DNA Templates (dna + templates)

Distribution by Scientific Domains


Selected Abstracts


Gel immobilization of acrylamide-modified single-stranded DNA template for pyrosequencing

ELECTROPHORESIS, Issue 12 2007
Pengfeng Xiao Dr.
Abstract A novel two-step process was developed to prepare ssDNA templates for pyrosequencing. First, PCR-amplified DNA templates modified with an acrylamide group and acrylamide monomers were copolymerized in 0.1,M NaOH solution to form polyacrylamide gel spots. Second, ssDNA templates for pyrosequencing were prepared by removing electrophoretically unbound complementary strands, unmodified PCR primers, inorganic pyrophosphate (PPi), and excess deoxyribonucleotides under alkali conditions. The results show that the 3-D polyacrylamide gel network has a high immobilization capacity and the modified PCR fragments are efficiently captured. After electrophoresis, gel spots copolymerized from 10,,L of the crude PCR products and the acrylamide monomers contain template molecules on the order of pmol, which generate enough light to be detected by a regular photomultiplier tube. The porous structure of gel spots facilitated the fast transportation of the enzyme, dNTPs and other reagents, and the solution-mimicking microenvironment guaranteed polymerase efficiency for pyrosequencing. Successful genotyping from the crude PCR products was demonstrated. This method can be applied in any laboratory; it is cheap, fast, simple, and has the potential to be incorporated into a DNA-chip format for high-throughput pyrosequencing analysis. [source]


Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regions

ENVIRONMENTAL TOXICOLOGY, Issue 3 2005
Youness Ouahid
Abstract Reliable cyanotoxin monitoring in water reservoirs is difficult because of, among other reasons, unpredictable changes in cyanobacteria biomass, toxin production, and inadequate sampling frequency. Therefore, it would be useful to identify potentially microcystin-producing strains of cyanobacterial populations in field samples. With this aim, we developed a methodology to distinguish microcystin-producing from non-producing Microcystis strains by amplifying six characteristic segments of the microcystin synthetase mcy cluster, three corresponding to the nonribosomal peptide synthetase, genes mcyA, mcyB, and mcyC, and three to the polyketide synthase, genes mcyD, mcyE, and mcyG. For this purpose five new primer sets were designed and tested using purified DNA, cultured cells, and field colonies as DNA sources. Simultaneous amplification of several genes in multipex PCR reactions was performed in this study. The results obtained showed that: (i) the expected specific amplicons were obtained with all microcystin-producing strains but not with nonproducing strains; (ii) cells could be directly used as DNA templates, 2000 cells being a sufficient number in most cases; (iii) simultaneous amplification of several gene regions is feasible both with cultured cells and with field colonies. Our data support the idea that the presence of various mcy genes in Microcystis could be used as a criterion for ascribing potential toxigenicity to field strains, and the possibility of applying whole-cell assays for the simultaneous amplification of various genes may contribute significantly to simplifying toxigenicity testing. © 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 235,242, 2005. [source]


Heterochromatin tells CENP-A where to go

BIOESSAYS, Issue 6 2008
Mickaël Durand-Dubief
The centromere is the region of the chromosome where the kinetochore forms. Kinetochores are the attachment sites for spindle microtubules that separate duplicated chromosomes in mitosis and meiosis. Kinetochore formation depends on a special chromatin structure containing the histone H3 variant CENP-A. The epigenetic mechanisms that maintain CENP-A chromatin throughout the cell cycle have been studied extensively but little is known about the mechanism that targets CENP-A to naked centromeric DNA templates. In a recent report published in Science,1 such de novo centromere assembly of CENP-A is shown to be dependent on heterochromatin and the RNA interference pathway. BioEssays 30:526,529, 2008. © 2008 Wiley Periodicals, Inc. [source]


Circular dichroism and the interactions of water soluble porphyrins with DNA,A minireview

CHIRALITY, Issue 4 2003
Robert F. Pasternack
Abstract The size, sign, and profile of induced circular dichroism (CD) features in the Soret region are reliable indicators of the binding modes of porphyrins and metalloporphyrins to DNA. Porphyrins shown (using such CD criteria) to be intercalators in monodispersed DNA duplexes prove extremely useful for the detection and characterization of organized, condensed forms of nucleic acids (,-condensates). In addition, certain select porphyrin derivatives can form extended assemblies on nonaggregated DNA templates. A combination of CD and resonance light scattering (RLS) measurements allows for sensitive detection and characterization of these porphyrin arrays. Chirality 15:329,332, 2003. © 2003 Wiley-Liss, Inc. [source]