Isolation DNA

The DNA isolated from different cells and viruses characteristically consists of two polynucleotide strands wound together to form a long, slender, helical molecule, the DNA double helix. The strands run in opposite directions that is, they are antiparallel and are held together in the double helical structure through interchain hydrogen bonds (Eigure 11.19). These H bonds pair the bases of nucleotides in one chain to complementary bases in the other, a phenomenon called base pairing. 

Samples of DNA isolated from different tissues of the same species have the same proportions of heterocyclic bases, but samples from different species often have greatly different proportions of bases. Human DNA, for example, contains about 30% each of adenine and thymine and about 20% each of guanine and cytosine. The bacterium Clostridium perfringens, however, contains about 37% each of adenine and thymine and only 13% each of guanine and cytosine. Note that in both examples the bases occur in pairs. Adenine and thymine are present in equal amounts, as are cytosine and guanine. Why ... 

In another series of experiments, a novel approach to the determination of nucleotide sequence was adopted by A. S. Jones, Stacey, and their co-workers. For example, when calf thymus DNA was treated with mercaptoacetic acid in the presence of zinc chloride and anhydrous sodium sulfate, it yielded aldehydo-apurinic acid bis(carboxymethyl) dithioacetal. When degraded with dilute alkali, this afforded dialyzable fragments, which were separated into at least 20 components. Some were identified, including mono-, di-, and tri-nucleotides, thereby revealing that DNA contain regions of at least three linked pyrimidine nucleotides. The same procedure was applied to the DNA isolated from M. phlei ... 

Figure 3.9 Cloning and DNA-isolation strategies commonly used in metagenomics.

The recently developed methodology for enzyme discovery that is based on random DNA isolation and subsequent screening (metagenome mining) is... 

Several standard DNA isolation kits are commercially available, including the QIAamp DNA Stool Mini Kit and the DNeasy Plant Mini Kit made by Qiagen. Both of these products are based on silica gel membrane technology and allow for the extraction of total DNA from processed foods and raw foodstuffs, respectively. In... 

Classical approaches to plant DNA isolation aim to produce large quantities of highly purified DNA. However, smaller quantities of crudely extracted plant DNA are often acceptable for PCR analysis. Another efficient method for preparation of plant DNA for PCR is a single-step protocol that involves heating a small amount of plant tissue in a simple solution. Several factors influence nucleic acid release from tissue salt, EDTA, pH, incubation time and temperature. These factors must be optimized for different sample substrates. EDTA in the sample solution binds the Mg + cofactor required by the Taq polymerase in the PCR, so the EDTA concentration in the solution, or the Mg + concentration in the PCR, must be carefully optimized. 

These reference genes demonstrate that the DNA isolated was of sufficient quality and quantity for PCR amplification. It is assumed that in the course of food processing, the species-specific reference gene and the transgene are degraded in a similar manner. It is also assumed that effects of the matrix on PCR amplification will be similar. The reduced amplification efficiency of both genes presumably has no effect on the ratio of their amounts, which reflects the ratio of modified and unmodified DNA. 

Faulkner SW, Leigh DA. Universal amplification of DNA isolated from small regions of paraffin-embedded, formalin-fixed tissue. BioTechniques 1998 24 47-50. 

Quantification of the DNA isolated from the product involves concurrent inclusion in the dot blot assay of a set of spots, containing known quantities of DNA, and being derived from the producer cell. After autoradiography, the intensity of the test spot is compared with the standards. 

PHA depolymerases, 20 253, 256 pH adjusting agents, 12 61-62 Phage cloning, DNA isolation for,... 

Varela-Alvarez E, Andreakis N, Lago-Leston A, Pearson GA, Serrao EA (2006) Genomic DNA isolation from green and brown algae (Caulerpales and Fucales) for microsatellite library construction. J Phycol 42 741-745... 

Ravanat JL, Cadet J (1995) Reaction of singlet oxygen with 2 -deoxyguanosine and DNA. Isolation and characterization of the main oxidation products. Chem Res Toxicol 8 379-388. 

Gabor, E.M., de Vries, E.J. and Janssen, D.B., Construction, characterization, and use of smaU-insert gene banks of DNA isolated from soil and enrichment cultures for the recovery of novel amidases. Environmental Microbiol., 2004, 6, 948-958. 

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