Nucleic acids isolation methods

M. Muller, Considerations for the scale-up of plasmid DNA purification in Nucleic Acid Isolation Methods Eds. B. Bowlen, P. Dtirre), American Scientific Publishers, New York, 2003. 

Bowien, B. and Durre, P. (2003) Nucleic Acids Isolation Methods, American Scientific Publishers, Stevenson Ranch, CA. 

Nucleic acid analysis. Methods that analyse the microbial community from nucleic acid composition are based on use of the polymerase chain reaction (PCR). Universal forward and reverse primers are used in combination with PCR to amplify species-specific DNA fragments (usually the 16S subunit of ribosomal DNA) from samples isolated directly from soil. Samples then are separated... 

Nivens D, Applegate B, Method for nucleic acid isolation using supercritical fluids. US patent 5922536, 1999. 

These sorbents may be used either for selective fixation of biological molecules, which must be isolated and purified, or for selective retention of contaminants. Selective fixation of biopolymers may be easily attained by regulation of eluent polarity on the basis of reversed-phase chromatography methods. Effective isolation of different nucleic acids (RNA, DNA-plasmid) was carried out [115, 116]. Adsorption of nucleosides, nucleotides, tRN A and DNA was investigated. It was shown that nucleosides and nucleotides were reversibly adsorbed on... 

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. 

In a number of methods, isolation of the nucleoprotein complex (stage 2) is avoided. In the isolation of ribonucleic acid from beef pancreas,1241 nuclear material and cell debris are removed from a normal-saline extract of the minced tissue, which is then brought to half-saturation with sodium chloride (to dissociate the protein from the nucleic acid). After removal of the protein, the nucleic acid is precipitated with alcohol. However, the suggestion has been made126 that it is more satisfactory to isolate the nucleoprotein first, and this has been carried out, for instance, in the extraction of the ribonucleic acid from fowl sarcoma GRCH 15.126 Nucleoprotein complexes have also been isolated from baker s yeast127 and have been separated into various fractions, the nucleic acids from which differ slightly in composition. In addition, nucleoproteins have been isolated by complex formation with cetyltrimethylammonium bromide.128... 

CNTs can conjugate with nucleic acids via non-covalent bond. ssDNA, short double-stranded DNA and total RNA molecules can attach to the surface of CNTs and can disperse CNTs in aqueous environment. The poly(30T) has the highest dispersion efficiency (Zheng et al., 2003). For example, 1 mg DNA molecules mix with lmg CNTs in 1ml water, yield at most 4mg/ml CNT solution. DNA-CNT complexes can be purified or isolated by electronic properties such as agarose gel electrophoresis and centrifuge method (Cui et al., 2004a Karajanagi et al., 2004). 

To solve this dilemma, a new method which allowed the separation of the transmembrane transporter not as the protein, but as the nucleic acid, followed by subsequent expression of the isolated nucleic acid and a screening of the functional activity of the encoded polypeptide was suggested. 

Determination of nucleic acid yield. Cleared lysate or ImuVert was diluted to a concentration which had an absorbance between 0.4 and 0.5 at 260 nm and the absorbances at 280 and 260 nm were measured. The nucleic acid concentration in solution was then calculated by the method of Warburg and Christian (18), The yield of nucleic acid was calculated by determining the percent of nucleic acid in the lysate that was isolated in the product. The acceptable range for ImuVert manufacturing is 10.5 + 1.5. 

In many cell types it is feasible to deliver nucleic acids and genes by a variety of methods when the cells are grown in tissue culture (Table 58.1). Nonetheless, some cells, such as pneumocytes and neurons, are not readily isolated from humans and do not grow well in vitro. Furthermore, for many diseases it is essential to alter the phenotype of a significant proportion of the total cell population, making ex vivo gene therapy of limited use. 

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