一种从雉类孵化卵壳中提取DNA的方法

从鸟类已孵化的卵壳中提取DNA属于一种非损伤性取样技术,在鸟类分子生态学研究中具有广阔的应用前景。本实验以河南董寨自然保护区白冠长尾雉(Syrmaticus reevesii)和环颈雉(Phasianuscolchicus)已孵化的卵壳为材料,利用红细胞破碎液、蛋白酶K及RNA酶等试剂,对卵壳膜内的总DNA进行了提取,建立了一种提取高质量DNA的新方法。琼脂糖凝胶电泳检测显示,采用新改进的方法提取出的DNA条带清晰。同时,利用聚合酶链式反应(PCR)技术成功地从总DNA中扩增出两种雉类的线粒体DNA控制区(CR)片段,测序后与GenBank中同一物种的CR序列进行比对,结果证实了PCR产物的真实性。文中利用卵壳膜提取出的DNA,对一窝环颈雉的雏鸟进行了性别鉴定,其结果与根据形态特征进行鉴定的结果完全一致,均为3雄4雌,从而证实了从卵壳膜中提取DNA的真实性。该种DNA提取方法在雉类研究中将具有广泛的应用。     

一种用于PCR的番茄DNA快速粗提方法

在对植物进行分子生物学研究时需要提取植物基因组DNA并对其进行PCR克隆,目前实验室常用CTAB法提取植物DNA,但其步骤相对繁琐,需要用有机溶剂反复抽提,在样本较多时,耗时较长。NaOH可以提取多种物种DNA,本研究优化了实验室条件下更加快速、经济、安全、高效的提取植物DNA的方式,主要包括以下步骤：利用液氮快速粉碎植物样品;采用碱裂解(0.5 mol/L NaOH)的方式一步提取DNA;提取的DNA利用TE缓冲液进行中和;以中和的粗提DNA作为模板进行PCR反应。本研究利用简单的DNA提取流程并结合PCR检测方式,完成了番茄转化子的快速初步鉴定。同时还证明了该NaOH法可用于番茄不同组织的DNA提取。本研究中整个操作过程步骤简单,耗时短,可在短时间内完成大量植物样品的DNA提取,无需使用氯仿、异丙醇等有毒物质,完成提取时间约为CTAB提取法的1/5,而且提取的不同组织DNA完整性较好,质量可观,满足常规的PCR检测,可用于基因克隆及转化子筛选鉴定,具有一定的利用价值和应用前景。     

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。     

一种用于PCR扩增的丝状真菌DNA快速提取方法

丝状真菌在工业、农业、医药以及基础生物学研究中具有重要作用。利用遗传转化技术对丝状真菌进行菌株改良和基因功能分析, 也越来越受到重视。然而, 丝状真菌DNA提取方法繁琐、费时, 难以满足利用PCR技术高通量筛选转化子的需要。本文以曲霉菌为例建立了一种快速提取丝状真菌DNA的实验方法, 微波处理置于10 × TE buffer中的菌丝即可得到DNA。RAPD试验和PCR扩增证明, 该方法提取的DNA能够达到PCR扩增的要求。研究结果为高通量快速筛选丝状真菌转化子奠定了基础。     

高通量测序技术在线虫多样性研究中的应用

土壤线虫多样性是土壤生态学研究的热点之一, 然而对土壤线虫群落组成及多样性的研究通常受到分类学和方法学的限制。当前, 分子生物学技术的快速发展丰富了我们对土壤线虫多样性的认识, 但也存在一定的局限性。本文综述了常用分子生物学技术如变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)、末端限制性片段长度多态性分析(terminal restriction fragment length polymorphism, T-RFLP)、实时荧光定量PCR (quantitative real-time PCR, qPCR)和高通量测序(high-throughput sequencing, HTS)技术近年来在线虫多样性研究中的应用, 重点从土壤线虫DNA提取方法、引物和数据库的选择、高通量测序技术和形态学鉴定结果的比较等方面阐述了高通量测序技术在线虫多样性研究中的优势与不足, 并提出选择合适的线虫DNA提取方法结合特定引物和数据库进行注释分析, 仍是今后使用高通量测序技术开展线虫多样性研究的重点。当研究目标是土壤线虫多样性时, 优先推荐富集线虫悬液提取DNA的方法, 因此, 研究人员应根据具体目标选择最优组合开展实验研究。     

DNA提取对微生物多样性测序分析的影响

运用高通量测序技术分析复杂样品中微生物种群的变化情况,已经成为目前微生物研究领域的热点问题之一。而微生物的样品准备,如DNA提取和16S可变区的扩增等,对于测序完成后的数据分析以及微生物原始群落组成的影响是至关重要的。采用国产试剂盒(天根土壤微生物基因组提取试剂盒)和进口试剂盒(MOBIO土壤微生物基因组提取试剂盒)分别对土壤样品和羊瘤胃食糜样品进行DNA提取。然后选取总DNA起始量为25ng,对16S V3可变区进行PCR扩增和文库构建,最后通过数据分析比较不同试剂盒提取的DNA对微生物多样性变化的影响,包括OTU数目、稀释曲线、微生物数量及物种种类等。研究发现,在相同DNA模板量和PCR条件下,进口试剂盒提取的DNA能够获得更多的微生物种类。     

基因工程菌里氏木霉染色体DNA的提取方法

研究基因工程菌株里氏木霉(Trichoderma Teesei)染包体DNA的提取方法。采用冷冻研磨CTAB法、氯化苄SDS法和冷冻研磨SDS法三种提取DNA的力法。通过琼脂糖凝胶电泳埘提取的DNA进行检测。结果表明：冷冻研磨CTAB法更适合此类DNA的提取。对冷冻研磨CTAB法提取条件进行了优化。用该法提取的DNA上的外源基因t—PAcDNA部分片段进行PCR扩增,获得良好结果。用本实验冷冻研磨CTAB法提取的DNA完全适用于PCR和其它的分子生物学操作的要求。     

一种简单、快速的苏云金芽胞杆菌基因组DNA提取方法

从培养时间、裂解溶液、抽提和沉淀时间等方面对苏云金芽胞杆菌(Bacillus thuringiensis,Bt)基因组DNA提取技术进行改进.提取8株对蛴螬有杀虫活性的野生Bt菌株的基因组DNA,分析其纯度,并进行PCR分析与酶切分析.试验结果表明,新的提取方法耗时36 min,明显短于旧方法所用时间(97 min);两种方法提取的基因组DNA A260/A280均大于1.8,无明显区别;琼脂糖凝胶电泳结果显示,上样量相同时,新方法提取的基因组DNA浓度是旧方法的5倍以上;新方法提取的基因组DNA能作为模板灵敏地扩增出测试基因,所提取的DNA能被限制性内切酶完全酶切,证明DNA具有很高的纯度.本研究改进的基因组DNA提取方法耗时短、产量高,并能满足PCR扩增、酶切等分子生物学需要.     

快速STR分型在法医DNA分析中的应用研究进展

STR分型是目前世界通用的DNA指纹分析方法.传统的分型方法包括DNA提取、DNA定量、PCR扩增和对扩增后的STR片段进行检测和分析,耗时较长(8¹0 h).一些情况下很难满足案件的实际需求.法医学家们在加快STR分型的方法研究一直不懈努力.现从快速提取DNA、无提取直接PCR、快速PCR、微流控芯片技术在STR快速分型方面的应用四方面,对近年来出现的可进行快速STR分型的新技术、新方法进行综述.     

一种从大熊猫粪便中提取DNA的改进方法

本研究描述一个改进的方法，使从大熊猫粪便中提取DNA用于PCR扩增变得更加容易。在粪便DNA的提取过程中采用一个新的预处理方法，将粪便用预冷的丙酮洗2～3次，除去粪便中含有的大量PCR抑制物，然后用蛋白酶K裂解、酚氯仿抽提，能提取到纯度很高的DNA供PCR扩增。本实验PCR扩增了大熊猫脑源性神经营养因子(BDNF)基因和线粒体细胞色素6基因片段，并进行测序分析，证实了提取的可靠性。对比本方法和未经丙酮预处理的方法提取的DNA进行PCR扩增，前者的扩增结果明显优于后者。     

以川陕哲罗鲑为目标物种的水样环境DNA分析流程的优化

水样环境DNA分析包括水样采集、DNA提取和分析等流程,已成为监测濒危水生生物种群分布调查的重要手段.为减少在监测目标物种尤其濒危物种中的不确定性,对水环境DNA分析流程的优化至关重要.本研究以川陕哲罗鲑为目标物种,采用滤膜法采集养殖池中的水样,设计了 250 mL、500 mL、1 L和2 L等4种水样采集量,分别采用 PoweWater DNA Isolation kit和DNeasy Tissue and Blood DNA extraction kit 提取水样环境DNA(eDNA),使用物种mtDNA D_loop区特异性引物进行PCR扩增,通过研究滤膜法、水样采集量和水样DNA提取方法对水样eDNA中目标基因检出率的影响,探索适宜的eDNA分析操作方案.结果表明: 使用DNeasy Tissue and Blood DNA extraction kit提取的水样DNA中目的基因的检出率为100%,效果明显优于PoweWater DNA Isolation kit(目标基因的检出率为0);目标基因扩增条带的亮度随水样采样量的增加而增加,其中2 L水样目标基因的扩增效果较理想;序列比对结果显示,本试验从水样DNA中成功扩增得到了川陕哲罗鲑mtDNA Dloop区部分序列.表明DNA提取方法和水样采集量对目标物种的检出率有显著的影响,滤膜法、2 L水样采集量、DNeasy Tissue and Blood DNA extraction kit更适宜进行水样的DNA分析,mtDNA D-loop区可作为川陕哲罗鲑识别的特异性分子标记.     

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.     

Whole genome sequencing of marine organisms by Oxford Nanopore Technologies: Assessment and optimization of HMW‐DNA extraction protocols

Marine habitats are Earth''s largest aquatic ecosystems, yet little is known about marine organism''s genomes. Molecular studies can unravel their genetics print, thus shedding light on specie''s adaptation and speciation with precise authentication. However, extracting high molecular weight DNA from marine organisms and subsequent DNA library preparation for whole genome sequencing is challenging. The challenges can be explained by excessive metabolites secretion that co‐precipitates with DNA and barricades their sequencing. In this work, we sought to resolve this issue by describing an optimized isolation method and comparing its performance with the most commonly reported protocols or commercial kits: SDS/phenol–chloroform method, Qiagen Genomic Tips kit, Qiagen DNeasy Plant mini kit, a modified protocol of Qiagen DNeasy Plant kit, Qiagen DNeasy Blood and Tissue kit, and Qiagen Qiamp DNA Stool mini kit. Our method proved to work significantly better for different marine species regardless of their shape, consistency, and sample preservation, improving Oxford Nanopore Technologies sequencing yield by 39 folds for Spirobranchus sp. and enabling generation of almost 10 GB data per flow cell/run for Chrysaora sp. and Palaemon sp. samples.     

A practical guide to sample preservation and pre‐PCR processing of aquatic environmental DNA

Environmental DNA (eDNA) is rapidly growing in popularity as a tool for community assessments and species detection. While eDNA approaches are now widely applied, there is not yet agreement on best practices for sample collection and processing. Investigators looking to integrate eDNA approaches into their research programme are required to examine a growing collection of disparate studies to make an often uncertain decision about which protocols best fit their needs. To promote the application of eDNA approaches and to encourage the generation of high‐quality data, here we review the most common techniques for the collection, preservation and extraction of metazoan eDNA from water samples. Specifically, we focus on experimental studies that compare various methods and outline the numerous challenges associated with eDNA. While the diverse applications of eDNA do not lend themselves to a one‐size‐fits‐all recommendation, in most cases, capture/concentration of eDNA on cellulose nitrate filters (with pore size determined by water turbidity), followed by storage of filters in Longmire's buffer and extraction with a DNeasy Blood & Tissue Kit (or similar) has been shown to provide sufficient, high‐quality DNA. However, we also emphasize the importance of testing and optimizing protocols for the system of interest.     

Comparison of three methods of DNA extraction from cold-smoked salmon and impact of physical treatments

AIMS: To compare three bacterial DNA extraction procedures on cold-smoked salmon (CSS) and assess the impact on their efficiency of two physical treatments of the food matrix, ionizing irradiation and freezing. METHODS AND RESULTS: As molecular methods for bacterial detection have become an important analytical tool, we compared bacterial DNA extraction procedures on CSS. Working with frozen and irradiated CSS, we obtained negative responses from samples known to be highly contaminated. Thus, we decided to study the impact of these two physical treatments on bacterial DNA extraction procedures. The efficiency of bacterial DNA extraction directly from the fish matrix suspension was measured by an rpoB PCR-based reaction. The results demonstrated that the DNeasy tissue extraction kit (Qiagen, Courtaboeuf, France) was the most efficient and reproducible method. We also showed that freezing and ionizing irradiation have a negative impact on DNA extraction. This was found probably not to be due to inhibition as the PCR reaction remained negative after adding BSA to the PCR mix reaction. CONCLUSIONS: The extraction kit was the most efficient method. Physical treatments were shown to hamper bacterial DNA extraction. SIGNIFICANCE AND IMPACT OF THE STUDY: Attention must be paid to molecular bacterial detection on food products subject to freezing or to ionizing irradiation.     

Evaluation of different DNA sampling techniques for the application of the real-time PCR method for the quantification of cyanobacteria in water

AIMS: To evaluate different types of sample storage and DNA extraction techniques for the real-time PCR quantification of cyanobacteria in water. METHODS AND RESULTS: Two different filter types for the cell harvest of Microcystis sp. and Planktothrix spp. that were either freeze-dried or stored frozen, and two different methods for DNA extraction were compared. DNA extraction was achieved by standard phenol-chloroform extraction or by a faster commercially available purification kit (DNeasy, QIAGEN). In general there was good agreement between the cell number equivalents of phycocyanin (PC) genotypes that were estimated using the Taq nuclease assay (TNA) between both filter types and the storing of samples. The standard DNA extraction procedure gave higher numbers of PC genotypes when compared with the DNeasy procedure. TNA results obtained from Planktothrix from natural samples extracted with the standard procedure revealed a significant correlation with the cell numbers estimated via the microscope. CONCLUSIONS: Freeze-drying of samples gives quantifiable data. The standard DNA extraction is considered to be the most reliable and accurate, although the DNeasy procedure is useful for early warning monitoring. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of quantitative genotype analysis in cyanobacteria from freeze-dried samples collected during recent and past sampling programmes.     

Estimating fish abundance and biomass from eDNA concentrations: variability among capture methods and environmental conditions

Environmental DNA (eDNA) promises to ease noninvasive quantification of fish biomass or abundance, but its integration within conservation and fisheries management is currently limited by a lack of understanding of the influence of eDNA collection method and environmental conditions on eDNA concentrations in water samples. Water temperature is known to influence the metabolism of fish and consequently could strongly affect eDNA release rate. As water temperature varies in temperate regions (both seasonally and geographically), the unknown effect of water temperature on eDNA concentrations poses practical limitations on quantifying fish populations using eDNA from water samples. This study aimed to clarify how water temperature and the eDNA capture method alter the relationships between eDNA concentration and fish abundance/biomass. Water samples (1 L) were collected from 30 aquaria including triplicate of 0, 5, 10, 15 and 20 Brook Charr specimens at two different temperatures (7 °C and 14 °C). Water samples were filtered with five different types of filters. The eDNA concentration obtained by quantitative PCR (qPCR) varied significantly with fish abundance and biomass and types of filters (mixed‐design ANOVA, P < 0.001). Results also show that fish released more eDNA in warm water than in cold water and that eDNA concentration better reflects fish abundance/biomass at high temperature. From a technical standpoint, higher levels of eDNA were captured with glass fibre (GF) filters than with mixed cellulose ester (MCE) filters and support the importance of adequate filters to quantify fish abundance based on the eDNA method. This study supports the importance of including water temperature in fish abundance/biomass prediction models based on eDNA.     

Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels

Identification of Fusarium species by traditional methods requires specific skill and experience and there is an increased interest for new molecular methods for identification and quantification of Fusarium from food and feed samples. Real-time PCR with probe technology (Taqman) can be used for the identification and quantification of several species of Fusarium from cereal grain samples. There are several critical steps that need to be considered when establishing a real-time PCR-based method for DNA quantification, including extraction of DNA from the samples. In this study, several DNA extraction methods were evaluated, including the DNeasy Plant Mini Spin Columns (Qiagen), the Bio robot EZ1 (Qiagen) with the DNeasy Blood and Tissue Kit (Qiagen), and the Fast-DNA Spin Kit for Soil (Qbiogene). Parameters such as DNA quality and stability, PCR inhibitors, and PCR efficiency were investigated. Our results showed that all methods gave good PCR efficiency (above 90%) and DNA stability whereas the DNeasy Plant Mini Spin Columns in combination with sonication gave the best results with respect to Fusarium DNA yield. The modified DNeasy Plant Mini Spin protocol was used to analyse 31 wheat samples for the presence of F. graminearum and F. culmorum. The DNA level of F. graminearum could be correlated to the level of DON (r(2) = 0.9) and ZEN (r(2) = 0.6) whereas no correlation was found between F. culmorum and DON/ZEA. This shows that F. graminearum and not F. culmorum, was the main producer of DON in Swedish wheat during 2006.     

Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.     

Evaluation of methods for extracting Xylella fastidiosa DNA from the glassy-winged sharpshooter

The recent spread of the plant pathogenic bacterium Xylclla fastidiosa Wells et al. by an invasive vector species, Homalodisca coagulata Say, in southern California has resulted in new epidemics of Pierce's disease of grapevine. Our goal is to develop an efficient method to detect low titers of X. fastidiosa in H. coagulata that is amenable to large sample sizes for epidemiological studies. Detection of the plant pathogenic bacterium X. fastidiosa in its insect vector is complicated by low titers of bacteria, difficulty in releasing it from the insect mouthparts and foregut, and the presence of substances in the insect that inhibit polymerase chain reaction (PCr). To select the optimal protocol for DNA extraction to be used with PCR, we compared three standard methods and 11 commercially available kits for relative efficiency of X. fastidiosa DNA extraction in the presence of insect tissue. All of the protocols tested were proficient at extracting DNA from pure bacterial culture (1 x 10(5) cells), and all but one protocol successfully extracted sufficient bacterial DNA in the presence of insect tissue. Three DNA extraction techniques, immunomagnetic separation, the DNeasy Tissue kit (Qiagen, Hercules, CA), and Genomic DNA Purification kit (Fermentus, Hanover, MD), were compared more closely using a dilution series of X. fastidiosa (5000-0 cells) with and without insect tissue present. The DNeasy Tissue kit was the best kit tested, allowing detection of 5 x 10(3) X. fastidiosa cells with an insect head background.