Improved Quantitative PCR Using Nested Primers Lawrence A. Haft Applied Biosystems Division, The Perkin-Elmer Corporation, Norwalk, Connecticut 06859

Quantitative PCR can often be improved by conducting the amplification with nested primers. First, fewer nonspeciflc amplification products, which could otherwise interfere with quantitation, are produced. Often, nonspeciflc products can be eliminated. In these cases, relatively simple nonspeciflc detection techniques are suitable for quantitation. In addition, nested primer PCR provides intrinsic PCR product carryover protection and generally improves the robustness and lower limit of detection of PCR. For a nested PCR to provide useful quantitative information, it is Important that the initial phase of amplification, performed with the outer pair of primers, takes place entirely in the exponential phase. This is generally achieved easily. The major consideration in designing a nested PCR protocol compatible with quantitation is to assure that the maximum concentration of PCR products produced by the outer primers does not exceed - 1 0 % the molarity of the outer primers. A simple formula can be used to determine the maximum number of thermal cycles that provide this assurance. Good correspondence was obtained between initial target concentration and final PCR product yield in a nested-primer HIV-1 PCR.

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PCR Methods and Applications

H iguchi et al. ~1~ described a powerful and c o n v e n i e n t m e t h o d to detect and quantify PCR amplification products by fluorescence e n h a n c e m e n t in e t h i d i u m bromide. These data can be treated in at least two ways to estimate the initial concentration of a target sequence. First, from data taken in the exponential amplification phase, the step cycle efficiency can be calculated and original target concentration easily calculated. (2~ Higuchi et al. ~1~ also demonstrated that the initial target sequence concentration could be calculated from data obtained in the n o n e x p o n e n t i a l phase (or plateau phase), based on the calculated n u m b e r of PCR cycles required for PCR product to reach an arbitrarily selected threshold concentration. A major technical advantage of this approach is that comparatively simple and inexpensive instrum e n t a t i o n can often provide adequate sensitivity to detect amplification products in the n o n e x p o n e n t i a l phase (typically, 10 -8 to 10 -7 M PCR product). A major advantage of the use of such nonspecific detection techniques is the complete e l i m i n a t i o n of the requirement for labeled DNA probes. However, a substantial objection to the wide i m p l e m e n t a t i o n of intercalating dye detection is its lack of specificity. Unless the PCR is very specific, nonspecific products are quantitated together with specific products, and a false estim a t i o n of initial copy n u m b e r m a y be obtained. This is particularly a p r o b l e m for amplifications starting with low copy n u m b e r s of targets. Higuchi et al. ~ noted that they were unable to quantify