General concepts for PCR primer design.

1Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; ZDepartment of Molecular Biology, Washington University, St. Louis, Missouri 63110; 3Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 PCR is a technology born of the modern molecular biology era. The enzyme used for PCR, Taq DNA polymerase, supplied with the 10x buffer, is purchased as a cloned product, and the nucleoside triphosphates are ultrapure, buffered, and available at a convenient concentration. Yet, with all of these commercially available starting materials, PCR still fails, particularly for the novice. Assuming that all of the reagents have been added in the proper concentrations, two critical PCR components are left to the researcher. The first is the nucleic acid template, which should be of sufficient quality and contain no inhibitors of Taq DNA polymerase (although when it comes to template purity, PCR is more permissive than many other molecular biology techniques). The second is the selection of the oligonucleotide primers. This process is often critical for the overall success of a PCR experiment, for without a functional primer set, there will be no PCR product. Although the selection of a single primer set may be trivial, the construction of primer sets for applications such as multiplex or nested PCR becomes more challenging. The manual selection of optimal PCR oligonucleotide primer sets can be quite tedious and thus lends itself very naturally to computer analysis. The primary factors that affect the function of the ol igonucleotides-their melting temperatures as well as possible homology among primers--are welldefined and straightforward tasks that are easily encoded in computer software. Once the computer has provided a small number of candidate primer sets, the task of selection can be (and still is) performed manually. In this approach, the researcher is taking advantage of the raw speed of computer calculations, trying all possible permutations of a primer's placement, length, and relation to the other primers that meet conditions specified by the user. From the thousands of combinations tested by the computer, a software program can present just those that are suitable for the needs of the experiment. Thus, the overall "quality" (as defined by the user in program parameters) of the primers selected is almost guaranteed to be better than the handful chosen and hand-tested by the research without computer assistance. As with any tool, understanding its function will make the end product more useful. A wide range of programs have been written to perform primer selection, varying significantly in selection criteria, comprehensiveness, interactive design, and user-friendliness. (1-1~ There are also commercial ly available specialty primer design software programs that offer enhanced user interfaces, additional features, and updated selection criteria, (1'2) as well as primer design options that have been added to larger, more general software packages. Although most people would agree that application of analytic computer software to a well-defined problem is a smart thing to do, not all researchers are convinced that PCR primer selection is a nontrivial task, or that the selection rules that make a primer amplify efficiently are even well defined. Even though many of the rules discussed have been fine-tuned by collective empirical wisdom, most are based on firm theoretical ground, if not c o m m o n sense. The purpose of this chapter is to explain basic rules of oligonucleotide primer design. With this understanding of primer selection criteria, the information deduced by primer design software can be rationally interpreted and manipulated to fit your experimental needs.