The enhancement of PCR amplification by low molecular-weight sulfones
Introduction
Polymerase chain reaction (PCR) is one of the most commonly used techniques in modern molecular biology. Its application, however, is often frustrated by inadequate yield of the target DNA sequence and the accompanying amplification of undesired nonspecific bands (Roux, 1995, Newton and Graham, 1994). These problems, in particular low yield, can be especially severe in the cases of targets with high GC contents (Varadaraj and Skinner, 1994, McDowell et al., 1998). Perhaps the most successful of the various methods of improving yield and specificity that have been attempted is the addition of certain organic additives, such as dimethylsulfoxide (DMSO), betaine, polyethylene glycol, glycerol and formamide, to the reaction mixture (Winship, 1989, Bachman et al., 1990, Pomp and Madrano, 1991, Smith et al., 1990, Weissensteiner and Lanchbury, 1996). Despite their general potency, the performance of these compounds in the case of GC-rich templates is quite unpredictable, with a particular compound often offering inadequate improvement over the control (Baskaran et al., 1996).
The availability of a larger selection of additives that are capable of improving PCR amplification of high GC targets will help make the amplification of particular targets much more tractable. To this end, we undertook a thorough investigation of the effects of a novel class of compounds, the low molecular-weight sulfones, on the amplification of GC-rich templates. Sulfones are similar to sulfoxides with the important difference that the sulfur atom is double-bonded to two oxygen atoms, instead of one. Given the effectiveness of DMSO in PCR optimization, we were interested in determining whether these related compounds are also effective, and whether they offer any selective advantages in the cases of high GC amplicons. The study incorporated three high GC targets and examined the following compounds: methyl sulfone, ethyl sulfone, n-propyl sulfone, tetramethylene sulfone (sulfolane), butadiene sulfone (sulfolene), 2,4-dimethylsulfolane, DMSO and betaine. We consider structure-activity correlations in the class of low molecular-weight sulfones and identify several of these compounds as effective enhancers of GC-rich template amplification.
Section snippets
PCRs
PCRs were carried out under the following conditions: 10 mM Tris–HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, 0.2 μM primers, 0.06 ng/μl template, 0.2 mM of each dNTP, and 0.04 U/μl Taq polymerase. The templates used were a 996 bp segment of human myeloid leukocyte c-jun cDNA, a 511 bp segment of human prostate-specific membrane antigen (PSM) cDNA, and bovine brain glycolipid transfer protein (GTP) cDNA (660 bp). cDNA synthesis was carried out using the First-Strand RT-PCR kit
Definition of terms
The effects of sulfone additives on PCR amplification were studied in this work using three different GC-rich templates. Description of the effectiveness of the various additives was achieved in the case of each target through the assignment of two densitometric quantities, termed potency and specificity, to each compound. The potency of an additive is defined as the maximum densitometric volume of target band amplification over the concentration gradient tested for that additive. Maximal
Discussion
The objectives of this investigation were twofold: (1) to identify novel enhancers of GC-rich template amplification that might function as well as or better than the current state of the art; and (2) to determine structure-activity correlations in PCR enhancement by the family of low molecular-weight sulfones. The data regarding the potency, best specificity and effective range of each of the additives, listed in Table 1, Table 2, form the basis for the discussion and analysis of both of these
Acknowledgements
R.C. wishes to thank the National Science Foundation for its support in offering a 3 year research fellowship. C.E.S. is supported by a grant from the National Institutes of Health (GM 44038).
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