一种从PAXgene全血RNA管内提取基因组DNA的方法

目的:从同一生物样本同步提取RNA和DNA,能提高样本的利用率,而且对于基因组学、转录组学和表观遗传学检测数据之间的比对和匹配分析也十分重要。本研究在不影响RNA样品制备的前提下,建立一种从PAXgene全血RNA管内提取基因组DNA的方法。方法:取一定量PAXgene全血RNA管血液样本,使用QIAamp DNA试剂盒提取血细胞基因组DNA,系统优化提取过程中的离心参数、洗脱量以及初始血液样本量等实验参数,并对提取的基因组DNA质量进行检测。结果:用PAXgene全血RNA管3 mL血液样本能够提取出8.918±1.100μg基因组DNA,紫外分光光度计检测DNA样品的OD 260/280比值为1.89±0.09,琼脂糖凝胶电泳结果显示DNA样品完整无降解。结论:利用本方法提取的DNA样品能够满足下游DNA芯片、DNA甲基化测序等实验要求。该方法有助于从有限的临床血液样本中获取全面的遗传信息,并且提高后续不同实验方法所生成数据之间的可比性和匹配度。     

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。     

枝干树皮宏基因组DNA的提取

旨在得到一种适用于提取多种枝干树皮的高质量DNA的方法。参考前人提取植物DNA的经验,综合了研磨时加PVP、缓冲液洗涤、SDS与CTAB结合使用、高浓度KAc低温冻融、高浓度Na Cl条件下异丙醇沉淀、DNA完全溶解后加微量RNase A处理等操作,所得5种基因组DNA浓度介于190-970 ng/μL,纯度都很高,皆能被限制性内切酶切割,都可用作模板扩增到细菌16S r RNA基因片段,以苹果树皮DNA为模板扩增到苹果BIP和PDI基因且苹果树皮基因组DNA经测序公司检测,质量满足高通量测序法研究微生物多样性对环境宏基因组的要求。     

PCR鉴定时的微量DNA快速制备

用基因组微量DNA快速提取方法,从胡萝卜转化再生株样品中快速提取了基因组DNA,并以此为模板进行胡萝卜转化再生株的PCR快速检测的结果表明,这是转基因时PCR检测的一种快速、简便、有效方法。     

血浆游离DNA全基因组甲基化测序的实用稳定性评估

随着液体活检技术的发展,血浆游离DNA成为当前的研究热点之一。血浆游离DNA的全基因组甲基化测序被认为在癌症检测等医学应用拥有巨大潜力,但目前尚缺乏针对该实验流程的实用稳定性评估。文中利用两名志愿者在不同时间采样的血浆游离DNA,在不同实验平台分别进行DNA甲基化的重亚硫酸盐转化前建库(Pre-BS)、转化后建库(Post-BS)和常规DNA建库,获取多因素影响下的测序数据样本。在此基础上,建立了一套血浆游离DNA测序数据分析的质量控制参考流程,综合评估了血液采集提取、游离DNA建库测序过程的实用稳定性,为血浆游离DNA全基因组甲基化测序应用于临床液体活检提供实用性的基础参考。     

人类肠道菌群DNA提取影响因素的研究

目的研究提取人类粪便中细菌基因组DNA的影响因素。方法采用溶菌酶和十二烷基磺酸钠(SDS)+石英砂+酚-氯仿抽提法提取粪便标本中细菌DNA,用PCR扩增细菌16SrDNA。比较不同粪便量,不同放置时间,不同放置温度下的粪便细菌DNA浓度及纯度改变;用Chao index评测高通量测序的结果。结果采用20mg粪便量提取出的细菌DNA的浓度最高;常温下放置3h后,提取的细菌DNA的浓度开始下降;在-20℃放置12h后,细菌DNA的浓度开始下降;-70℃放置48h后细菌DNA的浓度开始下降;样品纯度均在1.8以上;Chao index曲线趋于平缓表明测序数据量足够大。结论提取肠道细菌基因组DNA时,粪便标本取样量以20mg为宜,常温下粪便标本放置不超过2h,本研究所使用的方法所提取的DNA浓度可以达到高通量测序的样本要求。     

煤地质环境微生物总基因组DNA提取方法的优化

高质量的DNA是进行分子生物学研究的基础。通过对传统DNA抽提方法(酚-氯仿法)进行改进,并以Rhodococcus sp.R04和煤粉为实验材料对细胞破碎条件进行优化,建立了一种可用于煤地质环境微生物基因组DNA高效提取的改良方法。以改良法和商业试剂盒提取煤地质环境微生物基因组DNA,通过琼脂糖凝胶电泳、细菌及古菌特异性片段的PCR扩增来评价所提取DNA的质量。改良法和试剂盒法均能获得煤地质环境微生物基因组DNA,并能用于多种特异性PCR扩增。与试剂盒提取的DNA相比,改良法获得的DNA片段主带明显,约占总DNA含量的50%,分子量大小接近23 kb,并且提取量大,约为试剂盒的5-10倍。同时,能用于如DNA文库构建和宏基因组测序等。此外,改良法所用试剂普通,价格便宜,提取的煤地质环境微生物基因组DNA质量较高,适于实验室和科学研究。     

基于宏基因组测序的植物叶表微生物富集及DNA提取方法

获得高质量的微生物基因组DNA是进行复杂微生物群落宏基因组学研究的基础和难点。植物叶表是一个微生物多样性丰富的复杂生态系统,这些微生物群落可以调节叶片功能性状,影响植物的适应性。深入了解叶表微生物群落的基本结构和功能原理,有助于在促进植物生长和植物保护方面发挥重要的应用价值。由于叶表严苛的环境,导致富集叶表微生物难度较大,严重限制了高质量叶表微生物基因组DNA的提取。基于现有DNA提取方法,加入表面活性剂Silwet L-77进行前处理,同时循环利用洗脱液,加强叶表微生物的富集,以提高叶表微生物的获取量。结合商业试剂盒方法进行提取得到高纯度、高浓度的基因组DNA。经过质量控制和建库测序验证,DNA质量达到宏基组建库的要求。通过此方法可以提高叶表微生物分离和收集效率的方法,提高叶表微生物DNA提取成功率,为应用高通量测序技术研究叶表微生物组成和其他植物分子生物学研究提供参考。     

慢性阻塞性肺疾病患者支气管肺泡灌洗液细菌宏基因组DNA的制备

【目的】优化稳定期慢性阻塞性肺疾病(chronic obstructive pulmonary diseases,COPD)患者支气管肺泡灌洗液(bronchoalveolar lavage fluids,BALF)细菌宏基因组DNA的提取方法,以便于高效提取微量的细菌DNA进行后续的PCR反应和测序。【方法】取稳定期COPD患者的BALF 5mL,离心收集细胞。为了有效提取样品中革兰氏阳性菌的基因组,对QIAGEN的DNA提取试剂盒的操作步骤进行优化:加入裂解缓冲液ATL后首先运用研磨珠和多功能生物样品匀质器破碎菌壁,再加入蛋白酶K孵育,然后加入裂解缓冲液AL振荡混匀。无水乙醇沉淀DNA后,将全部溶液过柱,用洗液AW1和AW2各洗柱一次,最后加50μL洗脱液洗脱DNA。提取的DNA定量后,运用PCR方法检测样本中的细菌16S rDNA量,并按照测序要求构建DNA文库进一步验证。【结果】试剂盒优化法提取的BALF的DNA总量为467.5(135.0-1697.5)ng,明显高于按照传统酚-氯仿法提取的DNA总量95.0(0-612.5)ng,并且所提取的DNA可以很好的扩增细菌的16S rDNA以及构建DNA文库,改良后的扩增产物明显增多(P=0.002)。【结论】使用DNA提取试剂盒结合研磨珠和多功能生物样品匀质器破菌壁的方法能够更高效的提取BALF中微量的宏基因组DNA,为进一步的测序和菌群分析打下基础。     

适于基因组测序的高纯度的大豆线粒体基因组DNA提取方法

为满足高通量二代测序要求,本研究采用大豆黄花苗为试材,结合差速离心、蔗糖密度梯度离心及超速离心方法提取高纯度大豆线粒体基因组DNA(mt DNA)。结果表明,差速离心能够有效去除核基因组掺杂;超速离心与蔗糖密度梯度离心结合能够有效去除叶绿体污染。提取的mt DNA经琼脂糖凝胶电泳、紫外光度计检测及叶绿体和细胞核特异性引物检测表明,该方法提取的大豆mt DNA无叶绿体DNA及核DNA污染,且纯度高,可满足测序等对线粒体高纯度的要求,为研究大豆线粒体相关性状的机理奠定了坚实基础。     

Strategies for accessing soil metagenome for desired applications

Most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches promise the accessibility of the genetic resources and their potential applications. Genetic resources from terrestrial environments can be accessed by exploring the soil metagenome. Soil metagenomic analyses are usually initiated by the isolation of environmental DNAs. Several methods have been described for the direct isolation of environmental DNAs from soil and sediments. Application of metagenomics largely depends on the construction of genomic DNA libraries and subsequent high-throughput sequencing or library screening. Thus, obtaining large quantities of pure cloneable DNA from the environment is a prerequisite. This review discusses the recent developments related to efficient extraction and purification of soil metagenome highlighting the considerations for various metagenomic applications.     

Laboratory contamination over time during low‐biomass sample analysis

Bacteria are not only ubiquitous on earth but can also be incredibly diverse within clean laboratories and reagents. The presence of both living and dead bacteria in laboratory environments and reagents is especially problematic when examining samples with low endogenous content (e.g., skin swabs, tissue biopsies, ice, water, degraded forensic samples or ancient material), where contaminants can outnumber endogenous microorganisms within samples. The contribution of contaminants within high‐throughput studies remains poorly understood because of the relatively low number of contaminant surveys. Here, we examined 144 negative control samples (extraction blank and no‐template amplification controls) collected in both typical molecular laboratories and an ultraclean ancient DNA laboratory over 5 years to characterize long‐term contaminant diversity. We additionally compared the contaminant content within a home‐made silica‐based extraction method, commonly used to analyse low endogenous content samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to modern molecular biology laboratories, and changed over time according to researcher, month and season. The commercial kit also contained higher microbial diversity and several human‐associated taxa in comparison to the home‐made silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low‐biomass metagenomic studies: (a) extraction blank controls should be included and sequenced with every batch of extractions and (b) the contributions of laboratory contamination should be assessed and reported in each high‐throughput metagenomic study.     

Statistical assessment of DNA extraction methodology for culture-independent analysis of microbial community associated with diverse environmental samples

Cost-effectiveness, quality, time-effectiveness and ease of the methodology are the most crucial factors in isolating quality DNA from wide variety of samples. Thus, research efforts focusing on the development of an efficient DNA extraction protocol is the need of the hour. The present study therefore, focuses on development of an efficient, rapid and free of inhibitory substances based methodology for extracting metagenomic DNA from diverse environmental samples viz. anaerobic biogas digesta, ruminant stomach, human feces, soil, and microbial starter cultures used for preparation of fermented food. PCR–DGGE based analysis and quality metagenomic library preparation, using DNA extraction methodology, validates the developed protocol. The developed protocol is cost effective, capable of isolating DNA from small sample size (100–1000 µl), time efficient (1.5–2.0 h protocol) and results in significantly higher DNA yield (4–8 times increased yield) when compared to previously available DNA extraction method and a commercial DNA extraction kit. The DNA extracted from the samples using different protocols was evaluated based on its ability to identify diverse microbial species using PCR–DGGE profiles targeting variable region within the 16S rRNA gene. The results of microbial community analysis revealed comparability of the developed protocol to commercial kits, in effectively identifying dominant representatives of the microbial community in different samples. Using the DNA extracted from the presented methodology, metagenomic libraries were prepared, which were found suitable for sequencing on Illumina platform.     

Extraction of high molecular weight DNA from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.     

Biocatalysts and small molecule products from metagenomic studies

The vast majority of bacteria present in environmental samples have never been cultured and therefore have not been exploited for the ability to produce useful biocatalysts or collections of biocatalysts generating interesting small molecules. Metagenomic libraries constructed using DNA extracted directly from natural bacterial communities offer access to the genetic information present in the genomes of these as yet uncultured bacteria. This review highlights recent efforts to recover both discrete enzymes and small molecules from metagenomic libraries.     

宏基因组学与动物病原微生物检测

宏基因组学（ metagenome）是直接从土壤、海水、人及动物胃肠道、口腔、呼吸道、皮肤等环境中获取样品DNA,利用载体将其克隆到替代宿主细胞中构建宏基因文库,以高通量检测为主要技术来研究特定环境中全部微生物的基因组及筛选活性物质和基因的新兴学科。利用宏基因组学技术不仅能够有效地检测特定环境的微生物群落结构,扩展了微生物资源的利用空间,发展了新兴的高通量检测技术,丰富了生物信息学内容。基于宏基因组学研究方法在环境微生物研究中的优势,对近年来相关领域、方法及其在人及动物病原微生物研究中的应用进行综述,以期将此方法用于实验动物病原微生物的调查分析及动物疫情、生物安全的监测。     

Metagenomic Sequencing of an In Vitro-Simulated Microbial Community

Background

Microbial life dominates the earth, but many species are difficult or even impossible to study under laboratory conditions. Sequencing DNA directly from the environment, a technique commonly referred to as metagenomics, is an important tool for cataloging microbial life. This culture-independent approach involves collecting samples that include microbes in them, extracting DNA from the samples, and sequencing the DNA. A sample may contain many different microorganisms, macroorganisms, and even free-floating environmental DNA. A fundamental challenge in metagenomics has been estimating the abundance of organisms in a sample based on the frequency with which the organism''s DNA was observed in reads generated via DNA sequencing.

Methodology/Principal Findings

We created mixtures of ten microbial species for which genome sequences are known. Each mixture contained an equal number of cells of each species. We then extracted DNA from the mixtures, sequenced the DNA, and measured the frequency with which genomic regions from each organism was observed in the sequenced DNA. We found that the observed frequency of reads mapping to each organism did not reflect the equal numbers of cells that were known to be included in each mixture. The relative organism abundances varied significantly depending on the DNA extraction and sequencing protocol utilized.

Conclusions/Significance

We describe a new data resource for measuring the accuracy of metagenomic binning methods, created by in vitro-simulation of a metagenomic community. Our in vitro simulation can be used to complement previous in silico benchmark studies. In constructing a synthetic community and sequencing its metagenome, we encountered several sources of observation bias that likely affect most metagenomic experiments to date and present challenges for comparative metagenomic studies. DNA preparation methods have a particularly profound effect in our study, implying that samples prepared with different protocols are not suitable for comparative metagenomics.     

Construction and validation of two metagenomic DNA libraries from Cerrado soil with high clay content

A challenge of metagenomic studies is in the extraction and purification of DNA from environmental samples. The soils of the Cerrado region of Brazil present several technical difficulties to DNA extraction: high clay content (>55% w/w), low pH (4.7) and high iron levels (146 ppm). Here we describe for the first time the efficient recovery and purification of microbial DNA associated with these unusual soil characteristics and the construction and validation of two metagenomic libraries: a 150,000 clones library with insert size of approximately 8 kb and a 65,000 clones library with insert size of approximately 35 kb. The construction of these metagenomic libraries will allow the biotechnological exploitation of the microbial community present in the soil from this endangered biome.     

A Metagenomic Framework for the Study of Airborne Microbial Communities

Understanding the microbial content of the air has important scientific, health, and economic implications. While studies have primarily characterized the taxonomic content of air samples by sequencing the 16S or 18S ribosomal RNA gene, direct analysis of the genomic content of airborne microorganisms has not been possible due to the extremely low density of biological material in airborne environments. We developed sampling and amplification methods to enable adequate DNA recovery to allow metagenomic profiling of air samples collected from indoor and outdoor environments. Air samples were collected from a large urban building, a medical center, a house, and a pier. Analyses of metagenomic data generated from these samples reveal airborne communities with a high degree of diversity and different genera abundance profiles. The identities of many of the taxonomic groups and protein families also allows for the identification of the likely sources of the sampled airborne bacteria.