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

• Published in: Journal of clinical virology 2001, Vol.20 (1), p.71-75
• Main Authors: Berg, Jörg, Nagl, Verena, Mühlbauer, Gerhard, Stekel, Herbert
• Format: Article
• Language: English
• Subjects: DNA - analysis; DNA Primers; Humans; LightCycler; Nested PCR; Polymerase chain reaction; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Rapid-cycle PCR; Sensitivity and Specificity; Translocation, Genetic; Tumor Cells, Cultured; Two-round PCR
• ISSN: 1386-6532; 1873-5967
• DOI: 10.1016/S1386-6532(00)00157-8

Background: For many diagnostic applications, the specificity and sensitivity 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™ instrument, amplification products must be collected from the capillaries by centrifugation, a procedure thought to be particularly prone to product carry-over. Objective: Development of a technique to perform two-round PCR with the LightCycler™ instrument in a single closed capillary. Study design: Silicone oil was used to separate the second-round primers from first-round PCR mixture during the first-round PCR. The feasibility of the principle was demonstrated using a semi-nested primer system for the PCR analysis of genomic DNA. The first-round PCR reaction mixture was loaded into the capillary and covered by oil. Then, the second-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 PCR mixture with the first-round products. Amplified products were visualized by fluorescence melting curve analysis. Results: When a dilution series of genomic DNA was used for the single-capillary two-round PCR, 0.1 ng of DNA could consistently be detected. This was a 10-fold increase of sensitivity in comparison with single-round PCR. With the new technique, the first-round reaction mixture was sufficiently separated from second-round primers by the oil layer. Conclusions: Two-round PCR on the LightCycler™ using a single closed capillary excluded the possibility of amplification product carry-over. This new technique can easily be adapted for numerous applications, and should show feasibility for many nested primer PCR applications currently in use to the clinical detection of virus-derived DNA.