Fig. 2. Schematic demonstrating the polymerization of acrylamide and bis-acrylamide monomers.
A series of chemically modified agaroses with smaller pore sizes were developed by scientists at FMC BioProducts. These modified gel matrices are prepared by melting and casting similarly to unmodified agarose, but because of the smaller pore sizes, they are very useful for DNA separations in the 100- to 1000-bp size range and are widely used in molecular diagnostics. These products are marketed under the trade names NuSieve® and MetaPhor® agaroses (Cambrex Bio Science Walkersville, Inc., Walkersville, MD).
3.2. THE GEL MATRIX: POLYACRYLAMIDE The advent of PCR has had profound effects on the clinical laboratory's ability to use the tools of molecular biology for clinical diagnostic testing. However, it is important to understand the effect that PCR technology has had on the availability and choice of electrophoretic techniques in the clinical laboratory. The principal effect of PCR on the practice of electrophoresis has been to shift the size of the analytes from large fragments of DNA, as used in the Southern transfer, to small fragments of DNA, typically from 100 to 1000 bp in length. Although the chemically modified agaroses (which have been optimized for the separation of low-molecular-weight DNA fragments) are the most commonly used matrixes for the analysis of PCR products, it is necessary to use polyacrylamide gels for very high resolution.
Polyacrylamide gels are prepared from a monomer, acry-lamide, and a crosslinker, typically bis-acrylamide. (Note: Acrylamide is a potent neurotoxin and is readily absorbed through the skin. When using acrylamide in aqueous solution, wear gloves and a lab coat. When weighing powdered acryl-amide, wear goggles and perform the weighing operation in a chemical hood.) The first step in preparing a polyacrylamide gel is to add a free-radical initiator to a solution of monomer and crosslinker. The most commonly used initiator system is tetra ethylene diamine (TEMED) and ammonium persulfate (APS) (Fig. 2). APS reacts with the TEMED to form a TEMED derivative with a free, or unpaired, electron. This type of molecule is termed a free radical and is highly reactive. The TEMED radical reacts with an acrylamide molecule, forming a TEMED-acrylamide radical. This first step of the polymerization process is termed "chain initiation." The next steps are chain elongation, in which the polymer chain grows by repetitive addition of acrylamide monomers to the growing chain with the free-radical terminus. Chain branching occurs when a bis-acrylamide molecule is added to the end of the chain. Chain termination occurs when two free radicals react, giving a stable compound with paired electrons. In order to achieve complete polymerization, it is important that compounds that quench free-radical reactions, such as alcohols or oxygen, be excluded from the reaction mixture. Oxygen is typically removed from the acrylamide/bis-acrylamide solution by degassing under vacuum for 15-30 min prior to the addition of the initiators. To avoid oxygen contact during the polymerization process, the gel is generally cast between two glass plates. After polymerization, the gel is run in a vertical format.
The pore size of the final polyacrylamide gel is determined by the concentration of the acrylamide monomer and the ratio of the crosslinker to monomer. These parameters are referred to as %T and %C, respectively, and are defined as follows: %T = mass of all monomers and crosslinkers per 100 mL volume and
%C = mass of the crosslinker - mass of all monomers and crosslinkers per 100 mL volume. The pore sizes in polyacry-lamide gels are typically much smaller than those in agarose gels. Thus, they are used for DNA fragments of smaller size. Gels with %C of approx 3-5% and %T ranging from 5 to 15% are the most often used gels in the DNA laboratory, giving superior separations in the 100- to 1000-bp size range.
A series of gel products with improved performance for specific applications were developed by scientists at AT Biochem (Malvern, PA). These materials use acrylamide as a monomer, but incorporate proprietary comonomers and novel crosslink-ers. Recently, this line of products has been acquired Cambrex Bio Science Walkersville, Inc. (Walkersville, MD). A gel matrix offering longer reads for DNA sequencing and a matrix optimized for the detection of conformational changes in singlestrand conformation polymorphism or heteroduplex analysis are currently marketed under the trade names Long Ranger® and Mutation Detection Enhancement Gel (or MDE®), respectively.
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