6921_27_core - Promega Notes Number 65, 1998, p. 27 Betaine...

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Promega Notes Number 65, 1998, p. 27 By Susan Frackman, Gary Kobs, Dan Simpson and Doug Storts Promega Corporation Corresponding author: email to sfrackma@promega.com To improve yield and specificity of difficult targets in PCR amplifications, researchers often include enhancing agents in the reaction. We discuss two commonly used PCR enhancing agents, betaine and dimethyl sulfoxide (DMSO), and the identification of betaine in commercially available additives. I NTRODUCTION PCR is a powerful and extremely robust procedure for most applications and usually requires little optimization. However, there are instances in which a particular DNA region proves difficult to amplify by PCR. The amplification of targets rich in GC content or ones that can form secondary structure often result in little or no yield of expected product. Furthermore, amplification may result in products derived from regions other than the target DNA region, indicated by multiple bands on a stained agarose gel. Optimization of magnesium concentration, buffer pH, denaturing and annealing times and temperatures, and cycle number is useful in some, but not all cases. Targets that are refractory to amplification, despite optimization attempts, can often be amplified if the appropriate additive is included in the amplification mix. A variety of additives and enhancing agents can be included in PCR amplifications to increase yield, specificity and consistency. Agents include: dimethyl sulfoxide (DMSO), N,N,N-trimethylglycine (betaine), formamide, glycerol, nonionic detergents, bovine serum albumin, polyethylene glycol and tetramethylammonium chloride. These additives have beneficial effects on some PCR amplifications; however, it is not possible to predict which agents might be useful for a particular target. There are reports of PCR amplifications in which specificity was improved by formamide, but not DMSO (1), and reactions in which DMSO was more effective than formamide at increasing yield and specificity (2). Several agents that facilitate product formation in PCR amplifications are now commercially available. These agents alter the melting characteristics of DNA. Their identities, however, are not revealed by the respective suppliers. E NHANCING AGENTS: B ETAINE AND DMSO Two PCR enhancing agents that deserve particular attention are betaine and DMSO. DMSO is probably the most commonly used enhancing agent and is frequently included as part of a standard optimization of PCR amplifications. Betaine is another agent that has been used successfully for increasing yield and specificity of PCR products (3-6). Figure 1 shows the structural formula of betaine. Both of these agents facilitate strand separation; DMSO disrupts base pairing whereas betaine, an isostabilizing agent, equalizes the
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This note was uploaded on 08/01/2009 for the course 221 22412 taught by Professor Maz during the Spring '09 term at A.T. Still University.

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6921_27_core - Promega Notes Number 65, 1998, p. 27 Betaine...

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