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.     

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.     

Noncryogenic Preservation of Mammalian Tissues for DNA Extraction: An Assessment of Storage Methods

Reliable field methods for the storage of tissues to be used for DNA extraction and amplification are critical to many studies employing molecular techniques. Protection from DNA degradation was compared among three commonly used methods of noncryogenic storage of tissues over a time scale of 2 years. All three methods prevented DNA degradation during storage for at least 6 months. DMSO (dimethyl sulfoxide)-salt solution provided the best protection from DNA degradation of tissues stored for up to 2 years. High molecular weight DNA was recovered from lysis buffer in which tissue was stored for 2 years, however, moderate amounts of degraded DNA was also present. High molecular weight DNA was recovered from tissues stored in ethanol for 2 years, however, the yield was relatively small compared to the other two noncryogenic storage techniques. Much of the DNA degradation in ethanol preserved tissues appeared to occur during the extraction procedure and can be reduced by soaking the tissue in lysis buffer for a few hours prior to beginning the extraction. The yield of PCR products was greatest from DNA extracted from DMSO-salt solution preserved tissues, whereas DNA from tissues stored in either lysis buffer or ethanol produced lower yields.     

High-quality plant DNA extraction for PCR: an easy approach

Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies. For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires maceration of plant tissue of about 1.0 cm² (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes, followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20–30 μg cm⁻² for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5–1.8. The DNA is quite suitable for PCR using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive.     

Relieving efforts in palm‐tree tissue sampling for population genetics analyses

1. The young leaves are the main source of nucleic acids for population genetic studies in palm‐trees; however, the access to this tissue may be limited by specific features of each species. Using root tissues as an alternative source of nucleic acids could facilitate the sampling in large populations.
2. This study tests root tissue viability as an alternative nucleic acid source (root versus. leaf) and explores different protocols (tissue storage and DNA extraction methods) to obtain high‐quality DNA samples.
3. The results showed no significant differences in DNA concentration (603.7 vs. 599.1 ng/μl) and quality ratios (A260/280:2.1 vs. 1.9, and A260/230:2.1 vs. 2.0) for the comparisons of tissue source (leaf vs. root) and DNA extraction method (manual vs. kit). For tissue storage method, DNA concentration was significantly higher for root tissues stored in 70% and 90% alcohol solutions (692.8 and 822.6 ng/μl, respectively) versus those obtained from leaf tissue (603.7 ng/μl); however, for the quality parameters, no differences were found.
4. Results showed the effective potential of using root tissue as an alternative source for nucleic acids, which could facilitate population sampling of palm‐tree species for future studies, and this methodological alternative could be applied to other plant systems with similar sampling challenges.

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陈旧标本DNA提取方法

对于自然环境中的或长期保存的动物标本,由于保存环境不良或保存时间过长,DNA提取的难度较大。受标本保存时间和损害程度等因素影响,导致实验结果的不稳定性加强,对于同一标本需要反复实验。为了提高DNA提取效率,节省实验成本,现对陈旧损坏标本的DNA提取方法进行综述。     

Non-toxic and efficient DNA extractions for soybean leaf and seed chips for high-throughput and large-scale genotyping

In applied soybean (Glycine max L.) breeding programs, marker-assisted selection has become a necessity to select value-added quantitative trait loci. The goal of this work was to improve marker-assisted selection workflow by developing a reliable, inexpensive, high-throughput DNA extraction protocol for soybean seed and leaf samples that does not generate hazardous waste. The DNA extraction protocol developed allows for the leverage of robust SNP genotyping platforms such as the Simple Probe Assay and KASPar v4.0 SNP Genotyping System to genotype thousands of seeds or leaves non-destructively in a single day with a 95 % success rate. This methodology makes it possible to run up to 150 SNP markers on the DNA extracted from a single seed chip or leaf sample.     

A high-throughput DNA extraction protocol for tropical molecular breeding programs

Liquid handling robotics and capillary electrophoresis genetic analyzers now offer high-throughput solutions for 2 of the 4 key steps in PCR-based DNA marker-assisted fingerprinting (DNA extraction, PCR amplification, electrophoresis, data analysis). Thus, DNA extraction remains the most significant bottleneck at the bench for large-scale applications in plant breeding and germplasm characterization. We report on a rapid and low-cost method for relatively high-throughput extraction of high-quality DNA from young and mature leaves of sorghum, pearl millet, chickpea, groundnut, and pigeonpea. The procedure uses a modified CTAB/β-mercaptoethanol method for DNA extraction in a 96-well plate. The quantity and quality of the DNA extracted per sample is adequate for more than 1000 PCR reactions. A relatively high throughput of 96–384 samples per person per day can be achieved, depending on the crop. A major timesaving aspect of the protocol is the absence of a manual sample-grinding step. Finally, the cost is a magnitude lower than commercial plate-based kits, and, as such, is likely to have substantial application in tropical molecular breeding programs.     

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.     

A method for the medium-term storage of plant tissue samples at room temperature and successive cycles of DNA extraction

Based on the protocol originally described by Stein et al. (2001), we have developed a method that allows for medium-term conservation at room temperature of wheat (Triticum aestivum) tissue samples to use for DNA extraction. DNA quality was suitable for analysis by PCR and Southern hybridization, even after 2 months of storage at room temperature. This method allows successive DNA re-extractions from a previously extracted sample and maximization of the DNA yield that can be recovered from precious samples. This method has applications for conservation of leaf samples and management of DNA extraction. Our method can help improve data recovery in many plant molecular genetics research projects.     

Efficacy of long-term coral tissue storage in ethanol for genotyping studies

With climate change threatening the future of coral reefs, there is an urgent need for effective coral tissue preservation and repositories from which DNA can be extracted. Most collections use 95 % ethanol as the storage medium, but its efficacy for long-term storage for short-fragment DNA use remains poorly documented. We conducted an accelerated DNA aging trial on three species of coral to ascertain whether ethanol-stored tissue and skeleton samples could yield fit-for-purpose DNA at time scales of 100+ yrs. We conclude that even using a crude DNA extraction technique, samples kept at 40 °C for 20 months yielded DNA of sufficient quality for Symbiodinium and coral host genotyping. If stored at ?20 °C, these samples are likely to still yield useable DNA after 100 yrs. Ethanol-stored samples compared favorably in terms of DNA quality, quantity and sample integrity with those stored in an analogue of the commercial storage buffer RNAlater ^®.     

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.     

A high-throughput protocol for extracting high-purity genomic DNA from plants and animals

DNA extraction techniques that employ the reversible binding of DNA to silica via chaotropic salts can deliver high-quality genomic DNA from plant and animal tissues, while avoiding the use of toxic organic solvents. Existing techniques that use this method are either prohibitively expensive, or are applicable to only a restricted set of taxa. Here we describe a cost-effective DNA extraction technique suitable for a wide range of plant and animal taxa that yields microgram quantities of high-molecular-weight genomic DNA at a throughput of 192 samples per day. Our technique is particularly robust for tissue samples that are insoluble or are rapidly discoloured or oxidized in standard DNA extraction buffers. We demonstrate the quality of DNA extracted using this method by applying the amplified fragment length polymorphism technique to plant species.     

Paper-based archiving of mammalian and plant samples for RNA analysis

The ability to archive biological samples for subsequent nucleic acid analysis is essential for tissue specimens and forensic samples. FTA Card is a chemically treated filter paper designed for the collection and room temperature storage of biological samples for subsequent DNA analysis. Its usefulness for the preservation of biological samples for subsequent RNA analysis was tested. Here, we demonstrate that RNA in biological samples stored on FTA Cards is stable and can be used successfully for RT-PCR and northern blot analysis. RNA stability depends on the storage temperature and the type of biological specimen. RNA in mammalian cells stored on FTA Cards is stable for over one year at temperatures at or below -20 degrees C and for two to three months in samples stored at room temperature. For plant leaf, longer storage times (> 5 days) require temperatures at or below -70 degrees C following sample application. FTA Cards may constitute a method not only for convenient collection and storage of biological samples but also for rapid RT-PCR analysis of tissue and cell samples.     

Buccal cell DNA extraction: yield, purity, and cost: a comparison of two methods

Simple and cost-effective methods are needed to extract DNA in order to use it in large-scale studies. Blood is an excellent DNA source; however, it is costly and invasive thus an alternative is needed. Several kits and chemical protocols using buccal cells have been proposed for DNA extraction. The objective of the study is to evaluate buccal NaOH chemical protocol and Nucleospin Tissue Kit (BD Biosciences, Macery-Nagel, Germany) for DNA extraction. DNA swab samples were collected from 300 voluntary participants. DNA yields and purity were measured by NaOH and Nucleospin Tissue Kit techniques; the cost and time consumption for DNA extraction per sample were assessed as well. Results have shown that DNA amount and purity extracted by NaOH procedure was compared to that of the kit (p = 0.164; p = 0.249, respectively). NaOH method was considered cheaper and less time consuming (0.06 versus 3.80 USD, and 1.33 versus 3.59 minutes per sample, p < 0.001). Buccal cell derived DNA extracted by NaOH protocol can be considered a feasible substitute for more expensive and time-consuming kits.     

Maxiprep genomic DNA extractions for molecular epidemiology studies and biorepositories

Molecular epidemiology and genomic characterisation studies require the screening of large numbers of individuals to achieve statistical significance. Although many of the novel DNA extraction methods offer convenient, high-throughput capabilities, their use for the processing of larger sample volumes becomes very expensive. We are currently compiling the Mexican Genomic DNA Collection in order to address specific health priorities through molecular techniques. Our approach employs a low-cost laundry detergent based DNA extraction technique that maximizes DNA yield and quality. We have optimised four different modalities (maxiprep, midiprep, miniprep and microprep) for two different sources (leukocyte concentrates and whole blood). Our optimised protocol produces 4.5 mg of DNA from 15 ml of blood-bank discarded leukocyte concentrates with spectrophotometric quality, genomic integrity and PCR suitability that rivals that of phenol–chloroform extracted samples. We present evidence of many PCR applications that we have carried out on samples extracted with this technique including Killer-cell Immunoglobulin-like Receptor genotyping, Short Tandem Repeat profiling as well as nucleic acid screening for hepatitis B and human immunodeficiency type-1 viruses. This paper highlights many of the advantages that this DNA extraction technique provides over existing methodologies, whether it is used to establish large genomic DNA collections (as was our main intention) or as a routine DNA extraction method for PCR applications.     

Estimation of sperm numbers in insects by fluorometry

Summary To facilitate the study of mating biology in the desert leaf-cutter antAcromyrmex versicolor, methods were developed that allowed storage and easy quantification of sperm samples collected from both male and female reproductive tracts. Sperm samples stored frozen were sonicated, stained with a fluorescent DNA stain, and the fluorescence emitted by the stained sperm heads was measured. The intensity of fluorescence was shown to be a linear function of the number of sperm in the sample as determined by counting.     

RNA preservation of Antarctic marine invertebrates

Fifteen species of marine invertebrate commonly occurring in the near-shore environment of Rothera base, Antarctica, were used to test tissue sample storage protocols with regard to preservation of RNA integrity. After animal collection, the tissues were either immediately extracted for RNA or stored at −80°C after having been, either directly flash frozen in liquid nitrogen or preserved in a commercial RNA storage solution, for extraction in the UK. In four cases, direct flash freezing produced enhanced RNA integrity compared with samples in the commercial storage solution. A subset of samples were further tested for the preferred temperature of storage in the commercial reagent. RNA integrity was well preserved at both +4 and −20°C over periods of 2 months, but degradation was rapid in tissues stored at room temperature. Eight out of the fifteen species only produced a single ribosomal band on gel electrophoresis. This survey provides a guide for tissue transport of Polar cold water marine invertebrates.     

A simple and economic preservation method for genomic bacterial DNA from clinically significant pathogens

Bacterial culture was allowed to dry to completeness on Columbia agar base with defibrinated horse blood. Following 6 months storage at room temperature, microbial DNA was extracted and successfully amplified by PCR. This storage technique has the advantage over other methods of not requiring (i) a DNA extraction protocol prior to storage and (ii) refrigeration and/or freezing. This technique maybe useful in the transportation of bacterial genomic DNA in nonviable cells as well as reliable method for the storage of DNA in underdeveloped countries.     

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.