保存方式和回软温度对蜜蜂标本DNA提取的影响北大核心CSCD

旨在探寻保存方式及制作干标本前回软温度对蜜蜂不同部位DNA的影响。采用酚-氯仿法对不同方式保存的蜜蜂以及不同温度回软后的蜜蜂干标本的总DNA进行提取,通过琼脂糖凝胶电泳和PCR扩增对DNA的提取结果进行鉴定。电泳结果显示,从新鲜标本、无水乙醇泡制或自然干燥保存半年的标本中均可提取到较高质量的总DNA,尤以头部与足部提取效果最佳。经不同水浴温度回软后保存半年的蜜蜂干标本总DNA提取结果显示,回软温度为65℃时对蜜蜂DNA的破坏性最小,DNA提取的最佳部位为蜜蜂足部。正交试验结果显示,55℃回软后的足部为蜜蜂干标本DNA提取的最优组合。PCR扩增结果显示,本实验提取的总DNA能成功地应用于蜜蜂线粒体基因16S rRNA和COI的扩增。从保存方式、提取部位和回软操作3个因素对蜜蜂标本DNA的提取进行了研究,提供了较好的选择方案。     

保存方式和回软温度对蜜蜂标本DNA提取的影响

旨在探寻保存方式及制作干标本前回软温度对蜜蜂不同部位DNA的影响。采用酚-氯仿法对不同方式保存的蜜蜂以及不同温度回软后的蜜蜂干标本的总DNA进行提取,通过琼脂糖凝胶电泳和PCR扩增对DNA的提取结果进行鉴定。电泳结果显示,从新鲜标本、无水乙醇泡制或自然干燥保存半年的标本中均可提取到较高质量的总DNA,尤以头部与足部提取效果最佳。经不同水浴温度回软后保存半年的蜜蜂干标本总DNA提取结果显示,回软温度为65℃时对蜜蜂DNA的破坏性最小,DNA提取的最佳部位为蜜蜂足部。正交试验结果显示,55℃回软后的足部为蜜蜂干标本DNA提取的最优组合。PCR扩增结果显示,本实验提取的总DNA能成功地应用于蜜蜂线粒体基因16S rRNA和COI的扩增。从保存方式、提取部位和回软操作3个因素对蜜蜂标本DNA的提取进行了研究,提供了较好的选择方案。     

福尔马林对固定标本DNA提取和扩增的影响

福尔马林被广泛应用于生物标本的长期保存。由于福尔马林可能影响标本DNA的质量,因此需要对福尔马林固定标本DNA的提取和扩增过程进行改进。影响从福尔马林保存标本中提取的DNA质量的主要因素包括福尔马林导致的DNA与蛋白质之间、蛋白质与蛋白质之间、DNA与DNA之间的交联,福尔马林溶液的化学成分、pH值及浓度,标本保存的时间和温度,标本保存部位等。本文总结了目前常用的对标本DNA提取和扩增过程的改进措施及其优点。     

粉蚧标本保存与DNA提取方法的比较

【目的】探讨适合粉蚧的标本保存及DNA提取方法。【方法】采用改良CTAB法、改良SDS法、Gen Mag Bio动物细胞组织/细胞基因组磁珠法以及Gene JET Genomic DNA纯化试剂盒法4种方法分别对新鲜活体4℃、无水乙醇﹣20℃和无水乙醇4℃3种保存方式且保存一年以上的扶桑绵粉蚧Phenacoccus solenopsis成虫进行DNA提取,并对不同提取方法所获取的DNA纯度与质量浓度进行分析比较验证。【结果】3种保存方式中,新鲜活体效果最好,其次为无水乙醇-20℃。无水乙醇4℃效果和无水乙醇﹣20℃无明显区别,均存在一定程度的降解。对于新鲜活体标本,以CTAB法提取的DNA质量最高,其次为Gen Mag Bio磁珠法和SDS法,Gene JET试剂盒法最差;对于无水乙醇﹣20℃和4℃保存时间较长的标本,磁珠法提取的DNA质量明显优于其余3种方法。【结论】无水乙醇﹣20℃可用于粉蚧长期保存,可满足后续分子研究需要;改良CTAB法对新鲜粉蚧成虫DNA提取效果较好,磁珠法对长时间保存DNA存在一定程度降解的粉蚧成虫效果较好。     

一种对甲螨无形态损伤的DNA提取技术

甲螨是一类重要的土壤动物,体型微小,一般具有较厚的体壁。本研究针对甲螨这一特定类群,探讨了一种无形态特征损伤的DNA提取技术。通过结合试剂盒DNA提取法,并适当改进实验条件,设计出一套行之有效的DNA提取流程。通过对提取DNA之后的标本进行形态学观察,发现其主要的分类学特征均保存完好,可以作为凭证标本长期保存。本研究所提供的DNA提取技术既可以提取出足够的DNA又可以保留凭证标本,因此能有效促进甲螨分子分类学相关研究。     

卷象科昆虫无损形态的DNA提取方法

提取DNA是所有分子生物学实验的第一步,但在昆虫分子生物学研究中,常规提取总DNA的方法不能保留昆虫所有的形态特征。显然,这不适用于对于体形较小的珍稀标本。以鞘翅目卷象科(Coleoptera:Attelabidae)昆虫为实验材料,提供了一种新的取材方法,取下整个腹部,消化其肌肉组织后收回骨化腹板。该方法可在提取昆虫总DNA的同时保存全部外形特征及其生殖器。     

松墨天牛成虫标本保存及其DNA提取质量比较

【目的】探讨适合DNA提取的天牛成虫标本保存方法。【方法】采用SDS-蛋白酶K消化法对液氮中冷冻保存、无水乙醇-20℃冷冻保存、无水乙醇室温保存和干标本室温保存且保存时间在2年以上的松墨天牛Monochamus alternates Hope成虫标本基因组DNA进行提取,并对不同保存方式提取的DNA样本进行了质量比较和分析。【结果】在上述常见的松墨天牛成虫标本4种保存方式中,以液氮中冷冻保存效果最佳,其次为无水乙醇-20℃冷冻保存,插针干标本室温保藏效果最差。利用昆虫线粒体基因CO I和CO II的通用引物从上述DNA中均能够成功扩增出目的片段,测序结果证实扩增片段符合预期。【结论】液氮和无水乙醇-20℃冷冻保存适合松墨天牛成虫标本长期保存,且不影响后续的PCR扩增和测序。     

不同方法保存的蜜蜂基因组DNA提取的比较

目的:找出适合DNA提取的昆虫标本保存方法。方法:用几种常用的昆虫标本保存方法对蜜蜂处理不同时间后,用蛋白酶K法对其基因组DNA进行提取和纯化,然后对提取产物做琼脂糖凝胶电泳及紫外吸收分析。结果:75%乙醇及冻存处理材料的基因组DNA得率较高,为7.13～8.85μg/g,电泳条带较亮;甲醛处理材料的基因组DNA得率较低,为1.50～3.21μg/g。结论:用75%乙醇及冻存处理蜜蜂较适合于其基因组DNA的提取,不宜用甲醛。     

一种高效快速的鱼类标本基因组DNA提取方法

以95%酒精保存的黄鳝(M onopterus albus)和斑鳢(Channa maculates)标本为材料,采用先沉降DNA再去除杂质的方法从鱼类标本中提取基因组DNA。基因组DNA的琼脂糖凝胶电泳和紫外分光光度法检测以及PCR扩增结果显示,本方法提取的鱼类基因组DNA的电泳主带清晰明亮;A260/A280值在1.7830-2.0144之间;PCR扩增产物条带清晰明亮,且单一整齐没有拖带,表明本方法可从酒精保存的鱼类标本中提取比较纯净的DNA,能够满足一般分子生物学试验需要。与传统苯酚/氯仿法相比,本方法操作简单快速,避免了苯酚等物质对后续实验的影响,可作为一种常规动物组织DNA提取方法。     

福尔马林保存鱼类标本中大片段DNA的提取

从福尔马林保存的鱼类标本中获得高质量DNA是比较困难的。我们对前人的方法进行了如下改进：1）在标本的前处理过程中,通过长时间的缓冲液浸泡、短暂的加温、真空干燥来消除福尔马林对样品的影响;2）在样品消化过程中,加入相对过量的蛋白酶K和还原剂;3）提取DNA后立即进行PCR反应,并增加反应的循环次数和提高退火温度。通过这些改进,我们成功地从福尔马林保存的鱼类标本中提取出了高质量DNA;通过对比不同方法（福尔马林、酒精及冰冻）处理过的标本的DNA测序结果,表明该方法是值得信赖的;标本从死亡到用福尔马林处理之间的时间延搁可能是影响所提取的DNA质量的重要因素。     

Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis

Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology.     

The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations

Background

High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.

Methods

Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.

Results

No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.

Conclusions

Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.     

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2‐week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.     

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.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

Membrane biofouling characterization: effects of sample preparation procedures on biofilm structure and the microbial community

Ensuring the quality and reproducibility of results from biofilm structure and microbial community analysis is essential to membrane biofouling studies. This study evaluated the impacts of three sample preparation factors (ie number of buffer rinses, storage time at 4°C, and DNA extraction method) on the downstream analysis of nitrifying biofilms grown on ultrafiltration membranes. Both rinse and storage affected biofilm structure, as suggested by their strong correlation with total biovolume, biofilm thickness, roughness and the spatial distribution of EPS. Significant variations in DNA yields and microbial community diversity were also observed among samples treated by different rinses, storage and DNA extraction methods. For the tested biofilms, two rinses, no storage and DNA extraction with both mechanical and chemical cell lysis from attached biofilm were the optimal sample preparation procedures for obtaining accurate information about biofilm structure, EPS distribution and the microbial community.     

Long-Term Frozen Storage of Urine Samples: A Trouble to Get PCR Results in Schistosoma spp. DNA Detection?

Background

Human schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples’ storage or conditions for handling and DNA preservation and extraction methods.

Methodology/Principal Findings

We evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patientś urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template.

Conclusions/Significance

Long-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of extraction used.     

Extraction of cellular DNA from human cells and tissues fixed in ethanol

DNA can be extracted from ethanol-fixed lymphoid cells and tissues. The fixation procedure is simple and rapid, and the DNA extraction itself is the same as that normally used for fresh tissue or cells. DNA extracted from ethanol-fixed material is indistinguishable from DNA extracted from fresh samples based on its purity, its ability to be digested with restriction endonucleases, and its ability to specifically hybridize to DNA probes. The capability to extract DNA from ethanol-fixed cells and tissues eliminates the need for stringent handling and storage requirements of fresh or frozen specimens.     

An improved semiautomated rapid method of extracting genomic DNA for molecular marker analysis in cocoa,<Emphasis Type="Italic">Theobroma cacao</Emphasis> L

DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.     

Comparison of DNA extraction methods for PCR amplification of mitochondrial cytochrome c oxidase subunit II (COII) DNA from primate fecal samples

Mitochondrial COII DNA was amplified by PCR from total DNA extracted from field collected primate fecal samples (n=24) which had been stored without refrigeration for over 30 days. High molecular weight DNA total DNA was obtained from samples stored in 70% (v/v) ethanol, SDS lysis buffer (LB) and guanidine isothiocyanate buffer (GTB) than from samples stored in 10% formalin. Fecal DNA quality and COII amplification varied according to storage solution (formalin, ethanol, LB and GTB), extraction method (LB-based and GTB-based) and primate species (chimpanzee, baboon, human). It is recommended that fecal samples be collected in LB for DNA analysis. However, GTB-based protocols are suitable when total RNA is needed for epidemiological studies of viral diseases or gene expression analysis.