Nucleic acid extraction

Nucleic acid extraction protocols using guanidine hydrochloride, sodium sarco-syl, and ethanol have been developed to quantify viral RNA by bDNA in lymph node tissue, liver tissue, and peripheral blood monocytes (Wilber and Urdea, 1995). 

Burtscher, C. Fall, P. A. Wilderer, P. A. Wuertz, S. Detection of Salmonella and Listeria monocytogenes in suspended organic waste by nucleic acid extraction and PCR. Appl. Environ. Microbiol. 1999, 65, 2235-2237. 

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. 

Nucleic acid extracted from purified virus using phenol or dodecyl sulfate is easily destioyed by the homologous nucleases present m normal sera or tissues. DNA is destroyed by the enzyme deoxyribonuclease RNA by ribonucleases, This provides one means of identifying the type of nucleic acid. The intact virus is not affected by these enzymes. 

You are given a sample of nucleic acid extracted from a virus. How would you determine whether the virus has an RNA or DNA genome and whether it is single- or double-stranded ... 

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. 

H. W. Sauer, K. L. Babcock and H. P. Rusch (1956). High molecular weight phosphorus compound in nucleic acid extracts of the slime mould Physarum polycephalum. J. Bacteriol., 99, 650-661. W. B. Schaefer and C. W. Lewis (1965). Effect of oleic acid on growth and cell structure of Mycobacteria. J. Bacteriol., 90,1438-1446. 

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-... 

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. 

Chaotropic agents guanidine hydrochloride use for study of protein denaturation GTIC is considered to be more effective than GuCl GTIC used for nucleic acid extraction. 

After initial tests, the next step is to optimize steps of the analytical process, including nucleic acid extraction, amplification, detection, and quantification. For amplification procedures, variables to be optimized may include buffer pH, nucleotide concentration, MgCb concentration, type of polymerase, primer concentration, and annealing temperature. 

Internal calibrators compensate for inhibitors and/or loss of nucleic acid during the nucleic acid extraction procedure, but they (like internal amplification controls ) can compete with the target sequence for assay components and result in lower quantified result or no result at all. Since internal calibrators have some variation in sequence compared with the target to enable specific detection, natural analytical materials do not make good internal calibrators. 

Interpretation of a negative result requires the consideration of assay sensitivity and efficiencies of nucleic acid extraction and amplification. A false-negative result may be caused by mhibition of or decreased efficiency of amplification, and proper controls for this have been described above. Insufficient sample, inappropriate specimen type, inappropriate timing of sample collection, and degradation of nucleic acid during transport and handling are other sources of false-negative results. 

GenProbe, San Diego, CA) NG 16S rRNA Target capture nucleic acid extraction... 

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. 

Riemann K, Adamzik M, Frauenrath S, Egensperger R, Schmid KW, Brockmeyer NH et al (2007) Comparison of manual and automated nucleic acid extraction from whole-blood samples. J Clin Lab Anal 21 244-248... 

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. 

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