Number 65, 1998, p. 27
By Susan Frackman, Gary Kobs, Dan Simpson and Doug Storts
Corresponding author: email to
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.
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.
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).
shows the structural formula of betaine. Both
of these agents facilitate strand separation; DMSO disrupts base pairing whereas betaine, an isostabilizing agent, equalizes the