古DNA提取技术对比及概述

从古代原始材料中提取古DNA的方法多种多样,但是古DNA的研究受限于降解严重,内源性古DNA含量低,微生物和现生人群DNA污染严重等因素的影响。能否从古代人类遗骸中成功获取可靠且足量的内源性古DNA,一直是古DNA研究领域面临的实际困难和挑战。控制污染最直接且简便的策略就是在古DNA提取阶段的有效排除,本文整理了古DNA提取常用的去除污染的方法,对比分析了每种方法表现出来的优缺点。介绍了通常使用的骨粉裂解时间,并研究了在常温环境下,不同的裂解时间对古DNA回收效率的影响,提出了常温裂解过程中最佳孵育时间。同时对常用的古DNA纯化方法及其原理和在实际应用中的表现进行了概述与讨论。本文对古DNA提取技术的概述和实践经验,为古DNA相关领域的研究提供借鉴与参考。     

植物染色体微切割过程中细胞质DNA污染及其控制的研究

染色体微切割和微克隆已成为复杂基因组研究的有效途径。但是操作过程中的核外DNA的污染一直是令人担心的问题,通过研究植物染色体微切割（微分离）和微切割的染色体DNA扩增过程中细胞质DNA的污染问题,表明目前常用的植物染色体微切割过程中,细胞质DNA的污染几乎难以避免,并提出了一个改进的降低细胞质DNA污染的方法。对如何控制细胞质DNA的污染进行了详细的讨论。     

植物染色体微切割过程中细胞质DNA污染及其控制的研究

染色体微切割和微克隆已成为复杂基因组研究的有效途径,但是操作过程中的核外DNA的污染一直是令人担心的问题.通过研究植物染色体微切割(微分离)和微切割的染色体DNA 扩增过程中细胞质DNA的污染问题,表明目前常用的植物染色体微切割过程中,细胞质DNA的污染几乎难以避免,并提出了一个改进的降低细胞质DNA污染的方法,对如何控制细胞质DNA的污染进行了详细的讨论.     

一种实用可靠的古代 DNA 获取方法

古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题．改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列．实验利用距今4 300～3 900年前的猪牙样本,将改进的古 DNA 获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下．这可以为开展大规模古代样本检测提供一种切实可行的 DNA 获取方法．     

DNA指纹技术在污染土壤生态毒理诊断中的应用

生物标记物能在细咆或分子水平上指示暴露．效应关系,是进行污染土壤生态毒理诊断的主要技术手段之一。随着分子生物学技术的飞速发展,出现了一系列以聚合酶链式反应为基础的、在分子水平上检测污染物质导致的生物体DNA损伤的DNA指纹技术。DNA指纹技术的主要类型有：随机扩增多态性DNA（RAPD）、聚合酶链式反应．单链构象多态性（PCR-SSCP）、扩增片段长度多态性（AFLP）、任意引物聚合酶链式反应（AP—PCR）、差异显示反转录聚合酶链式反应（DDRT）、短DNA重复序列（SSR）及限制片段长度多态性（RFLP）等。这些技术与检测基因突变、染色体畸变和损伤为主的一系列经典研究方法如彗星分析、微核实验等相比具有简便、快速、灵敏等优点。本文着重介绍了随机扩增多态性DNA、聚合酶链式反应．单链构象多态性、扩增片段长度多态性3种重要的DNA指纹技术在污染土壤诊断中的应用。     

古DNA 及其在生物系统与进化研究中的应用

古DNA是指从已经死亡的古代生物的遗体和遗迹中得到的DNA。本文回顾了近20年古DNA研究所经历的3个阶段, 从早期参与研究的科学家较少并主要利用克隆技术, 到后来由于PCR技术的出现以及提取化石DNA技术的成熟从而出现大量有关古DNA的报道; 近几年由于发现不少问题, 并引起激烈的争论, 科学家们因此而开始考虑古DNA的真实性问题, 并且提出了开展古DNA研究的严格标准。本文还讨论了古DNA在人类起源、系统发育重建、动植物驯化及考古研究中的重要意义以及现状, 表明古DNA的研究给某些原先的观点如人类的非洲起源说提供了重要证据, 也对某些观点提出了挑战; 古DNA研究还提供了某些已经灭绝生物的形态学和分子资料, 为从序列上确定古代材料的系统位置并有效地补充仅用现代DNA建立起来的谱系提供了来自古生物的依据。在动植物驯化及考古方面, 古DNA证据也为科学家提供了许多有价值的信息。最后, 本文还对古DNA研究的应用前景进行了展望。     

古DNA技术在人类墓葬遗骸研究中的应用及进展

考古工作中得到的生物遗骸由于长期的风化,自然侵蚀等因素的影响,遗骸本身含有的古代生物的DNA的大部分会分解,使得对遗骸中的生物遗传信息研究变得非常困难.可将现代生物工程的PCR技术应用到考古工作中,该技术能够对残存的微量DNA进行大量的生物体外扩增,得到更多的古代生物的遗传信息,提高时遗骸种属鉴定的准确性.通过对出土的人类遗骸中微量DNA的扩增、测试和遗骸间DNA序列的对比,在计算机软件的帮助下与已知的人类DNA序列进行比较,能确定同一墓葬中不同遗骸的亲缘关系和该遗骸群体在整个人类进化体系中的位置.对这一试验过程的一些方法、技术、研究进展和目前仍然面临的一些问题作了介绍.     

古DNA及其在生物系统与进化研究中的应用

古DNA是指从已经死亡的古代生物的遗体和遗迹中得到的DNA.本文回顾了近20年古DNA研究所经历的3个阶段,从早期参与研究的科学家较少并主要利用克隆技术,到后来由于PCR技术的出现以及提取化石DNA技术的成熟从而出现大量有关古DNA的报道;近几年由于发现不少问题,并引起激烈的争论,科学家们因此而开始考虑古DNA的真实性问题,并且提出了开展古DNA研究的严格标准.本文还讨论了古DNA在人类起源、系统发育重建、动植物驯化及考古研究中的重要意义以及现状,表明古DNA的研究给某些原先的观点如人类的非洲起源说提供了重要证据,也对某些观点提出了挑战;古DNA研究还提供了某些已经灭绝生物的形态学和分子资料,为从序列上确定古代材料的系统位置并有效地补充仅用现代DNA建立起来的谱系提供了来自古生物的依据.在动植物驯化及考古方面,古DNA证据也为科学家提供了许多有价值的信息.最后,本文还对古DNA研究的应用前景进行了展望.     

用于(医学)诊断的DNA探针

用DNA作诊断试验是非常迅速、灵敏和特异的,容易进行并容易分析。DNA杂交试验的非放射性检测方法出现,对现代的重组DNA技术从研究实验室进入更接近临床的实验室起到了相应大的促进作用。在这里要谈到这项新颖技术的基本原则及其应用。 DNA探针是一种能够识别并能特异地与互补DNA片段,即使这些互补的DNA序列是在其它完全无关的DNA序列中。     

重金属污染土壤总DNA提取方法的研究——土壤预处理和硫酸铵铝在DNA提取中的应用

【目的】从土壤中获得高纯度、高得率和完整性好的总DNA,为重金属污染土壤微生物群落多样性的分析奠定基础。【方法】将一定浓度的硫酸铵铝增补入DNA提取液中,分别联合不同方式的土壤预处理,对所提取的土壤总DNA进行完整性、纯度和得率分析。【结果】TENP-AlNH4(SO4)2法、ABG-AlNH4(SO4)2法、Wash-AlNH4(SO4)2法和试剂盒4种提取方法获得的DNA片段均在23 kb左右,总DNA完整;Wash-AlNH4(SO4)2法提取的DNA纯度较高,A260/A230达到2.00,A260/A280达到1.62;ABG-AlNH4(SO4)2法的DNA得率最高,达到1 010μg/g土壤;这两种方法提取的DNA在纯度和浓度上均达到后续PCR等分子实验要求。通过扩增提取的土壤总DNA中16S rRNA基因,表明合适浓度的硫酸铵铝能有效去除土壤中的PCR干扰因子。【结论】本研究将土壤的预处理和一定浓度的硫酸铵铝联合使用,获得理想的重金属污染土壤的总DNA。     

Arsenic-Rich Iron Floc Deposits in Seeps Downgradient of Solid Waste Landfills

Iron flocculate or “floc” deposits are commonly observed in groundwater discharge zones downgradient of unlined solid waste landfills. Bright orange in color, and composed predominantly of amorphous iron oxyhydroxides, these deposits generally have been regarded as aesthetically undesirable but environmentally benign. In recent years, there has been increased awareness of the widespread occurrence of elevated arsenic in reducing groundwaters. Research carried out at municipal landfills in New England indicates that naturally occurring arsenic exhibits redox-mediated mobility and is frequently associated with reduced iron as a dissolved constituent in leachate-impacted groundwaters. If iron precipitates in discharge zones where reduced groundwaters are exposed to atmospheric oxygen, it follows that arsenic may co-precipitate with iron in these areas. To assess the prevalence of arsenic as a constituent of iron floc deposits, samples were collected at seven landfills and at one natural mineral spring in the lower Hudson Valley of southeastern New York State. At six of seven landfill sites, arsenic concentrations exceeded 33 mg/kg, which represents the “severe effects level” for aquatic life as identified in New York State regulatory guidance for screening contaminated sediments. These results indicate that arsenic contamination is of potential concern for downgradient of landfills wherever iron-stained leachate discharges are observed. Sampling and analysis of iron flocs associated with such leachates could also provide a means of identifying landfills that may present risks of arsenic contamination to downgradient water supply wells, especially in cases where groundwater monitoring wells are not available for sampling.     

古DNA提取技术新进展

古DNA是揭示古代生物生长状态以及生物千百万年来进化情况的最重要的信息载体,在治疗人类遗传性的疑难病症及牲畜饲养和粮食作物种植等方面都有重大的贡献。古DNA提取技术作为获得该重要的信息载体的最重要手段,长久以来受到了世界各地的考古学家以及医学研究学者们的高度重视。随着科学的发展,古DNA提取技术已经形成多种核心方法:Chelex-100法、酚-氯仿抽提法、二氧化硅(硅粒)法、NaOH法、硅离心柱法试剂盒、磁珠法试剂盒等方法。本文将根据最新的研究成果对以上提到的几种方法进行分析比较,以期能够为将来古DNA提取技术的发展创新提供新的思路与方向。     

Mitochondrial cytochrome b of the Lyakhov mammoth (Proboscidea,Mammalia): new data and phylogenetic analyses of Elephantidae

The phylogenetic relationships between recent Elephantidae (Proboscidea, Mammalia), that is to say extant elephants (Asian and African) and extinct woolly mammoth, have remained unclear to date. The prevailing morphological scheme (mammoth grouped with Asian elephant) is either supported or questioned by the molecular results. Recently, the monophyly of woolly mammoths on mitochondrial grounds has been demonstrated (Thomas, et al., 2000), but it conflicts with previous studies (Barriel et al., 1999; Derenko et al., 1997). Here, we report the partial sequencing of two mitochondrial genes: 128 bp of 12S rDNA and 561 bp of cytochrome b for the Lyakhov mammoth, a 49,000-year-old Siberian individual. We use the most comprehensive sample of mammoth (11 sequences) to determine whether the sequences achieved by former studies were congruent or not. The monophyly of a major subset of mammoths sequences (including ours) is recovered. Such a result is assumed to be a good criterion for ascertaining the origin of ancient DNA. Our sequence is incongruent with that of Yang et al. (1996), though obtained for the same individual. As far as the latter sequence is concerned, a contamination by non-identified exogenous DNA is suspected. The robustness and reliability of the sister group relation between Mammuthus primigenius and Loxodonta africana are examined: down-weighting saturated substitutions has no impact on the topology; analyzing data partitions proves that the support of this clade can be assigned to the most conservative phylogenetic signal; insufficient taxonomic and/or characters sampling contributed to former discordant conclusions. We therefore assume the monophyly of "real mammoth sequences" and the (Mammuthus, Loxodonta) clade.     

Attachment ofSalmonella typhimurium andListeria monocytogenes to glass as affected by surface film thickness,cell density,and bacterial motility

Summary With equal cell densities, surface film thickness did not influence the numbers ofSalmonella typhimurium andListeria monocytogenes cells which attached to glass. MotileL. monocytogenes cells had a greater cell surface charge and generally attached in higher numbers than non-motile cells.Mention of a trade name, proprietary product or specific equipment is necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

古DNA提取技术新进展

古DNA 是揭示古代生物生长状态以及生物千百万年来进化情况的最重要的信息载体,在治疗人类遗传性的疑难病症及牲畜饲养和粮食作物种植等方面都有重大的贡献。古DNA提取技术作为获得该重要的信息载体的最重要手段,长久以来受到了世界各地的考古学家以及医学研究学者们的高度重视。随着科学的发展,古DNA提取技术已经形成多种核心方法：Chelex-100 法、酚-氯仿抽提法、二氧化硅（硅粒）法、NaOH 法、硅离心柱法试剂盒、磁珠法试剂盒等方法。本文将根据最新的研究成果对以上提到的几种方法进行分析比较,以期能够为将来古DNA提取技术的发展创新提供新的思路与方向。     

Frequency and recency of infection and their relationship with disgust and contamination sensitivity

Both disgust and contamination sensitivity likely evolved to protect us from infectious disease. Paradoxically, disgust may be reduced by frequent exposure to disgust-inducing cues — cues most likely to occur in disease-rich environments. In this study, we examined whether more frequent or recent illness might act to reverse this process. To test this, we surveyed 616 adults, obtaining illness frequency and recency data, disgust and contamination sensitivity, and a variety of control measures. Heightened contamination sensitivity was associated with more frequent infectious illness, but not with recency of infection. We also found that participants who had heightened contamination sensitivity and who were also more disgust sensitive had significantly fewer recent infections. These findings suggest that frequent illness may up-regulate contamination sensitivity potentially counteracting the effects of exposure on disgust. More importantly, these data provide the first direct evidence of a protective effect of contamination and disgust, against infectious disease.     

Ancient DNA studies and new bioethic problems

The development of the ancient DNA technique allows the genetic study of vanished human populations, some of them, as Fueguians and Tasmanians, of great anthropological interest. This possibility, however, involves some new bioethic problems, concerning the origin of the skeletal samples that can be analyzed and the moral authority of living aboriginals with respect to human remains of perhaps several thousand years of antiquity. It is my opinion that, despite some skeletal collections might be unethically gathered by the 19th century anthropologists, the vanished human populations must be genetically studied. If not, after contribute to their disappearance, we will be now ignoring their contribution to the humankind diversity.     

The human skeletons of herculaneum

Skeletons of 139 Herculaneans trapped by the vulcanic eruption of Mt. Vesuvius in A.D. 79 were studied by observation, measurement and chemical analysis. A cross section of the population suggests that reproduction may not have been sufficient to maintain population numbers, a hypothesis corroborated by parity statistics as well as contemporaty literature. In general, this population had excellent teeth with few lesions and edge-bite occlusion. Twenty-seven percent had some degree of hypoplastic lines in the dental enamel, suggesting that childhood illnesses were common. The ancient population was taller than modern Neopolitans, but shorter than modern Americans. Also, their children grew at a slower rate than Americans of the same ages. Biochemical analysis suggests that their diet was more dependent on sea fish than on red meat. Lead analysis shows slightly higher values for the adult male population than for the females. Some degree of arthritis was apparent in 42% of the population. Traumata occurred to 22.7% of these people. Signs of healed anemia in any degree are present in 34.1%; etiology could have been nutritional deficiency or heterozygotic thalassemia. Two individuals and their pathologies are presented: one case of congenital bilateral hip dysplasia and the other of healed rickets.     

Speciation and Environmental Fate of Chromium in Rivers Contaminated with Tannery Effluents

Redox and size speciation of chromium in rivers contaminated with tannery wastewater was carried out to provide insight into its transport and removal mechanisms. Total chromium was determined with Inductively Coupled Plasma‐Mass Spectrometry and Cr (VI) with Catalytic Adsorption Stripping Voltammetry. For the size speciation, particles were retained with a cartridge filter (cut‐off 1.2 μm) and the total filterable fraction was further fractionated with Tangential Flow Filtration to determine the concentrations of chromium associated with the High Molecular Weight Colloidal (HMWC), Low Molecular Weight Colloidal (LMWC) and Truly Dissolved (TD) fractions. Two fluvial systems of similar sizes, but located in contrasting climatic zones, were selected for comparison: the Sebou‐Fez system in Morocco and Dunajec River‐Czorsztyn Reservoir system in Poland. Particulate Cr dominated in the Sebou‐Fez system (about 90 %); while in the Dunajec‐Czorsztyn system, it represented only 17–53 % of the total chromium in raw water. Still, the partition coefficients [K_d] were of the same magnitude. Chromium (III) was the only form detected in Sebou‐Fez, whereas in Dunajec‐Czorsztyn Cr (VI) was also present with its proportion increasing downstream from the input of tannery wastewater due to the preferential removal of Cr (III). In the filtered water in Morocco a large fraction of Cr occurred in the HMWC fraction (50–70 %) at the two most contaminated sites, while the LMWC and TD forms prevailed at the non‐contaminated sites in the Sebou River. At a very high concentration, in the water in the proximity of tanneries (well above the theoretical saturation level) Cr precipitated as polynuclear Cr‐hydroxide. In Dunajec‐Czorsztyn, the partition of Cr (III) was approximately equal between the HMWC, LMWC and TD fractions, in contrast to Cr (VI) which occurred almost exclusively in the TD fraction. In both systems, Cr (III) was rapidly removed from the water to the sediments. The confluence of the Sebou with the Fez and the Czorsztyn reservoir trapped efficiently Cr (III) preventing its spreading over long distances. Cr (VI) showed conservative behavior and bypassed the Czorsztyn Reservoir. This study provides a first set of data on the partitioning of Cr (III) and Cr (VI) between the particulate, the colloidal and truly dissolved fractions in fluvial systems contaminated with tannery effluents. It also suggests that, in these systems, truly dissolved Cr (III) can be adequately modeled from the total filterable concentrations.