Comparable sensitivity and specificity of nested PCR and single-stage PCR using a thermally activated DNA polymerase.

Abstract

To augment sensitivity and specificity of polymerase chain reaction (PCR), a nested PCR protocol is used (1). This procedure allows detection of even single copies of specific template sequences (5,6) but requires additional manipulations (such as opening of reaction tubes and transfer of PCR products), which may increase the risk of contamination and lead to false-positive results. To avoid this disadvantage, attempts are being made to increase the sensitivity of single-stage PCR protocols. One way to approach this problem is to use AmpliTaq Gold, a modified form of AmpliTaq DNA Polymerase (both from Perkin-Elmer, Norwalk, CT, USA). AmpliTaq Gold is inactive at room temperature. However, the big advantage of this enzyme is that, despite raising the temperature, it will not start to extend primers that have annealed at nonspecific sites during the first cycle, thus eliminating nonspecific PCR products. Because heat restores the polymerase activity (2), it can be used for hot-start methodology (3) without additional manipulations. AmpliTaq Gold has been shown to increase the sensitivity and amplification specificity of PCR (2). We compared the sensitivities and specificities of conventional singlestage PCR (with unmodified AmpliTaq DNA polymerase) and nested PCR with a single-stage PCR protocol using the new DNA polymerase AmpliTaq Gold. As a model system, we used three different primer systems specific for gag, pol and env genes of human immunodeficiency virus type 1 (HIV-1). Crude lysates of 5 × 104 peripheral blood mononuclear cells [PBMCs (6)] as nonspecific background were mixed with 30, 10, 3, 1 and 0.3 (for nested and AmpliTaq Gold PCR) or 1000, 300, 100, 30, 10 and 3 (for conventional PCR) copies of pBH10 containing the HIV-1 DNA sequence (4). The samples were subjected to PCR or nested PCR in a total volume of 50 μL containing 1 U of AmpliTaq or AmpliTaq Gold, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% (wt/vol) gelatin, 200 μM of each dNTP and the respective primers. Samples were overlaid with mineral oil and amplified in a GeneAmp PCR System 2400 (PerkinElmer) according to the following protocols. For conventional PCR with AmpliTaq (active at room temperature), the samples were amplified in a singlestage PCR for 45 cycles with 500 ng each of the respective primers and with the following thermal cycling profile: 30 s at 94°C, 30 s at 60°C for gag (SK38/SK39) and env (SK68/SK69) or 55°C for pol (JA18/JA19), 45 s at 72°C with a final extension at 72°C for 1 min. For PCR with AmpliTaq Gold (inactive at room temperature), a heat-activation step for 10 min at 94°C preceeded the 45-cycle amplification. For nested PCR, the samples were amplified for 20 cycles in first-stage PCR with 100 ng primers for 30 s at 94°C, 30 s at 55°C for gag (GA1/GA2), 45°C for pol (JA17/JA20) or 50°C for env (JA13/

Topics

    10 Figures and Tables

    Download Full PDF Version (Non-Commercial Use)