Single-tube two-round polymerase chain reaction using the LightCycler (TM)instrument

Background: For many diagnostic applications, the specificity and sensitivi ty of polymerase chain reaction (PCR) is markedly enhanced by applying two rounds of PCR with nested or semi-nested pairs of primers. In two-round PCR protocols on the LightCycler(TM) instrument, amplification products must b e collected from the capillaries by centrifugation, a procedure thought to be particularly prone to product carry-over. Objective: Development of a te chnique to perform two-round PCR with the LightCycler(TM) instrument in a s ingle closed capillary. Study design: Silicone oil was used to separate the second-round primers from first-round PCR mixture during the first-round P CR. The feasibility of the principle was demonstrated using a semi-nested p rimer system for the PCR analysis of genomic DNA. The first-round PCR react ion mixture was loaded into the capillary and covered by oil. Then, the sec ond-round PCR reaction mixture was layered on top of it. PCR was run in two rounds separated by a centrifugation step that combined the second-round P CR mixture with the first-round products. Amplified products were visualize d by fluorescence melting curve analysis. Results: When a dilution series o f genomic DNA was used for the single-capillary two-round PCR, 0.1 ng of DN A could consistently be detected. This was a 10-fold increase of sensitivit y in comparison with single-round PCR. With the new technique, the first-ro und reaction mixture was sufficiently separated from second-round primers b y the oil layer. Conclusions: Two-round PCR on the LightCycler(TM) using a single closed capillary excluded the possibility of amplification product c arry-over. This new technique can easily be adapted for numerous applicatio ns, and should show feasibility for many nested primer PCR applications cur rently in use to the clinical detection of virus-derived DNA. (C) 2001 Else vier Science B.V. All rights reserved.