Purification of total cellular DNA from a single plant

A procedure is outlined for purifying DNA from a single plant. A crude organelle pellet consisting of nuclei, chromatin, chloroplasts, and mitochondria is prepared, suspended, and immediately lysed with detergents. The DNA is separated from RNA, protein, and polysaccharides by banding it in CsCl density equilibrium gradients. Ethidium bromide is included in all buffers to act as an inhibitor of DNAase activity. The DNA prepared in this manner can be digested with restriction endonucleases, separated by gel electrophoresis, and used to identify specific genes by hybridization of cloned DNA sequences.These experiments were supported by Grant DEB79-2298 from the National Science Foundation and Grant 59-2133-0-1-489-0 from the USDA Competitive Research Grants Program.     

On-line recovery of large molecules from mixtures using reciprocating size exclusion chromatography

Blue Dextran, a standard large molecule, was successfully recovered on-line from the aqueous mixture solution with nickel nitrate using a novel reciprocating size exclusion chromatography. After 7 cycles of repeating operations of frontal mode, 70% of Blue Dextran in the feed was isolated as a pure solution. On-line recovery of large molecules from the mixture is an unusual trial, comparing to the routine practice of filtration where small molecule is isolated from the mixture.     

Isolation of plant DNA: A fast,inexpensive, and reliable method

We describe here a simple method to isolate DNA of high molecular weight from a wide variety of plant materials, such as trees, herbaceous plants, cell suspension cultures, calli, seeds, dried embryos, ferns and lichens. The crucial step of the extraction is the use of an acidic extraction medium. When necessary, the sample was separated on a fast RPC-5 column providing us with highly purified DNA suitable not only for restriction endonuclease analyses but also for PCR experiments, RLFP analyses, or detection of adducts.     

一种快速微量提取植物叶片DNA的方法

介绍了一种快速提取微量DNA的方法。该方法简单易行,无需任何特殊设备,所需样品量少。提取的DNA纯度高,D260nm/D280nm在1．9-2．1之间,可满足RAPD、SSR、转基因植株的PCR检测等以PCR扩增为基础的实验需要。     

On-line recovery of large molecules and concentration of small molecules using reciprocating size exclusion chromatography with temperature swing

The reciprocating size exclusion chromatography (RSEC) was operated with swing between two temperatures in a synchronous way with flow direction to recover a large solute on-line from the mixture, in addition to the small solute concentration. The concentration of small solutes in RSEC with a temperature swing was made possible by taking advantage of the temperature-dependent swelling properties of the porous gel. After 7 cycles of frontal mode operation, 76% of Blue Dextran in the feed was recovered and nickel nitrate in the feed reservoir was concentrated by 13%.     

A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue

We describe an inexpensive method for dehydration of plant tissue and extraction of high molecular weight DNA. Tissue is dried for 12 to 24 hours in a food dehydrator and subsequently powdered for DNA extraction. Dicot tissue can be powdered in centrifuge tubesen masse using a commercial paint mixer and glass beads. With the use of the paint mixer, tissue never touches common surfaces that might lead to cross contamination, a potential benefit when the DNA is to be used for PCR reactions. The DNA is of a quality equal to that obtained from either lyophilized or fresh frozen tissue (commonly used in many labs). The advantages of the described procedure are that it is fast, does not require expensive equipment (e.g., lyophilizer) and can be used in situations where large numbers of samples must be extracted.     

Age estimation in a long‐lived seabird (Ardenna tenuirostris) using DNA methylation‐based biomarkers

Age structure is a fundamental aspect of animal population biology. Age is strongly related to individual physiological condition, reproductive potential and mortality rate. Currently, there are no robust molecular methods for age estimation in birds. Instead, individuals must be ringed as chicks to establish known‐age populations, which is a labour‐intensive and expensive process. The estimation of chronological age using DNA methylation (DNAm) is emerging as a robust approach in mammals including humans, mice and some non‐model species. Here, we quantified DNAm in whole blood samples from a total of 71 known‐age Short‐tailed shearwaters (Ardenna tenuirostris) using digital restriction enzyme analysis of methylation (DREAM). The DREAM method measures DNAm levels at thousands of CpG dinucleotides throughout the genome. We identified seven CpG sites with DNAm levels that correlated with age. A model based on these relationships estimated age with a mean difference of 2.8 years to known age, based on validation estimates from models created by repeated sampling of training and validation data subsets. Longitudinal observation of individuals re‐sampled over 1 or 2 years generally showed an increase in estimated age (6/7 cases). For the first time, we have shown that epigenetic changes with age can be detected in a wild bird. This approach should be of broad interest to researchers studying age biomarkers in non‐model species and will allow identification of markers that can be assessed using targeted techniques for accurate age estimation in large population studies.     

基于植物DNA条形码rbcLa序列的日本条螽食谱分析

日本条螽Ducetia japonica广泛分布于我国绝大部分区域,并在城区绿地大量发生。据此假设其食物谱可能具有较高的物种多样性,但目前对其在自然环境下取食的植物种类仍缺乏了解。本研究对来自河北保定城区绿地和城郊荒地两种生境共计75头日本条螽肠道内容物进行DNA提取、rbcLa基因片段扩增及测序,并利用BOLD和NCBI数据库进行分类鉴定。结果显示：日本条螽在野外至少取食10科、13种植物。在城区绿地和城郊荒地两种生境中,取食频率最高的植物分别是卫矛科冬青卫矛Euonymus japonicus(47.37%)和大麻科葎草Humulus scandens(81.08%)。只有1条rbcLa基因序列未鉴定至种级水平,表明利用植物DNA条形码rbcLa基因序列可对植食性昆虫取食的植物种类进行快速鉴定。同时,研究结果也初步证实日本条螽的食物谱包含较高的物种多样性,为城市绿地螽斯类鸣虫保护和繁育提供依据。     

一种用于PCR模板制备的电泳产物简易回收方法

为了探索一种简便、有效而且能从琼脂糖凝胶中大量回收用于第2次PCR扩增的DNA电泳条带的方法,采用刀片切胶法和牙签插胶法从琼脂糖中回收DNA,并进行了两种方法的比较．结果显示牙签插胶法回收的DNA用作第2次PCR的模板,获得了清晰、稳定的PCR产物电泳条带,用该法成功地制备了一批DNA微阵列探针．由此可见牙签插胶法是一种简便、快速、有效的用于PCR模板的DNA琼脂糖凝胶回收法．     

Land use is a determinant of plant pathogen alpha‐ but not beta‐diversity

Little is known about the diversity patterns of plant pathogens and how they change with land use at a broad scale. We employed DNA metabarcoding to describe the diversity and composition of putative plant pathogen communities in three substrates (soil, roots, and leaves) across five major land uses at a national scale. Almost all plant pathogen communities (fungi, oomycetes, and bacteria) showed strong responses to land use and substrate type. Land use category could explain up to 24% of the variance in composition between communities. Alpha‐diversity (richness) of plant pathogens was consistently lower in natural forests than in agricultural systems. In planted forests, there was also generally low pathogen alpha‐diversity in soil and roots, but alpha‐diversity in leaves was high compared with most other land uses. In contrast to alpha‐diversity, differences in within‐land use beta‐diversity of plant pathogens (the predictability of plant pathogen communities within land use) were subtle. Our results show that large‐scale patterns and distributions of putative plant pathogens can be determined using metabarcoding, allowing some of the first landscape level insights into these critically important communities.     

Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue

This report summarizes major changes in previously published protocols for DNA extraction to improve the quality of DNA extracted from plants. Here, we highlight the critical modifications in the original protocols. The efficiency of these changes results in high-quality DNA ready to use in a variety of phytogenetically distant plant families, in particular species with mucopolysaccharides. The DNA obtained can be used without further purification in various molecular biology assays, including direct sequencing and AFLP and RAPD (random-amplified polymorphic DNA) analyses. The effectiveness of this method is proven by the amplification and sequencing of PCR products of up to 1 kb with DNA extracted from herbarium tissue ≥60 years old. This versatility is not usually found in DNA extraction protocols. In addition, this method is quick, adaptable to standard laboratories, and most important, safer and more cost-effective.     

Detection of recombinant growth hormone by evanescent cascaded waveguide coupler on silica‐on‐silicon

An evanescent wave based biosensor is developed on the silica‐on‐silicon (SOS) with a cascaded waveguide coupler for the detection of recombinant growth hormone. So far, U ‐bends and tapered waveguides are demonstrated for increasing the penetration depth and enhancing sensitivity of the evanescent wave sensor. In this work, a monolithically integrated sensor platform containing a cascaded waveguide coupler with optical power splitters and combiners designed with S ‐bends and tapper waveguides is demonstrated for an enhanced detection of recombinant growth hormone. In the cascaded waveguide coupler, a large surface area to bind the antibody with increased penetration depth of evanescent wave to excite the tagged‐rbST is obtained by splitting the waveguide into multiple paths using Y splitters designed with s ‐bends and subsequently combining them back to a single waveguide through tapered waveguide and combiners. Hence a highly sensitive fluoroimmunoassay sensor is realized. Using the 2D FDTD (Finite‐difference time‐domain method) simulation of waveguide with a point source in Rsoft FullWAVE, the fluorescence coupling efficiency of straight and bend section of waveguide is analyzed. The sensor is demonstrated for the detection of fluorescently‐tagged recombinant growth hormone with the detection limit as low as 25 ng/ml. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

One-step isolation of plant DNA suitable for PCR amplification

We report a one-step extraction technique for the isolation of plant DNA, DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm² in less than 20 min, with no tube changes. The method was tested on several plant specA00AK020ies. The described method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated by the novel method to PCR products generated using standard DNA isolation techniques.     

DNA barcodes from century‐old type specimens using next‐generation sequencing

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next‐generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century‐old type specimens of Lepidoptera by attempting to recover 164‐bp and 94‐bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories – high (164‐bp sequence), medium (94‐bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR‐based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.     

Tousled‐like kinase 2 regulates recovery from a DNA damage‐induced G2 arrest

In order to maintain a stable genome, cells need to detect and repair DNA damage before they complete the division cycle. To this end, cell cycle checkpoints prevent entry into the next cell cycle phase until the damage is fully repaired. Proper reentry into the cell cycle, known as checkpoint recovery, requires that a cell retains its original cell cycle state during the arrest. Here, we have identified Tousled‐like kinase 2 (Tlk2) as an important regulator of recovery after DNA damage in G2. We show that Tlk2 regulates the Asf1A histone chaperone in response to DNA damage and that depletion of Asf1A also produces a recovery defect. Both Tlk2 and Asf1A are required to restore histone H3 incorporation into damaged chromatin. Failure to do so affects expression of pro‐mitotic genes and compromises the cellular competence to recover from damage‐induced cell cycle arrests. Our results demonstrate that Tlk2 promotes Asf1A function during the DNA damage response in G2 to allow for proper restoration of chromatin structure at the break site and subsequent recovery from the arrest.