bisulfite sequencing from both freshly prepared and archived dried blood spots, using a combination of high purity DNA extraction and efficient bisulfite conversion. With the number of new technologies available for DNA methylation studies, we have extended this analysis and have successfully used a high-throughput mass spectrometry method for DNA methylation analysis on these samples. This provides a new source of material for epigenetic analysis of birth samples and provides an invaluable reference point to track temporal change in epigenetic profiles possibly linked with health and disease.     

The Use of Fur Collected in the Field for Studying the Genetic Diversity of Wild Ungulates

The methods of studying the genetic diversity of wild animals using tissue samples collected without harming the studied populations are discussed for the red deer (Cervus elaphus) fur samples. The methods are proposed for the collection and storage of fur samples in field conditions. The optimal methods of DNA isolation from the fur and other tissues (dry, alcohol-fixed, and frozen skin and muscle) of the red deer and other ungulates were selected, and the conditions of PCR for amplification of the mitochondrial DNA fragments were determined. The comparison between the pure-blood red deers from Central England and the Voronezh State Reserve (Russia) was performed using a cytochrome b fragment of the mitochondrial DNA.     

Francisella tularensis live vaccine strain: proteomic analysis of membrane proteins enriched fraction

Proteome analysis of Gram-negative facultative intracellular pathogen Francisella tularensis (F. tularensis) live vaccine strain has been performed only on whole-cell extracts so far. This is the first study dealing with the analysis of the membrane subproteome of this microorganism. A fraction enriched in membrane proteins obtained by carbonate extraction was separated using two-dimensional electrophoresis and all visualized spots were identified by mass spectrometry. The reference map is the basis for further comparative analyses of virulent and non-virulent F. tularensis strains.     

Imported Malaria in United Arab Emirates: Evaluation of a New DNA Extraction Technique Using Nested PCR

Local malaria transmission in the United Arab Emirates (UAE) came to an end in 1997. Nevertheless, UAE has been subjected to substantial importation of malaria cases from abroad, concerning both UAE nationals and immigrants from malarious countries with a total number of 2,119 cases in 2007. To evaluate a new DNA extraction technique using nested PCR, blood samples were collected from 132 individuals who presented to Infectious Diseases Department in Rashid Hospital, Dubai, and Central Department of Malaria Control with fever and persistent headache. Giemsa-stained blood films and ELISA test for malaria antibodies were carried out for detection of Plasmodium infection. Plasmodium infections were identified with the genus-specific primer set and species differentiation using nested PCR. A rapid procedure for diagnosis of malaria infections directly from dried blood spots using for the first time DNA extract from FTA Elute cards was evaluated in contrast to extraction techniques using FTA classic cards and rapid boiling technique. Our new simple technique for DNA extraction using FTA Elute cards was very sensitive giving a sensitivity of 100% compared to 94% using FTA classic cards and 62% in the rapid boiling technique. No complex preparation of blood samples was required prior to the amplification. The production cost of DNA isolation in our PCR assay was much less in comparable to that of other DNA extraction protocols. The nested PCR detected plasmodial infection and could differentiate P. falciparum from P. vivax, and also detected the mixed infection.     

Comparison of three methods for DNA extraction from bone remains

Extraction of amplifiable DNA is a frequent problem when working with degraded specimens like bone samples. The possibility of obtaining as much information as possible from these samples has a particular significance in many forensic investigations. The present investigation was aimed to assess the efficiency of three organic extraction methods for purifying amplifiable DNA from bone samples. The amount of nucleic acids obtained, the success rate in the amplification of DNA microsatellite (STR) markers and amelogenin by PCR, the influence of PCR inhibitors and environmental conditions, and where the samples were found before their processing in the laboratory, were all evaluated in this investigation for the three methods. Results showed that method A (a modification of FBI method for DNA extraction) performed better in producing not a higher amount but a better quality amplifiable DNA, in comparison with the other two methods evaluated. It was also demonstrated that the quality of the DNA to be amplified by PCR was influenced by the presence of inhibitors and/or contaminants and the environmental conditions where the bone sample was taken from. The worst conditions were observed from aquatic environments. The results suggest that the implementation of some specific modifications in the method A (use of purification columns, reliable quantification methods and different dilutions) would help to obtain better DNA extracts intended to be used in different molecular identification tests.