Extraction of DNA

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. 

EXTRACTION OF DNA/RNA FROM FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE BASED ON THE ANTIGEN RETRIEVAL PRINCIPLE... 

Shibata D. Extraction of DNA from paraffin-embedded tissue for analysis by polymerase chain reaction new tricks from an old friend. Hum. Pathol. 1994 25 561-563. 

Klintschar M, Neuhuber F. Evaluation of an alkaline lysis method for the extraction of DNA from whole blood and forensic stains for STR analysis. J. Forensic Sci. 2000 45 669-673. 

DeSantis et have reported the discovery of new nitrilases through the screening of genomic libraries created by the extraction of DNA from various environments (metagenomics). In preliminary experiments, using 25 mM mandelonitrile in pH 8 buffer containing 10% methanol and 0.12 g mL of one of these nitrilases, the acid was produced quantitatively with 98 % ee within 10 min. The product was subsequently shown to be (7 )-mandelic acid after isolation in 86 % yield. In a parallel reaction, (/ )-2-chloromande-lic acid was produced at a seventeenth of the rate (Scheme 1.44). 

Saliva. The extraction of DNA from mouthwash samples provides high-yield, good-quality DNA that is also suitable for whole-genome amplification (23). The advantage of this sample source over blood is that the procedure for collecting samples is noninvasive, and the mouthwash samples are stable at room temperature for extended periods of time. 

Commercial kits are available for DNA extraction from the majority of sources, including blood, mouthwash, plasma, serum, frozen tissue, and formalin-fixed tissue. Most kits work very weU if carried out in accordance with the manufacturer s instructions. In addition, they reduce the hkeh-hood of variabihty in the quality and quantity of DNA between batches of samples. Published protocols for the extraction of DNA from paraffin-embedded tissue are also available (37). 

Ceo RN, Kazerouni MR, Rengan K. 1993. Chelex 100 as a Medium for Simple Extraction of DNA for PCR-based T3rping from Forensic Material. Biotechnique 1993 10 506-513. 

C4CiIm][PPg], a widespread IL, was recently used for fhe direcf exfraction of double-stranded DNA (dsDNA) [30]. The authors demonstrated that DNA may be extracted with high efficiency, >95%, while profeins and mefal-loproteins do nof inferfere extraction. The back-extraction of DNA info fhe aqueous phase wifh fhe efficiency of 30% was performed in fhe presence of phosphafe-cifrafe buffer solution (pH 4). 

Lam, C.-W., Casanova, M. Heck, H.D A. (1986) Decreased extractability of DNA from proteins in the rat nasal mucosa after acetaldehyde exposure. Fundam. appl. Toxicol., 6, 541— 550... 

Another major problem associated with the extraction of DNA from archaeological specimens is that the procedure often co-extracts impurities that can later complicate, or prevent, the study of the extracted DNA by inhibiting PCR amplification (reviewed by 5). Commonly encountered inhibitory substances found in aDNA extracted from teeth, bones, mummified tissue, and coprolites include humic acids, ftilvic acids, tannins, porphyrin products, phenolic compounds, hematin, and collagen type I (37—42). The formation of Maillard products, commonly encountered in coprolite samples, can also prevent PCR amplification by causing DNA to become inaccessibly trapped in these sugar-derived condensation products (12). As the negative results in many aDNA studies are attributed to the presence of PCR inhibitors, our extraction method outlined below pays particular attention to the problem and offers a simple test for the presence of PCR inhibitors in DNA extracts. 

Turbett, G. T., Barnett, T. C., Dillon, E. K., and Sellner, L. N. 1996. Single-tube protocol for the extraction of DNA or RNA from paraffin-embedded tissues using a starch-based adhesive. Biotechniques 20 846-853. 

Since then much progress has been made in sample preparation techniques that reduce sample complexity. An overview of the sequence of extraction, isolation, and purification of nucleic acids is presented in Figure 8.1. It can be categorized in several unit steps beginning with the extraction of DNA until its sizing and sequencing. The different options within each step are listed in Table 8.1 and are described in this chapter. The technique best suited in a given application depends on ... 

With the need to provide PCR-amplifiable DNA, multiple approaches for incorporation of the extraction protocol onto microchips were examined. Recent development includes the implementation of a solid-phase extraction of DNA on a microchip [49]. The extraction procedure utilized was based on adsorption of the DNA onto bare silica. The silica beads were immobilized into the channel using a sol-gel network. This method made possible the extraction and elution of DNA in a pressure-driven system. 

The extraction of DNA from mummified and fossilized plant tissue is one of the most exciting developments in molecular systematics. Mummified plant tissues up to 44,600 years old have yielded analyzable DNA.13-19 The recent publications of rbcL sequences from Miocene fossils (17-20 million years old) of Magnolia20 and Taxodium21 and the amplification of similar-aged DNA from Platanus and Pseudofagus21 open a new source for land plant tissue and offer enormous possibilities for research in molecular systematics and evolution. The latter report clearly invalidates the objections22 to the authenticity of the Miocene fossil plant DNA and hence of their systematic utility. 

Ambient temperatures are not always sufficient for successful extraction of DNA from hair shafts. In the case of plucked human hairs left in envelopes in a desk drawer for over 1 year, the rate of successful mtDNA amplification was low (< 10%) compared to recently plucked hairs prepared and amplified using the same protocols. We therefore recommend that, once hairs and feathers are taken, they should be frozen at—70° until extracted for nucleic acids. 

Fast and simple methods for extraction of DNA or RNA from cells, whole blood, and other fluids have been reviewed by Bloch24 and Kawasaki.25 In general, target DNA is released from the cells or virus by proteinase K digestion in the presence of detergent followed by heat inactivation of the proteinase K. A small aliquot of the cell lysate is then used directly as the template in the amplification reaction. For cell culture supernatant fractions, the proviral DNA released from the lysed cells can be directly... 

Tian H, Huhmer AF, Landers JP. Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format. 

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