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High Molecular Weight DNA (high + molecular_weight_dna)
Selected AbstractsIsolation of polymorphic microsatellite markers from the malaria vector Anopheles darlingiMOLECULAR ECOLOGY RESOURCES, Issue 4 2001Jan E. Conn Abstract High molecular weight DNA was extracted from the primary Neotropical malaria vector, Anopheles darlingi from Capanema, Pará, Brazil, to create a small insert genomic library, and then a phagemid library. Enriched sublibraries were constructed from the phagemid library using a microsatellite oligo primed second strand synthesis protocol. The resulting 242 760 individual clones were screened. The mean clone size of the positive clones was 302 bp. Flanking primers were designed for each suitable microsatellite sequence. Eight polymorphic loci were optimized and characterized. The allele size ranges are based on 253 samples of A. darlingi from eastern Amazonian and central Brazil. [source] Xanthogenate nucleic acid isolation from cultured and environmental cyanobacteriaJOURNAL OF PHYCOLOGY, Issue 1 2000Daniel Tillett The isolation of high-quality nucleic acids from cyanobacterial strains, in particular environmental isolates, has proven far from trivial. We present novel techniques for the extraction of high molecular weight DNA and RNA from a range of cultured and environmental cyanobacteria, including stains belonging to the genera Microcystis, Lyngbya, Pseudanabaena, Aphanizomenon, Nodularia, Anabaena, and Nostoc, based on the use of the nontoxic polysaccharide solubilizing compound xanthogenate. These methods are rapid, require no enzymatic or mechanical cell disruption, and have been used to isolate both DNA and RNA free of enzyme inhibitors or nucleases. In addition, these procedures have proven critical in the molecular analysis of bloom-forming and other environmental cyanobacterial isolates. Finally, these techniques are of general microbiological utility for a diverse range of noncyanobacterial microorganisms, including Gram-positive and Gram-negative bacteria and the Archea. [source] Fluorometric Analysis of DNA Unwinding (FADU) as a Method for Detecting Repair-induced DNA Strand Breaks in UV-irradiated Mammalian Cells,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2000Christa Baumstark-Khan ABSTRACT Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray,induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20°C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation). [source] Rapid extraction of high-quality genomic DNA from Porphyra yezoensis (Bangiales, Rhodophyta)PHYCOLOGICAL RESEARCH, Issue 1 2000Maiko Nakajima SUMMARY We developed a simple, rapid and stable method for extraction of high molecular weight DNA from the marine red alga Porphyra yezoensis Ueda using both guanidium treatment and QIAGENÔ kit (Funakoshi, Tokyo, Japan). The method does not require expensive equipment and complex steps. The DNA yield averaged 1.5 ,g 100 mg,1 of Porphyra tissue and the A260/A280 and A230/A260 ratios of the DNA were approximately 1.8 and 0.4, respectively. It was of sufficient quality to be used for not only polymerase chain reactions but also other DNA manipulation techniques such as restriction digestion and construction of genomic libraries. [source] | ||||||||||