Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing.

We describe a simple and cost-effective method for the synthesis of an internal fluorescently labeled DNA standard for fragment sizing using an automated DNA sequencer. A set of primer pairs labeled with ROX was developed to amplify 12 DNA fragments, 58-417 bp, derived from a conserved region of plant chloroplast DNA. These amplified fragments were mixed together, constituting a fluorescent internal DNA size marker. The precision of the size standard was evaluated by estimating the size of 20 alleles that were amplified at four dinucleotide microsatellite loci with the synthesized size standard and the commercial internal sizing standard, GeneScan Rox500. A number of intra-gel and inter-gel comparisons were run, and an analysis of variance was carried out. No significant difference was observed between the size estimates obtained with the synthesized DNA standard and the commercial standard. This facile and general PCR-based method for the synthesis of internal standards allows for significant savings in the implementation of large genotyping experiments using microsatellite or AFLP markers.     

High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88–100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.     

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。     

A microsatellite based method for quantification of fungi in decomposing plant material elucidates the role of Fusarium graminearum DON production in the saprophytic competition with Trichoderma atroviride in maize tissue microcosms

Common PCR assays for quantification of fungi in living plants cannot be used to study saprophytic colonization of fungi because plant decomposition releases PCR-inhibiting substances and saprophytes degrade the plant DNA which could serve as internal standard. The microsatellite PCR assays presented here overcome these problems by spiking samples prior to DNA extraction with mycelium of a reference strain. PCR with fluorescent primers co-amplifies microsatellite fragments of different length from target and reference strains. These fragments were separated in a capillary sequencer with fluorescence detection. The target/reference ratio of fluorescence signal was used to calculate target biomass in the sample. Such PCR assays were developed for the mycotoxin deoxynivalenol (DON)-producing wheat and maize pathogen Fusarium graminearum and the biocontrol agent Trichoderma atroviride, using new microsatellite markers. In contrast to real-time PCR assays, the novel PCR assays showed reliable fungal biomass quantification in samples with differentially decomposed plant tissue. The PCR assays were used to quantify the two fungi after competitive colonization of autoclaved maize leaf tissue in microcosms. Using a DON-producing F. graminearum wild-type strain and its nontoxigenic mutant we found no evidence for a role of DON production in F. graminearum defense against T. atroviride. The presence of T. atroviride resulted in a 36% lower wild-type DON production per biomass.     

Quantitative PCR assessment of microsatellite and SNP genotyping with variable quality DNA extracts

In this study we developed eight quantitative PCR (qPCR) assays to evaluate the starting copy number of nuclear and mitochondrial DNA fragments ranging from 75 to 350 base-pairs in DNA extracts from Chinook salmon tissues with varying quality. Samples were genotyped with 13 microsatellite and 29 SNP assays and average genotyping success for good, intermediate, and poor quality samples was 96%, 24%, and 24% for microsatellite loci, and 98%, 97%, and 79% for SNPs, respectively. As measured by qPCR, good quality samples had a consistently high number of starting copies across all fragment sizes with little change between the smallest and largest size. In contrast, the intermediate and poor quality samples displayed decreases in starting copy number as fragment size increased, and was most pronounced with poor samples. Logistic regression of genotyping success by starting copy number indicated that in order to achieve at least 90% genotyping success, approximately 1,000 starting copies of nuclear DNA are necessary for microsatellite loci, and as few as 14 starting copies for SNP assays (but we recommend at least 50 copies to reduce genotyping error). While these guidelines apply specifically to Chinook salmon and the genetic markers included in this study, the principles are transferable to other species and markers due to the underlying process associated with template quantity and PCR amplification.     

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

ABSTRACT Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS = 50%, LS = 15%; P = 0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5–25%, 6.25–62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%–10%, 4.7–25 ng/swab, 10% amplified at 1,000 bp). Paired blood and fresh field-swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples.     

Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations

Alterations of the mitochondrial DNA (mtDNA) are implicated in various pathological conditions. In this study, we used denaturing high performance liquid chromatography (DHPLC) as a method to rapidly screen the entire mtDNA for mutations. Overlapping DNA fragments, amplified by one single cycling protocol from frozen pre-formulated PCR mixes, were subjected to DHPLC analysis. Single DHPLC injections of fragments yielded straightforward interpretation of results with a detection limit down to 1% mtDNA heteroplasmy. Furthermore, collection and re-amplification of low degree heteroduplex peak-fractions allowed sequence analysis of mtDNA mutations down to the detection limit of the DHPLC method. In order to demonstrate that the method has diagnostic value, we analyzed and confirmed known mtDNA mutations in patient samples.     

Evaluation of different storage methods to characterize the fecal bacterial communities of captive western lowland gorillas (Gorilla gorilla gorilla)

Freezing is considered to be the best method for long-term storage of bacterial DNA from feces; however this method cannot be usually applied for samples of wild primates collected in the challenging conditions of the tropical forest. In order to find an alternative conservation method of fecal samples from wild great apes, we compared freezing with other fixation methods. Fecal samples from 11 captive gorillas (Gorilla gorilla gorilla) from three Czech Zoos were stored using freezing, RNA Stabilization Reagent (RNAlater), and 96% ethanol. Subsequently, the samples were examined using culture-independent methods (PCR-DGGE, and Real-time PCR) to qualitatively and quantitatively assess fecal microbiota composition and to compare differences among the storage methods. Noticeably, freezing samples resulted in the highest recoveries of DNA. No significant differences in DNA recovery were found between freezing and using RNAlater; however, significantly lower DNA concentrations were recovered from samples stored in 96% ethanol. Using PCR-DGGE we found that either 96% ethanol, RNAlater or freezing were suitable for preserving bacterial DNA; however fingerprints obtained from RNAlater storage were more similar to those obtained from the frozen method; in comparison to the patterns resulting from storing samples in ethanol. Using qPCR, frozen samples yielded the highest values of bacterial counts, with the exception of Enterobacteriaceae, which showed the highest numbers using samples stored in ethanol. Sequences of amplicons obtained from PCR-DGGE belonged to the families Clostridiaceae, Lactobacillaceae, Staphylococcaceae, and Lachnospiraceae, phylum Firmicutes; however most amplicons showed sequence similarity to previously uncultured microorganisms. Bacteria belonging to the phylum Firmicutes were the most frequently identified species in the fecal bacterial communities of captive western gorillas. The study showed that RNAlater is an optimal storage method when freezing is not possible.     

从非变性聚丙烯酰胺凝胶回收纯化DNA样本

介绍一种从非变性聚丙烯酰胺凝胶中回收和纯化目标DNA片段的方法,经比较回收纯化前后PCR产物的电泳结果,表明该方法具有简单快捷和效率高的优点。     

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.     

An improved high yield method to obtain microsatellite genotypes from red deer antlers up to 200 years old

Analysis of DNA from older samples, such as museum specimen, is a promising approach to studying genetics of populations and ecological processes across several generations. Here, we present a method for extracting high quality nuclear DNA for microsatellite analysis from antlers of red deer (Cervus elaphus). The genotyping of individuals was based on nine microsatellite loci. Because the amount of DNA found in antlers was high, we could reduce the amount of sample and chemicals used and shorten the decalcification time in comparison to other methods. Using these methods, we obtained genotypes from antlers up to 200 years old.     

豚鼠基因组26个多态性微卫星标记的筛选

目的筛选豚鼠基因组的多态性微卫星标记,为豚鼠遗传质量控制及基因定位等工作奠定基础。方法采用磁珠富集法和豚鼠基因组数据库筛选法获取微卫星位点序列,通过分析和初步筛选,挑选部分候选位点,根据其序列设计引物,对5种不同来源的豚鼠基因组DNA标本进行PCR扩增,以期获得多态性分子标记。结果本实验采用磁珠富集法共获得微卫星序列304个,设计引物125对,最终获得多态性位点1个,暂未发现多态性的特异性位点17个;用数据库筛选法共获得微卫星序列292个,设计并合成相应引物178对,最终发现多态性位点25个,暂未发现多态性的特异性位点28个。结论本实验获得26个多态性微卫星标记,45个潜在的候选标记,为微卫星标记在豚鼠遗传质量监测及突变基因定位等工作的应用奠定了基础。     

The effect of sample age and storage method on DNA yield and microsatellite amplification from baboon (Papio ursinus) faecal samples

Sampling methods that allow DNA collection without the physical handling of animals are popular in conservation genetics, but DNA isolated from faecal samples may be degraded, potentially leading to erroneous microsatellite genotyping results. We collected baboon faecal samples from fresh to 1-week post-defecation in a controlled sampling environment and preserved these using three storage techniques. After DNA isolation and quantification, the samples were genotyped at eight microsatellite loci. Quantitatively, DNA yield was highest when using silica as storage medium. However, microsatellite amplification from samples stored in 95% ETOH were most successful, with 100% success for fresh samples and dropping only marginally to 87.5% of loci for samples collected after 1 week. This disparity between quantitative and qualitative results suggests that total DNA concentrations do not necessarily provide a reliable indication of the amount of target DNA present in a DNA isolate. Our results nevertheless confirm that microsatellite fragments can successfully be amplified from faecal samples up to 1 week post-defecation, with careful selection of storage protocols and loci.     

Amplified Fragment Length Microsatellites (AFLM) might be used to develop microsatellite markers in organisms with limited amounts of DNA applied to Arbuscular Mycorrhizal (AM) fungi

Developing microsatellite markers for organisms with limited amounts of DNA can be difficult because sequence information is needed. To overcome this problem in the arbuscular mycorrhizal (AM) fungi Glomus etunicatum and Gigaspora gigantea, global amplification of the genomes of each species was performed with linker-adaptor-PCR from single spores. Amplified fragments were enriched for microsatellite motifs with 5'-biotinylated oligonucleotides and recovered by magnetic streptavidin beads. The recovered fragments were reamplified and separated on denaturing polyacrylamide gels, and 16 selected bands were excised, cloned and sequenced. Seven microsatellite motifs were detected from six clones (efficiency rate of 43.8%). Primers were designed for all putative microsatellite loci and most were successfully amplified from three single-spore preparations and from pools of five, 10 and 20 spores after global amplification. This approach, termed amplified fragment-length micosatellites (AFLM), might aid investigations of organisms that cannot or are not readily cultured in vitro and where DNA is a limiting factor for genetic studies. However, the technique also can be used to isolate microsatellite loci in any organism.     

A test of the efficacy of whole‐genome amplification on DNA obtained from low‐yield samples

Conservation and population genetic studies are sometimes hampered by insufficient quantities of high quality DNA. One potential way to overcome this problem is through the use of whole genome amplification (WGA) kits. We performed rolling circle WGA on DNA obtained from matched hair and tissue samples of North American red squirrels (Tamiasciurus hudsonicus). Following polymerase chain reaction (PCR) at four microsatellite loci, we compared genotyping success for DNA from different source tissues, both pre‐ and post‐WGA. Genotypes obtained with tissue were robust, whether or not DNA had been subjected to WGA. DNA extracted from hair produced results that were largely concordant with matched tissue samples, although amplification success was reduced and some allelic dropout was observed. WGA of hair samples resulted in a low genotyping success rate and an unacceptably high rate of allelic dropout and genotyping error. The problem was not rectified by conducting PCR of WGA hair samples in triplicate. Therefore, we conclude that WGA is only an effective method of enhancing template DNA quantity when the initial sample is from high‐yield material.     

A new method for the determination of critical polyethylene glycol concentration for selective precipitation of DNA fragments

Separation strategies based on size-selective precipitation of DNA fragments with polyethylene glycol (PEG) have been used for achieving desired DNA interval in automated sample preparation for next-generation sequencing. By varying PEG concentration, DNA fragments of different sizes can be precipitated onto surfaces of carboxyl-coated paramagnetic particles selectively, and therefore, the desired DNA interval can be obtained. However, one of the crucial points in this approach is to determine the critical PEG concentration for DNA fragment of a certain size. The aim of this work was to develop a convenient and reliable method for accurately determining the critical PEG concentration. In our method, at a fixed concentration of sodium chloride (NaCl), recovered DNA samples obtained with different PEG concentrations were directly quantified, and their concentrations as a function of the PEG concentration were fitted by the logistic function. The critical PEG value was easily and accurately determined from the fitted logistic function. The repeatability and stability of the critical PEG value were assessed, showing an excellent reliability of the method. Based on this method, critical PEG values of different-size DNA fragments were determined at different NaCl concentrations. The effectiveness of the method was also demonstrated by selective precipitation of DNA fragments.     

Dynamics of sperm DNA fragmentation in domestic animals III. Ram

From a biological viewpoint spermatozoa are ejaculated by the male and received into the female while maintaining roughly constant temperature, which in most mammals is below the temperature of the soma. When ejaculated spermatozoa are used for artificial reproductive purposes a temperature excursion episode is produced, because the spermatozoa are often stored as frozen or chilled samples and the biological temperature is only recovered after insemination. In this study we have analyzed the effects of cooling (to 15 degrees C) and freezing ram spermatozoa on the subsequent sperm DNA fragmentation index (sDFI) during a varying period of storage at 37 degrees C. The aim was to emulate in vivo processes that cooled or frozen-thawed spermatozoa experience after insemination. The study was performed using commercial semen samples derived from rams regularly used for reproductive purposes. Semen samples were studied after a cooling or cryopreservation episode followed by biological temperature recovery and incubation up to 48h. The results indicated that when spermatozoa experience a severe (frozen) or mild (cooled) temperature excursion episode, major effects on sperm viability and DNA fragmentation are induced and cause the subsequent rapid decline of ram sperm quality. This effect could be detected just at the onset of the biological temperature recovery. Sperm DNA damage in cooled samples was observed after 5h of incubation at 37 degrees C, while this time was reduced to less than 60min in frozen-thaw samples. The dynamics of sDFI in different animals, analyzed under the same experimental conditions, was different from one sample to another, regardless of the method used for storage. Sperm viability was better preserved in cooled rather than in frozen samples. While for the frozen-thawed samples sperm viability was almost abolished after 5h of incubation, a stable proportion of viable spermatozoa (ranging from 20% to 60%) was observed in the cooled samples at the corresponding time points. Finally, with respect to the prevalence of sDFI in ram, the level commonly found was lower than 5% at the onset of the experiment. However, sDFI was higher than 5% in 25% of the samples and in 15% of rams this index exceeded 10%.     

Isolation of High Molecular Weight DNA from Marine Sponge Bacteria for BAC Library Construction

Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery. DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes, and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library demonstrated that the recovered HMW DNA is suitable for metagenomic library construction.     

An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonucleasePalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi includingGossypium hirsucum (cotton),Pseudomonas, Bacillus, Streptomyces, andColletotrichum. Up to 100 g DNA/g (wet weight) of soil and 400 g DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonucleasePalI, contained 12–20 DNA fragments, appearing as sample fingerprints. Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.     

Characterisation of insect and plant origins using DNA extracted from small volumes of bee honey

A DNA-based tool was validated that potentially enables the characterisation of both plant and insect of origin of small (approximately 1 ml) samples of bee honey. Using this method, mitochondrial, nuclear and chloroplast DNA (mtDNA, nuDNA, cpDNA) markers were successfully extracted, PCR amplified, and sequenced from a range of honeys, and the relative amount of plant nuDNA and cpDNA, and bee mtDNA in the samples was quantified using quantitative real-time PCR. Short, but taxonomically informative lengths of insect and plant organelle DNA could be routinely recovered from all honey samples tested, and longer organelle, and nuclear DNA sequences can be recovered from many. The data also enabled preliminary characterisation of the quality of these different DNA sources in honey. Although the absolute quantity of the different genetic markers varied considerably between sample, a general trend was observed of insect mtDNA dominating over plant organelle DNA, and with plant nuclear DNA at the lowest levels. Furthermore there was a clear correlation between the plant DNA content and the success of the PCR assays. To maximise successful characterisation of samples, future studies are recommended to focus on the use of organelle markers, and limit the size of PCR amplicons targeted, although with appropriate sample selection and assay optimisation, other approaches may be possible.