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.     

Protocols for dry DNA storage and shipment at room temperature

The globalization of DNA barcoding will require core analytical facilities to develop cost‐effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry‐state DNA stabilization systems: commercial Biomatrica^® DNAstable^® plates, home‐made trehalose and polyvinyl alcohol (PVA) plates on 96‐well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at ?20 °C. PCR and selective sequencing were performed over a 4‐year interval to test the condition of DNA extracts. Biomatrica^® provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica^® at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at ?20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long‐term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities.     

PCR反应混合液的冻干处理及其应用

本研究将除模板DNA以外的PCR反应混合液,经过真空浓缩冻成干粉状后,分别置于室温和4℃贮存,经过一定的贮藏时间后进行PCR反应,发现经冻干处理的样品能在较长的贮存时间内保持扩增活性,这将给实际工作带来很大的便利。 Abstract:In this paper we introduce a new storage method of PCR ingredient.PCR mixture except DNA template has been frozen to dry powder by the DNA-Plus system.This kind of powder was stored at room temperature or 4℃.PCR has been run in different period of storage.It was discovered that the samples of lyophilization could keep activity for a long time.     

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.     

Preparation of DNA from Silica Gel Dried Mini-amount of Leaves of Oryza rufipogon for RAPD Study and Total DNA Bank Construction

Gene resources of Oryza rufipogon Griff. play a crucial role in rice breeding, and hence to study their conservation is of utter importance. The authors describe a method for preparation of DNA from mini- amotmt of the silica-gel-dried leaves of Oryza rufipogon. The high molecular weight DNAs of 1 168 individuals representing 44 populations have been obtained with high yields, which could be used for RAPD PCR and construction of total DNA bank of this species. The template DNA from silica-gel-dried leaves stored for one year at room temperature gave the same RAPD results as that from the newly prepared silica-gel-dried leaves. The optional template DNA concentrations for amplification ranged from 3.1 ng to 50 ng. In addition, the quality and quantity of the template DNAs that affect RAPD results are also discussed.     

DNA synthesis in excised tobacco leaves after bromodeoxyuridine incorporation: immunohistochemical detection in semi-thin spurr sections

We describe a method for localizing replicating cells in detached tobacco leaves allowed to root. The proposed protocol has shown that formalin fixation and Spurr embedding of petiole bases can be used for demonstrating DNA synthesis after bromodeoxyuridine (BrdU) incorporation. The incorporated BrdU was immunologically visualized. After resin removal, different procedures of DNA denaturation and protease digestion were tested. Combined hydrolysis with 4 N HCl for 10 min at room temperature and digestion with 0.4% pepsin for 15 min at 37 degrees C led to the best reproducible results, with either the peroxidase or the gold detection system. The method is rapid and sensitive, with precise resolution. It can be used at the light and electron microscopic levels. Its potential application is to elucidate in the same organ the role of cytokinins, a class of plant growth regulators, in dividing cells and to define the chronology of their biosynthesis in roots in relation to DNA synthesis.     

从普通野生稻硅胶干燥的小量叶片中制备DNA用于RAPD分析和总DNA库的建立(英文)

普通野生稻（ Ｏｒｙｚａｒｕｆｉｐｏｇｏｎ Ｇｒｉｆｆ．）的基因资源对水稻的育种起着至关重要的作用。报道了从其硅胶干燥的小量叶片中制备ＤＮＡ的方法。用此方法制备的ＤＮＡ分子量大（４０～４５ ｋｂ） ,产率也较高（５０ ～２００ μｇ／ｇ） ,且成功地进行了ＲＡＰＤ扩增。用制备的４４ 个居群,１１６８ 个个体的总ＤＮＡ 建立了中国普通野生稻的总ＤＮＡ 库作长期冷冻保存,可用于基于ＰＣＲ 的ＤＮＡ水平上的各种目的的研究。根据实验结果,从在室温下贮存１ 周、３ 个月、６ 个月、１ 年的硅胶干燥的叶片中提取的ＤＮＡ 用于ＲＡＰＤ扩增所得的扩增产物没有差异;模板ＤＮＡ浓度在３ ．１ ～５０ ｎｇ 的范围内均得到很好的ＲＡＰＤ扩增结果。这说明了从硅胶干燥的叶片中提取的普通野生稻的ＤＮＡ 用于ＲＡＰＤ扩增的产物很稳定,将其用于群体遗传分析具有很好的可比性和可靠性。同时也讨论了模板ＤＮＡ的纯度和浓度对ＲＡＰＤ扩增的影响     

Detection of plant genes using a rapid, nonorganic DNA purification method

We have developed a simple procedure for the preparation of plant genomic DNA using FTA paper. Plant leaves were crushed against FTA paper, and the genomic DNA was purified using simple, nonorganic reagents. The 18S rRNA gene and the gene encoding the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from the chloroplast genome were detected by PCR amplification of DNA on FTA paper. DNA amplification was successful using extracts from 16 dicot and monocot plants. Studies of specific plant extracts revealed that extracts of leaf samples could be collected and stored at room temperature on FTA paper without a decrease in the DNA amplification success rate for more than a month. Both the 18S RNA gene and the rbcL gene were detected in the genomic DNA isolated from various soybean cultivars stored in this manner. Furthermore, by modestly increasing the number of cycles of DNA amplification, we were able to detect the uidA gene in transgenic tobacco and rice leaves as well as a single copy gene linked to the resistance gene of cyst nematode race 3 using genomic DNA isolated on FTA paper. These results demonstrate that genomic DNA isolated using FTA paper can be used for the detection of plant genes, from a wide range of plants with either high or low gene copy number and of either nuclear or cytoplasmic origin.     

A rapid and efficient assay for extracting DNA from fungi

AIMS: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. METHODS AND RESULTS: Tissue (< 3.0 mg) was scraped from freshly-grown fungal isolates. The tissue was suspended in buffer AP1 and subjected to seven rounds of freeze/thaw using a crushed dry ice/ethanol bath and a boiling water bath. After a 30 min boiling step, the tissue was quickly ground against the wall of the microfuge tube using a sterile pipette tip. The Qiagen DNeasy Plant Tissue Kit protocol was then used to purify the DNA for PCR/sequencing applications. CONCLUSIONS: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.     

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.     

Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces

Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34%+/-9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers.     

The use of activated charcoal for the removal of PCR inhibitors from oyster samples

Activated charcoal is a carbonaceous adsorbent with a high internal porosity, and hence a large internal surface area. Cells of a strain of Escherichia coli O157:H7 seeded into oyster tissue homogenates were completely bound to untreated charcoal after an incubation period of 15 min at room temperature. In contrast, activated charcoal particles coated with cells of Pseudomonas fluorescens resulted in 92.6%+/-3.7 recovery of E. coli O157:H7. This allowed the successful use of the coated activated charcoal for the absorption of PCR inhibitors from seeded tissue samples. With coated charcoal, real-time PCR was able to detect 1x10(3) CFU of E. coli 0157:H7/g of tissue which was equivalent to 50 genomic targets per real-time PCR. In contrast, without the use of treated charcoal, the real-time PCR failed to detect 10(7) CFU/g. This is a promising, and convenient technology that can be applied to increase the sensitivity of the PCR assay without selective enrichment, for the detection of low numbers of pathogenic microorganisms in complex matrices such as foods, clinical, and environmental samples, which frequently exhibit high levels of PCR inhibition.     

The influence of time and temperature on molecular gut content analysis： Adalia bipunctata fed with Rhopalosiphum padi

Gut content analysis is a useful tool when studying arthropod predator-prey interactions. We used polymerase chain reaction （PCR） technique to examine how detection of prey DNA in the gut content of predators was influenced by digestion time and temperature. Such knowledge is critical before applying PCR-based gut content analysis to field collected predators. Larvae of the two-spotted ladybeetle （Adalia bipunctata L.） were fed with the bird cherry-oat aphid （Rhopalosiphum padi L.） at either 21℃ or 14℃. After consuming one aphid, the predators were allowed to digest the prey for a range of time periods up to 24 hours. The influence of temperature on A. bipunctata feeding behavior was also recorded. From the fed larvae, total DNA was extracted and PCR reactions with R. padi specific primers were run. The number ofA. bipunctata that tested positive for R. padi DNA was negatively related to the length of digestion time. Temperature influenced larval feeding behavior but did not have a significant effect on R. padi DNA detection. After pooling the data from both temperature treatments we estimated the time point when R. padi DNA could be amplified from 50% of the fed A. bipunctata by PCR to be 4.87 hours. With such a rapid decrease in prey DNA detection success, positive PCR reactions will most likely be the result of predation events occurring shortly before capture. If a defined digestion temperature range has proven not to influence prey detection, PCR data obtained from predators collected within that particular range can be interpreted in the same way.     

A simple method for the preparation of paraffin-embedded cell blocks from fine needle aspirates, effusions and brushings

A simple method for the preparation of paraffin-embedded cell blocks from cytologic specimens obtained by fine needle aspiration, by brushing or from effusions is described. The cells are fixed in suspension in 50% ethanol for one hour and pelleted by centrifugation in a 50-mL plastic tube. The fixative is removed, and the pellet is suspended in 3 mL of acetone for dehydration for ten minutes and thereafter repelleted. The acetone is then removed, and the cell pellet is dried at 60 degrees C for one hour. Melted paraffin is added onto the dry warmed cell mass and allowed to solidify at room temperature. A conical paraffin block with the cells in the top is obtained and can be handled as a routine tissue block.     

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.     

一种快速提取葱属植物 DNA 的方法

目的：针对分子育种工作的繁琐性及葱属植物次生代谢物较多的特点,研究一种利用普通试剂及简单仪器高效快速提取葱属植物DNA的方法。方法：对碱处理法稍加改进,在碱性环境中高温裂解细胞,将释放的DNA保存在Tris缓冲液中,用PCR检测提取质量并分析DNA的保存时间。结果：与用试剂盒提取的DNA相比,扩增产物无显著差异,利用此方法提取DNA并分析了13个洋葱品种的细胞质雄性不育类型;保存时间分析显示,DNA在常温下只能保存5d,在4℃或-20℃可至少保存2个月。结论：该方法方便快速、成本低、适于田间操作,得到的DNA可满足分子生物学实验的基本要求,可用于以PCR为基础的分子标记辅助育种。     

Re-probing DNA blots: Wet is better than dry storage of uncharged nylon membranes after removing probes

DNA immobilized on a nylon membrane can be re-probed multiple times with different probes. Protocols typically recommend that DNA blots be stored either dry at room temperature or wet at 4 or −20°C after a probe is removed. This study shows substantial differences in the effect of these storage options on the performance of uncharged nylon membranes in subsequent hybridizations. Uncharged membranes, air-dried and stored at room temperature after probe removal, could not be successfully re-probed. However, excellent rehybridization results were obtained following probe removal when wet membranes were wrapped in plastic and stored at −20°C.     

Acetone preservation: a practical technique for molecular analysis

In attempts to establish a convenient and reliable method for field collection and archival preservation of insects and their endosymbiotic microorganisms for molecular analysis, acetone, ethanol, and other organic solvents were tested for DNA preservability of the pea aphid Acyrthosiphon pisum and its intracellular symbiotic bacterium Buchnera sp. After 6 months' storage, not only the band of high-molecular-size DNA but also the bands of rRNA were well preserved in acetone, ethanol, 2-propanol, diethyl ether and ethyl acetate. Polymerase chain reaction (PCR) assays confirmed that the DNA of both the insects and their symbionts was well preserved in these solvents. In contrast, methanol and chloroform showed poor DNA preservability. When water-containing series of acetone and ethanol were examined for DNA preservability, acetone was apparently more robust against water contamination than ethanol. Considering that most biological materials contain high amounts of water, acetone may be a more recommendable preservative for DNA analysis than ethanol which has been widely used for this purpose. The DNA of various insects could be preserved in acetone at room temperature in good condition for several years. In addition to the DNA of the host insects, the DNA of their endosymbionts, including Buchnera and other mycetocyte symbionts, Wolbachia, and gut bacteria, was amplified by PCR after several years of acetone storage. The RNA and protein of the pea aphid and its endosymbiont were also preserved for several years in acetone. After 2 years' storage in acetone, proteins of A. pisum could be analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and the endosymbiotic bacteria were successfully detected by immunohistochemistry and in situ hybridization on the tissue sections.     

改良CTAB法提取番石榴叶片总DNA

目的:从番石榴叶片中快速提取高质量的总DNA。方法:改良CTAB法。主要改进之处在于不用液氮,而是直接研磨硅胶干燥样品;用高浓度CTAB、低浓度乙醇与NaCl盐析相结合等方法去除多糖。结果:应用改良后的方法可以快速提取番石榴叶片总DNA,有效去除组织中的多糖、蛋白质,抑制提取过程中的组织褐变。提取的DNA可用于限制性内切酶酶切和PCR扩增。结论:传统CTAB法经过改良,可用于快速提取番石榴高质量DNA。     

A simple and reliable method for DNA extraction from bivalve mantle

A simple and reliable method was developed for extracting genomic DNA from preserved mantle tissues of Pacific oyster Crassostrea gigas for reproducible PCR amplification. The method was based on destruction of the tissue using Proteinase K, Chelex 100 resin, detergents, and urea, followed by preferential capturing of genomic DNA with silica particles. Approximately 5 mg of mantle tissue provided a sufficient quality and quantity of DNA for several hundreds of PCR reactions amplifying the hypervariable mitochondrial DNA intergenic spacer, which is a useful genetic marker for population structure analysis of Pacific oyster. The method can be applied for DNA preparation from not only fresh and frozen but also ethanol-preserved mantle tissues, so this rapid and economical method can serve for investigating a large number of bivalve specimens collected in the field and next transported in ethanol at ambient temperature.