风沙土微生物总DNA不同提取和纯化方法比较

为筛选和建立风沙土中总DNA的提取和纯化方法,选取了5种直接提取法、1种间接提取法和2种纯化法分别对风沙土中总DNA进行了提取和纯化,并对其质量和产量进行了比较.结果表明：6种方法均可从风沙土中提取到大小为23 kb左右的总DNA,其中改进后的高盐提取法（用40%聚乙二醇8000和4 mol·L^-1 NaCl沉淀DNA）效果最好,纯化后总DNA的纯度最高,可进行16S rDNA的PCR扩增,且产量仅稍低于试剂盒提取法;电泳加柱回收纯化法的纯化效果较好,经该方法纯化后的总DNA大部分可进行PCR扩增,可满足后续分子操作对DNA纯度的要求.     

两种外周血 DNA 提取方法的比较

目的:外周血DNA的提取是研究乙型肝炎病毒相关临床疾病的基础,所提取DNA的质与量直接关乎下游研究的成败,经济、高效、便捷的外周血DNA提取方法对于疾病分子水平的研究尤为重要,本实验旨在比较两种外周血DNA提取方法,从而为临床研究提供有力的参考。方法:以外周抗凝血为试验样本,分别采用改良盐析法和DNA提取试剂盒法(硅胶柱纯化)进行基因组DNA的提取,通过分光光度仪测量DNA浓度和纯度,并进行PCR扩增及电泳实验。比较改良盐析法与试剂盒提取法(硅胶柱纯化)的效果。结果:试剂盒提取法(硅胶柱纯化)标本用量甚微,省时,提取DNA纯度高,步骤繁琐,PCR条带单一、亮度差;改良盐析法操作步骤少,提取DNA浓度高,PCR条带亮度佳、杂带多,耗时长。结论:两组方法各有优缺点,试剂盒提取法(硅胶柱纯化)可靠、快速,但所获DNA量少、极易降解,改良盐析法耗时,但所获DNA浓度高、量多,可根据实验时间与经费,实验所需的DNA纯度与浓度,提供的样本体积等不同的临床研究需求及条件来综合选择适宜的提取方法。     

接触DNA常规提取纯化方法的比较探讨

接触DNA的提取纯化是法医物证工作的重点和难点之一。接触性检材上的脱落细胞本就量小体微,所以运用有效的方法对这些潜在的微量脱落细胞中的DNA进行提取纯化就显得十分重要。文章拟对接触DNA的常规提取纯化方法及影响因素进行简要阐述,并对其进行横向的比较探讨,以期得出最有效的接触DNA提取纯化方法。     

用于分子生态学研究的土壤微生物DNA提取方法

利用SDS高盐法和变性剂加SDS高盐法对土壤微生物总DNA进行了提取,然后通过电泳加树脂柱回收和连续2次树脂柱回收方法进行了纯化.结果表明,变性剂加SDS高盐法的DNA提取效率明显高于前者,电泳加树脂柱法的纯化效果更好.通过PCR扩增表明,经过纯化后的DNA,都可以进行16SrDNA扩增和nirK、nosZ、nifH等功能基因的扩增.因此,变性剂加SDS高盐法是一种更为高效、可靠且适合于环境微生物分子生态学研究的DNA提取方法.     

狭叶坡垒基因组总DNA的提取和纯化方法研究

以狭叶坡垒叶片为材料,对基因组总DNA的提取和纯化方法进行了研究,并对改良CTAB法、高盐低pH法和改良SDS法进行比较.结果表明,改良的CTAB法更适合狭叶坡垒基因组总DNA的提取,且硅胶干燥30 d的叶样和新鲜叶样所得的DNA几乎没有区别.再对改良CTAB法的水浴时间进行探索,发现150 min是较为合适的水浴时间.对比酚纯化法和试剂盒纯化法,发现试剂盒纯化损失的DNA少,得率高,是一种简便、快速、安全的纯化方法.     

CTAB结合DNA凝胶回收试剂盒提取食用菌DNA

目的:为探索从食用菌子实体中快速分离和纯化DNA的方法.方法:以三种栽培的食用菌为材料,采用CTAB结合DNA凝胶回收试剂盒进行基因组DNA的分离和纯化.结果:与对照相比,结合法提取DNA样品的电泳条带更规则、清晰,并且DNA的酶切效率显著高于对照.结论:该结合法是一种快捷、高效提取纯化食用菌子实体DNA的方法.     

土壤微生物总DNA的提取和纯化

本文建立了从土壤中提取总DNA的方法,并通过改进使适合于对革兰氏阳性菌的提取。用9种性质不同的土壤进行验证,均提取到了DNA,每克干土的DNA提取量从3.30μg～13.41μg,通过透析袋回收进行纯化,纯化回收率达到65.34%,纯化后的土壤DNA可以直接扩增出16S rDNA。9种土壤的提取率从60.51%～93.45%,可以从每g干土添加362个菌体的土壤中扩增到目的条带。     

泰泽病原体基因组DNA提取方法的建立

目的　提取泰泽病原体基因组DNA ,为建立该菌基因组文库奠定基础。方法　使用密度梯度离心结合酶解消化方法、酶解消化方法、本研究建立方法即过滤盐析离心法 ,从感染肝脏组织纯化泰泽病原体 ,并比较三种方法纯化泰泽病原体效果 ;采用氯化苄法、试剂盒、酚法提取泰泽病原体基因组DNA ,并比较三种方法提取基因组DNA质量 ;鉴定酚法提取泰泽病原体基因组DNA特异性。结果　使用过滤盐析离心法从感染肝脏组织纯化泰泽病原体 ,采用酚法提取其基因组DNA ,所获得的基因组DNA特异性好、纯度高、DNA片段长度大于 5 0kb ,且均一性好 ,无降解。结论　本研究首次成功提取泰泽病原体基因组DNA ,可用于多种分子生物学实验     

一种快速简单的质粒DNA纯化方法

本文介绍一种快速获取高纯度质粒DNA的方法。本法对煮沸提取法进行改良后,快速提取质粒DNA粗制品,然后用国产滤纸从琼脂糖凝胶中回收纯化质粒DNA。同时对本法所提取的质粒DNA的回收率、浓度、纯度及可能的用途进行验证和讨论,证明此法简易,所得样品纯度高,可直接用于转化、酶切和基因克隆。     

4种黑曲霉基因组DNA提取方法的比较

目的:筛选适合提取曲霉DNA的方法.方法:比较2个菌落培养时间段(3d内和10d左右)提取DNA质量的差异;运用氯化苄法、石英砂+CTAB法、Biospin法和微波法分别提取黑曲霉基因组DNA,然后用直接电泳、浓度测定、PCR扩增等方法比较所提DNA的浓度和质量.结果:培养3d内的菌落提取的DNA纯度较高,无需纯化即可用于后续实验;4种方法制备的DNA均可用于PCR等后续实验,其中以石英砂+CTAB法提取的DNA纯度好,产率最高.结论石英砂+CTAB法是一种适用于曲霉DNA提取的简便方法.     

DNA extraction from hair shafts of wild Brazilian felids and canids

Wild felids and canids are usually the main predators in the food chains where they dwell and are almost invisible to behavior and ecology researchers. Due to their grooming behavior, they tend to swallow shed hair, which shows up in the feces. DNA found in hair shafts can be used in molecular studies that can unravel, for instance, genetic variability, reproductive mode and family structure, and in some species, it is even possible to estimate migration and dispersion rates in given populations. First, however, DNA must be extracted from hair. We extracted successfully and dependably hair shaft DNA from eight wild Brazilian felids, ocelot, margay, oncilla, Geoffroy's cat, pampas cat, jaguarundi, puma, and jaguar, as well as the domestic cat and from three wild Brazilian canids, maned wolf, crab-eating fox, and hoary fox, as well as the domestic dog. Hair samples came mostly from feces collected at the S?o Paulo Zoo and were also gathered from non-sedated pet or from recently dead wild animals and were also collected from museum specimens. Fractions of hair samples were stained before DNA extraction, while most samples were not. Our extraction protocol is based on a feather DNA extraction technique, based in the phenol:chloroform:isoamyl alcohol general method, with proteinase K as digestive enzyme.     

基于毛干蛋白质组的族群推断技术的建立与验证

毛干是一种案件现场常见的生物物证,由于核DNA含量极少且高度降解,难以采用现有的短串联重复序列(short tandem repeat,STR)检验方法进行个人识别鉴定,目前仅使用线粒体DNA检验进行母系亲缘关系的判定,利用率非常低.毛干中蛋白质非常稳定,而且具有遗传多态性,表现为基因组中的非同义单核苷酸多态性(non-synonymous single nucleotide polymorphisms,ns SNPs),转录翻译后形成蛋白质序列中的单氨基酸多态性(single amino acid polymorphisms,SAPs).充分利用毛干蛋白质中蕴含的遗传信息,为案件提供线索和证据,是实际公安业务的迫切需求,具有重要的应用价值.本文选取了104份中国汉族的毛干样本进行蛋白质组的检测,共获得了703个SAP位点,位于460个蛋白质上,共推导出552个nsSNP位点.进一步筛选在所有样本中检出率超过15%的位点,获得了88个nsSNP位点,使用毛干样本对应的口腔拭子DNA对88个ns SNP位点进行一代测序验证.为评估发现的nsSNP位点对于人群的区分能力,以千人数据库(1 000 Genome Project)为参考数据库,采用聚类分析和群体匹配概率等方法对检测的19份毛干样本进行人群来源推断.结果显示,通过检测毛干蛋白质组中的ns SNP可以实现东亚、欧洲、非洲三大洲际人群的区分.     

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT "point samples" obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair''s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.     

A Simple Method to Extract DNA from Hair Shafts Using Enzymatic Laundry Powder

A simple method to extract DNA from hair shafts was developed by using enzymatic laundry powder at the first step of the process. The whole extraction can be finished in less than 2 hours. The simple extraction reagent proposed here contains only two cheap components: ordinary enzymatic laundry powder and PCR buffer. After extraction, an ultra sensitive fluorescent nucleic acid stain, PicoGreen, was used for quantifying trace amount of double-stranded DNA in the solution extracted. For further validation of DNA extraction, four primers were employed to amplify DNA microsatellite loci. Both fluorescence spectroscopy and PCR results suggested that this method can extract DNA from hair shafts with good efficiency and repeatability. The study will greatly facilitate the use of hair shafts in future for DNA analyses on genome-wide scale.     

利用改进的酚-氯仿法从猪毛囊中提取基因组DNA

为提高从猪毛囊组织中提取基因组DNA的效率, 文章在借鉴从其他组织提取基因组DNA方法的基础上, 对经典的酚-氯仿法的反应体系和步骤进行了改进。利用改进的酚－氯仿抽提法, 从猪的毛囊组织中快速、高效地提取了高质量基因组DNA。利用该方法从1~6根猪毛囊中提取的基因组DNA可满足基于PCR技术的相关分子生物学实验需要。     

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.     

Differentiating Inbred Mouse Strains from Each Other and Those with Single Gene Mutations Using Hair Proteomics

Mutant laboratory mice with distinctive hair phenotypes are useful for identifying genes responsible for hair diseases. The work presented here demonstrates that shotgun proteomic profiling can distinguish hair shafts from different inbred mouse strains. For this purpose, analyzing the total hair shaft provided better discrimination than analyzing the isolated solubilized and particulate (cross-linked) fractions. Over 100 proteins exhibited significant differences among the 11 strains and 5 mutant stocks across the wide spectrum of strains surveyed. Effects on the profile of single gene mutations causing hair shaft defects were profound. Since the hair shaft provides a discrete sampling of the species proteome, with constituents serving important functions in epidermal appendages and throughout the body, this work provides a foundation for non-invasive diagnosis of genetic diseases of hair and perhaps other tissues.     

Age, gender and caste variations in scalp hair micro-morphological variables among Brahmins and Banias of Punjab, India

A cross-sectional sample of 3136 scalp hair drawn from 392 individuals aged 10 to 60 years and belonging to the Bania (n = 201) and Brahmin (n = 191) caste groups of Punjab State of India were examined for diameters of hair shaft and medulla, scale count, medulla type, hair index, medullary index and scale-count index, employing standard procedures. The mean hair shaft diameter, medullary diameter, incidence of medullation and scale-count index was higher in males, while the mean scale count was higher in females. However, with a few exceptions, the gender differences were not statistically significant (p < 0.05). The Brahmins showed significantly (p < 0.05) higher mean values of hair shaft diameter and scale count than the Banias. The mean scale-count index was higher among the Banias. The fragmentary medulla was the most common medullary type among the Banias, while the continuous medulla was the most frequent type of medulla among the Brahmins. The frequency of medullation was significantly more among the Banias. On the average, the hair shaft diameter and diameter of medulla increased up to 30 years. Some age variations in medullation were noticed in both the caste groups. No clear age trend was noticeable in the scale count. By and large, the mean hair shaft diameter of the Banias was less than that of other populations. The mean hair shaft diameter of the Brahmin males was greater than that of the Banias, the Onges, and the Bengalees. The Brahmin females showed greater mean hair shaft diameter than that of the Banias, the Australian Aborigines, the Juangs and the Onges. Human scalp hair shows some age, gender and population variations in micro-morphological variables which have the potential of being useful for anthropological and forensic investigations.