Nucleic acids extraction methods

The specimen will be the basis for the analytic analysis. Is it RNA or DNA What is the origin of the tissue Amniocentesis Was it a spontaneous product of conception Were anatomic pathology slides or tissue blocks prepared Are cell lines involved Are these primary or immortalized Was a chorionic villus sampling procedure done Is the sample properly collected peripheral blood The answers to each of these questions should be noted, and considered part of the validation of a useful nucleic acid extraction method. A molecular diagnostics laboratory should adhere to the highest standards in providing services, and prior validation of applicable nucleic acid extraction procedures is a must to ensure high-quality service. 

Dellunde, S., et al. 2002. A fast and sensitive nucleic acid extraction method for the detection of Cryptosporidium by PCR in environmental water samples. Water Supply 2 95. 

Other nucleic acid extraction methods, which may have been integrated in a microsystem, will be described in subsequent sections. 

The collection of plant tissue is quite different from animal tissue collection. The discussion of collection of plant and animal tissue by Dessauer et al.2S is detailed and helpful. However, the recommendations for procedures unique to plant tissue collection are somewhat misleading and outdated, especially when tropical collections are involved. Plant tissue can now be collected and transported as either fresh tissue (leaves and/or shoot cuttings) or preserved tissue the latter either as cryopreserved tissue (liquid nitrogen or dry ice) or as dried tissue (air-dried, herbarium-dried, lyophilized, or chemically dried). Ambient-temperature liquid chemical preservation techniques (such as those routinely done for herbarium plant specimens in the tropics) so far have been ineffective in maintaining adequate yields of high-quality DNA.15 It should be stressed again that the manner of collecting plant tissue is dictated by several other factors what macromolecule (DNA, RNA, or isozymes) will be examined, what type of nucleic acid extraction method will be used (or, more impor-... 

DNA PGR assay (neither of which has been cleared by the FDA). GMV PGR LDTs that use standard and real-time PGR methods are also widely used in clinical laboratories. These LDTs use various specimen types, nucleic acid extraction methods, target genes, calibrators, and detection methods. As a result, viral load values obtained with the different assays may not always agree. This makes it very difficult to compare results among clinical studies that use these assays and to establish concentrations of GMV DNA that correlate with clinical disease. 

To extract DNA and RNA from serum samples or from culture supernatant fractions which contain a low level of proviral DNA, the IsoQuick extraction kit (a modified guanidinium salt-organic solvent extraction method) by MicroProbe Corporation (Bothell, WA) has been used successfully by our group. Following the manufacturer s recommended procedures for total nucleic acid extraction, the final DNA-RNA suspension can be amplified directly to detect proviral DNA. Using cDNA templates synthesized from total nucleic acids with random hexamer priming generally increases the PCR product yield. 

Fast blot methods to minimize nucleic acid extraction and immobilization steps have been developed. Those with nylon as a solid phase can take advantage of the ability of NaOH to dissociate cells, denature DNA and immobilize DNA. Nitrocellulose membranes have a lower binding capacity and co-immobilization of nucleic acid and protein from neutral solutions can be a problem. Bresser et al. (1983) used hot concentrated Nal to inhibit protein immobilization, to denature DNA and to irreversibly bind the nucleic acid to nitrocellulose (no baking required). This method can also be used for RNA. About 10 cells are minimally required for a unique DNA sequence, whereas > 0.01% of total mRNA can be detected by the Nal methods. 

A variety of well-established macroscale SPE methods for nucleic acid extraction have been successfully transferred to microscale devices [10, 31-57]. Although the physical principles of these methods may be different (e.g., chaotropic interactions, electrostatic interactions, affinity interactions, etc.), micro-SPE protocols typically consist of three steps (1) selective adsorption of nucleic acids onto a solid phase (2) removal of contaminants by a washing step and (3) elution of the preconcentrated nucleic acids from the solid support using water or a low salt buffer [31]. Like their macroscale counterparts, micro-SPE devices possess a loading level of target material that is dependent upon the available surface area within the extraction bed and, thus, are manufactured either by packing the solid phase... 

Regardless of sample type, the first step in our process is nucleic add amplification. The prestep is therefore nucleic acid extraction and/or purification and is highly dependent upon the sample type and matrix (blood, saliva, dirt, etc.) in which the organism(s) of interest is (are) found. Thus, nucleic acid sample preparation is somewhat outside the scope of our methods, but numerous kits and protocols are commercially available for various applications. Subsequent steps comprising PCR,... 

The previous biomarkers relate to phenotypic assessments of microbial diversity and most will probably measure a restricted part of the total microbial pool, since not alt markers will be expressed uniformly by every cell. In contrast, methods involving the detection of nucleic acids may be directly applicable to all microorganisms provided that the complete extraction of DNA (lysis of cells) or permea-bilization of cells can be achieved. 

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. 

Recently, the use of AR has extended into several other areas, yielding interesting information for cytology, fresh cell/tissue sections, and fluorescence IHC (fluorescence in situ hybridization [FISH]), in addition to adaptations of the method for extraction of nucleic acids and proteins from FFPE tissues for use with modern methods of molecular analysis. In this chapter, the emphasis is on expanded applications in diagnostic cytology, fresh frozen cell/... 

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... 

---