Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.     

Adenoviruses in lymphocytes of the human gastro-intestinal tract

Objective

Persistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects.

Methods

The presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA.

Results

Intestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested.

Conclusion

Adenoviral DNA is highly prevalent in lymphocytes from the gastro-intestinal tract indicating that adenoviruses may be part of the normal gut flora.     

Extraction, amplification and characterization of wood DNA from dipterocarpaceae

A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations of wood tissue. Genotypes, the origin of sampled material, and species can be identified based on an investigation of wood if suitable information on genetic variation patterns within and among species is available. Potential applications are in forensics and in the control of the timber and wood trade. We extracted DNA from wood of Dipterocarpaceae, a family that dominates rainforests and comprises many important timber species in Southeast Asia. Several different DNA isolation techniques were compared and optimized for wood samples from natural populations and from wood processing enterprises. The quality of the DNA was tested by spectrophotometry, PCR amplification, and PCR inhibitor tests. An average DNA yield of 2.2 μg was obtained per 50–100 mg of dried wood sample. Chloroplast DNA (cpDNA) regions of different length were amenable to PCR amplification from the extracted DNA. Modification of DNA isolation techniques by the addition of polyvinylpyrrolidone (PVP) addition up to 3.1% into lysis buffer reduced PCR inhibition effectively. In order to evaluate the extraction method, we analyzed leaves and wood from the same tree by PCR amplification, genotyping and sequencing of chloroplast microsatellites.     

Chelex without boiling, a rapid and easy technique to obtain stable amplifiable DNA from small amounts of ethanol-stored spiders

DNA barcoding projects require high-throughput generation of sequence data to assemble the comprehensive reference databases that are required to perform large-scale biodiversity inventories and molecular ecology studies. With the advent of new sequencing technologies, the extraction step, which often requires a considerable amount of time and money, represents a significant bottleneck in many studies. Here, we present a one-step Chelex double-stranded DNA extraction protocol that is quick, cheap, easy and works with a small quantity of ethanol-stored tissue. We developed this protocol by removing the denaturation step appearing in classic methods. This modification reduces the number of handling steps to one, thus simplifying the extraction procedure and reducing the risk of sample contamination, and yields double-stranded DNA instead of the single-stranded form that classical Chelex extraction protocols usually release. DNA obtained through our method is then suitable for long-term conservation (over 1.5 years). We tested our protocol on a highly diverse genus of spiders comprised of mainly very small species. We also apply the method to two other genera of spiders, one with average size species, the other one with giant species, to test the efficacy of the method with varying amounts of input tissue. We also discuss the advantages and limitations of this DNA extraction technique when working with arthropods.     

High-resolution melting analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine

The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D. pinea in Austrian pine (Pinus nigra). HRMA exploits differences in the melting behaviour of PCR products to rapidly identify DNA sequence variants without the need for cumbersome post-PCR methods. We developed a HRMA method to detect specific fungal sequences in the mitochondrial small subunit ribosome gene (mt SSU rDNA). The reliability of this technique was firstly assessed on DNA extracted from pure cultures of D. pinea and closely related species. Amplicon differences were screened by HRMA and the results confirmed by direct DNA sequencing. Subsequently, HRMA was tested on DNA from symptomatic and symptomless pine shoots, and the presence of the fungus was also confirmed by both conventional and molecular quantitative approaches. The HRMA allowed the distinction of D.?pinea from closely related species, showing specific melting profiles for the each pathogen. This new molecular technique, here tested in a plant-fungus pathosystem for the first time, was very reliable in both symptomatic and symptomless shoots. HRMA is therefore a highly effective and accurate technique that permits the rapid screening of pathogens in the host.     

Skin swabbing as a new efficient DNA sampling technique in amphibians, and 14 new microsatellite markers in the alpine newt (Ichthyosaura alpestris)

This study introduces a novel DNA sampling method in amphibians using skin swabs. We assessed the relevancy of skin swabs relevancy for genetic studies by amplifying a set of 17 microsatellite markers in the alpine newt Ichthyosaura alpestris, including 14 new polymorphic loci, and a set of 11 microsatellite markers in Hyla arborea, from DNA collected with buccal swabs (the standard swab method), dorsal skin swabs and ventral skin swabs. We tested for quality and quantity of collected DNA with each method by comparing electrophoresis migration patterns. The consistency between genotypes obtained from skin swabs and buccal swabs was assessed. Dorsal swabs performed better than ventral swabs in both species, possibly due to differences in skin structure. Skin swabbing proved to be a useful alternative to buccal swabbing for small or vulnerable animals: by drastically limiting handling, this method may improve the trade-off between the scientific value of collected data, individual welfare and species conservation. In addition, the 14 new polymorphic microsatellites for the alpine newt will increase the power of genetic studies in this species. In four populations from France (n=19-25), the number of alleles per locus varied from 2 to 16 and expected heterozygosities ranged from 0.04 to 0.91. Presence of null alleles was detected in two markers and two pairs displayed gametic disequilibrium. No locus appeared to be sex-linked.     

DNA sampling from eggshell swabbing is widely applicable in wild bird populations as demonstrated in 23 species

There is increasing interest in noninvasive DNA sampling techniques. In birds, there are several methods proposed for sampling DNA, and of these, the use of eggshell swabbing is potentially applicable to a wide range of species. We estimated the effectiveness of this method in the wild by sampling the eggs of 23 bird species. Sampling of eggs was performed twice per nest, soon after the clutch was laid and again at the end of egg incubation. We genotyped DNA samples using a set of five conserved microsatellite markers, which included a Z-linked locus and a sex-typing marker. We successfully collected avian DNA from the eggs of all species tested and from 88.48% of the samples. In most of the cases, the DNA concentration was low (ca. 10 ng/μL). The number of microsatellite loci amplified per sample (0-5) was used as a measure of the genotyping success of the sample. On average, we genotyped 3.01 ± 0.12 loci per sample (mean ± SE), and time of sampling did not seem to have an effect; however, genotyping success differed among species and was greater in those species that used feather material for lining their nest cups. We also checked for the occurrence of possible genotyping errors derived from using samples with very low DNA quantities (i.e. allelic dropout or false alleles) and for DNA contamination from individuals other than the mother, which appeared at a moderate rate (in 44% of the PCR replicates and in 17.36% of samples, respectively). Additionally, we investigated whether the DNA on eggshells corresponded to maternal DNA by comparing the genotypes obtained from the eggshells to those obtained from blood samples of all the nestlings for six nests of magpies. In five of the six magpie nests, we found evidence that the swab genotypes were a mixture of genotypes from both parents and this finding was independent of the time of incubation. Thus, our results broadly confirm that the swabbing of eggshells can be used as a noninvasive method for obtaining DNA and is applicable across a wide range of bird species. Nonetheless, genotyping errors should be properly estimated for each species by using a suite of highly polymorphic loci. These errors may be resolved by sampling only recently laid eggs (to avoid non-maternal DNA contamination) or by performing several PCR replicates per sample (to avoid allelic dropout and false alleles) and/or by increasing the amount of DNA used in the PCR through increasing the volume of the PCR or increasing the concentration of template DNA.     

A DNA extraction method for RAPD analysis from plants rich in soluble polysaccharides

A simple procedure for the isolation of DNA from mature leaf tissue was developed. This procedure purified DNA using Sephacryl S-1000 column and PEG 8000 precipitation. Polysaccharide-like components were successfully removed from DNA samples from species in which polysaccharides were found to be difficult to remove by phenol/chloroform extraction. The DNA obtained by this method was suitable for PCR, RAPD, enzyme digestion, and Southern-blot analyses.     

Development of real-time PCR assays for discrimination and quantification of two Perkinsus spp. in the Manila clam Ruditapes philippinarum

The Manila clam Ruditapes philippinarum is infected with 2 Perkinsus species, Perkinsus olseni and P. honshuensis, in Japan. The latter was described as a new species in Mie Prefecture, Japan, in 2006. Ray's Fluid Thioglycollate Medium (RFTM) assay has been most commonly used to quantify Perkinsus infection. However, this assay cannot discriminate between species that resemble one another morphologically. We developed real-time PCR assays for the specific quantification of P. olseni and P. honshuensis. DNA was extracted using Chelex resin. Cultured P. olseni and P. honshuensis cells were counted and spiked into uninfected clam gill tissue prior to DNA extraction to generate standard curves, which allowed quantification based on the PCR cycle threshold values. We compared the RFTM assay with both real-time PCR assays by quantifying Perkinsus spp. in gill tissue samples from the same individual clams obtained from various localities in Japan. Infection intensities estimated by both assays were significantly correlated (r2 = 0.70). Our results suggest that the prevalence and infection intensity of P. honshuensis are much lower than for P. olseni in Manila clams.     

DNA isolation protocol for red seaweed (rhodophyta)

We report a DNA isolation protocol for red seaweed. The method is a modification of the Dellaporta et al. (1983) protocol for land plants. Our simplified version can be used to process large sample numbers and to minimise polysaccharide co-isolation. The protocol was applied to 12 red seaweed species as well as one green alga and one land plant. The protocol yields about 5 μg of high molecular weight DNA from 10 mg of dried material, with no RNA. No sign of degradation was observed after agarose gel electrophoresis for both freshly extracted DNA and DNA stored for 18 months at 4°C. DNA isolated by our protocol was suitable for genomic library construction (tested for one species), endonuclease restriction, and PCR amplification for all species.     

Faecal DNA amplification in Pacific walruses (Odobenus rosmarus divergens)

Dietary information is critical for assessing the population status of seals, sea lions and walruses—and is determined for most species of pinnipeds using non-invasive methods. However, diets of walruses continue to be described from the stomach contents of dead individuals. Our goal was to assess whether DNA could be extracted from the faeces of Pacific walruses (Odobenus rosmarus divergens) collected at haulout sites, and whether potential prey species or taxa could be amplified from that DNA. We extracted DNA from 70 faecal samples collected from ice pans in the Bering Sea during the spring of 2008 and 2009 (with between 4.6 and 308.9 ng/μl of DNA in every sample). We also extracted DNA from 12 potential prey species or taxa collected by bottom-grabs in 2009 to identify positive controls for primers and to test the ability of previously published taxon-specific and species-specific primers to correctly identify the prey using conventional PCR. We tested primers that successfully amplified DNA from the tissue of at least one potential prey species or taxon on all 70 walrus faecal samples. We found that two sets of primers successfully amplified many of the potential prey species or taxa using DNA from their tissue, and that one of these primer sets produced positive amplification in 4 of the 70 faecal samples. The band size that was produced for prey organisms and in the faecal samples was consistent with expectations, although prey identities were not verified with sequencing. Our pilot study demonstrates that DNA can be successfully extracted and amplified from walrus faeces, providing a stepping stone towards describing the diets of walruses from faecal DNA.     

Proboscidean DNA from Museum and Fossil Specimens: An Assessment of Ancient DNA Extraction and Amplification Techniques

Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.     

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.     

Extraction of DNA from milligram amounts of fresh,herbarium and mummified plant tissues

Summary We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3–200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22–118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.     

Identification of pathogenic yeast species by polymerase chain reaction amplification of the RPS0 gene intron fragment

Aims: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (10⁷–10⁵ cells). Conclusion: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.     

A rapid and efficient method for the extraction of total DNA from mature leaves of the data palm (Phoenix dactylifera L.)

A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may be applicable to other species of palms.     

A sensitive and specific multiplex PCR approach for sex identification of ursine and tremarctine bears suitable for non‐invasive samples

We report a new approach for molecular sex identification of extant Ursinae and Tremarctinae bears. Two Y‐specific fragments (SMCY and 318.2) and one X‐specific fragment (ZFX) are amplified in a multiplex PCR, yielding a double test for male‐specific amplification and an internal positive control. The primers were designed and tested to be bear‐specific, thereby minimizing the risk of cross‐amplification in other species including humans. The high sensitivity and small amplicon sizes (100, 124, 160 base pairs) facilitate analysis of non‐invasively obtained DNA material. DNA from tissue and blood as well as from 30 non‐invasively collected hair and faeces yielded clear and easily interpretable results. The fragments were detected both by standard gel electrophoresis and automated capillary electrophoresis.     

High polymorphism and resolution in targeted fingerprinting with combined β-tubulin introns

Tubulin-Based-Polymorphism (TBP) was originally introduced as a novel method for assaying genetic diversity in plants. TBP is based on polymorphism resulting from the PCR-mediated amplification of the first intron in the coding region of the β-tubulin gene family. Although, the method was successful in genetic assessment of some plant species and varieties, it suffered from low number of molecular markers due to limited variation in the first intron of β-tubulin gene family. We have now rectified this limitation by introducing the second intron of the β-tubulin genes as a valuable source of molecular markers. We show that the combined use of the two introns substantially increases the number of molecular markers and results in a reliable assessment of species/varieties relationships. After a preliminary validation on Brassica, this new combinatorial method was tested on species of Eleusine and Arachis. For both, reliable assessment of species relationships were obtained that were consistent with recently published studies resulting from more elaborated methods including DNA sequencing. Combinatorial TBP is a reliable, reproducible, simple, fast, and easy to score method that is very useful for breeding programs and species and variety assessments.     

一种改良的植物基因组DNA通用提取方法

高质量的基因组DNA是分子生物学研究的基础,而从富含糖类和次生代谢物且异质性强的植物材料中分离DNA相对困难。本方法在CTAB法和商业DNA提取试剂盒的基础上,在裂解细胞之前,对植物材料进行预处理．去除干扰DNA提取的代谢物,并在后续步骤中进行了一些优化。该方法适于多种不同的植物种类,所提取的基因组DNA质量较好,能满足下一步基因操作的要求,是一种通用的植物基因组DNA提取方法。