Solidphase Extraction

Solid-phase extraction methods for DNA have been developed in recent years in formats that enable high-throughput DNA extractions. One of the most active efforts in this area is with silica-based extraction methods and products from QIAGEN, Inc. (Valencia, CA). QIAamp spin columns have also proven effective as a means of DNA isolation (Greenspoon et al. 1998). In this approach, nucleic acids selectively absorb to a silica support, such as small glass beads, in the presence of high concentrations of chaotropic salts such as guanidine hydrochloride, guanidine isothiocyanate, sodium iodide, and sodium perchlorate (Vogelstein and Gillespie 1979, Boom et al. 1990, Duncan et al. 2003). These chaotropic salts disrupt hydrogen-bonding networks in liquid water and thereby make denatured proteins and nucleic acids more thermodynamically stable than their correctly folded or structured counterparts (Tereba et al. 2004).

If the solution pH is less than 7.5, DNA adsorption to the silica is typically around 95% and unwanted impurities can be washed away. Under alkaline conditions and low salt concentrations, the DNA will efficiently elute from the silica material. This solid-phase extraction approach can be performed with centrifugation or vacuum manifolds in single tube or 96-well plate formats (Hanselle et al. 2003, Yasuda et al. 2003) and is even being developed into formats that will work on a microchip (Wolfe et al. 2002).

Another solid-phase extraction approach is the DNA IQ™ system marketed by Promega Corporation. The DNA IQ™ system utilizes the same silica-based DNA binding and elution chemistries as QIAGEN kits but with silica-coated paramagnetic resin (Tereba et al. 2004). With this approach, DNA isolation can be performed in a single tube by simply adding and removing solutions. First, the DNA molecules are reversibly bound to the magnetic beads in solution with a solution pH of less than 7.5 (see above). A magnet is used to draw the silica-coated magnetic beads to the bottom or side of the tube leaving any impurities in solution. These solution impurities (proteins, cell debris, etc.,) can easily be removed by drawing the liquid off of the beads. The magnetic particles with DNA attached can be washed multiple times to more thoroughly clean the DNA. Finally, a defined amount of DNA can be released into solution via heating for a few minutes.

The quantity of DNA isolated with this approach is based on the number and capacity of the magnetic particles used. Since flow-through vacuum filtration or centrifugation steps are not used, magnetic bead procedures enable simple, rapid, and automated methods. This extraction method has been automated on the Beckman 2000 robot workstation and implemented into forensic casework by the Virginia Division of Forensic Science (Greenspoon et al. 2004).

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Stammering Its Cause and Its Cure

Stammering Its Cause and Its Cure

This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.

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