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DNA Suitable (dna + suitable)
Selected AbstractsDetection of Phytophthora nicotianae in Soil with Real-time Quantitative PCRJOURNAL OF PHYTOPATHOLOGY, Issue 1 2010Junli Huang Abstract Phytophthora nicotianae is one of the most important soil-borne plant pathogens. A rapid, specific and sensitive real-time polymerase chain reaction (PCR) detection method for P. nicotianae was established, which used primers targeting the internal transcribed spacer (ITS) regions of rDNA genes of Phytophthora spp. Based on the nucleotide sequences of ITS2 of 15 different species of Phytophthora, the primers and probe were designed specifically to amplify DNA from P. nicotianae. With a series of 10-fold DNA dilutions extracted from P. nicotianae pure cultures, the detection limit was 10 pg/,l in conventional PCR, whereas in SYBR Green I PCR the detection limit was 0.12 fg/,l and in TaqMan PCR 1.2 fg/,l, and real-time PCR was 104,105 times more sensitive than conventional PCR. The simple and rapid procedures maximized the yield and quality of recovered DNA from soil and allowed the processing of many samples in a short time. The direct DNA extractions from soil were utilized to yield DNA suitable for PCR. By combining this protocol with the real-time PCR procedure it has been possible to specifically detect P. nicotianae in soil, and the degree of sensitivity was 1.0 pg/,l. The system was applied to survey soil samples from tobacco field sites in China for the presence of P. nicotianae and the analyses of naturally infested soil showed the reliability of the real-time PCR method. [source] The use of biomagnetic separation to recover DNA suitable for PCR from Claviceps species ,LETTERS IN APPLIED MICROBIOLOGY, Issue 2 2000D.L. Scott Jr DNA analysis of agriculturally important fungi using polymerase chain reaction (PCR)-based methods is becoming routine in research and for diagnostic purposes. Rapid, small-scale DNA isolation methods that take advantage of the sensitivity, speed and automation potential of PCR technology are needed for timely analysis of important plant pathogens. DNA isolated from Claviceps africana (causal agent of ergot of sorghum) using several standard DNA extraction protocols was found to be unamplifiable using PCR. The standard methods apparently failed to separate DNA from substances inhibitory to the Taq polymerase enzyme. We obtained DNA amenable to PCR analysis using a novel method involving magnetic beads and high salt extraction buffer. The biomagnetic purification method allowed us to obtain reliable PCR amplification of the internal transcribed spacer (ITS) regions of rDNA of Claviceps africana, making genetic comparisons possible. [source] Optimization of DNA Extraction from a Scleractinian Coral for the Detection of Thymine Dimers by Immunoassay,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007Anastazia T. Banaszak ABSTRACT Ultraviolet (UV)-B is known to cause DNA damage, principally by the formation of thymine dimers, but little research has been conducted in coral reef environments where UV doses are high. The majority of tropical reef-dwelling corals form a mutualistic symbiosis with the dinoflagellate Symbiodinium but few studies have been conducted on in situ DNA damage in corals and none have investigated the symbiotic components separately. The aim of this research was to quantify DNA damage in both the coral host and the dinoflagellate symbiont. The first step in this investigation was to optimize the extraction of DNA from the host, Porites astreoides, as well as the symbiont. The optimization was divided into a series of steps: the preservation of the samples, separation of the coral tissue from the skeleton, separation of the host tissue from the algal cells to prevent cross contamination as well as the extraction and purification of genomic DNA from the algae that are located intracellularly within the invertebrate animal tissue. The best preservation method was freezing at low temperatures without ethanol. After scraping with a razor blade, the coral tissue can be divided into host and algal components and the DNA extracted using modifications of published techniques yielding DNA suitable for the quantification of thymine dimer formation using antibodies. Preliminary data suggest that in P. astreoides collected from 1 m depth, thymine dimers form approximately 2.8 times more frequently in the host DNA than in the DNA of its symbionts. [source] Noninvasive collection of fresh hairs from free-ranging howler monkeys for DNA extractionAMERICAN JOURNAL OF PRIMATOLOGY, Issue 4 2009Monica Améndola-Pimenta Abstract The use of noninvasive collected samples as source of DNA in studies of wild primate populations has increased in recent years. Fresh-plucked hairs represent an important source of DNA, with relatively high quality and concentration. In this study, we describe a low-cost noninvasive technique for collecting fresh-plucked hairs used to obtain DNA samples from free-ranging black howler monkey populations (Alouatta pigra). We designed and manufactured darts made of wooden dowels, with the anterior part smeared with glue, which were projected with blowpipes to trap howler monkey hairs. All of the materials to make the darts are inexpensive and are available locally. We collected 89 samples from 76 individuals residing in 15 troops, and the total number of hairs obtained was 754. We found no differences in the number of hairs collected among sex,age classes or among localities but the percentage of darts recovered with sample varied among localities. Preliminary results indicate that over 96% of samples yielded DNA suitable for polymerase chain reaction-based microsatellite marker analysis. The technique proved successful for collecting fresh-plucked hairs of free-ranging black howler monkeys without any trauma to the animals and can be easily adapted to obtain samples from other wild primate and mammal species. Am. J. Primatol. 71:359,363, 2009. © 2009 Wiley-Liss, Inc. [source] | ||||||||||