Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Berg, J; Nagl, V; Mühlbauer, G; Stekel, H.
Single-tube two-round polymerase chain reaction using the LightCycler instrument.
J Clin Virol. 2001; 20(1-2):71-75
Doi: 10.1016/S1386-6532(00)00157-8
Web of Science
PubMed
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- Führende Autor*innen der Med Uni Graz
- Berg Jörg
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- Abstract:
- 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.
- Find related publications in this database (using NLM MeSH Indexing)
- DNA - analysis
- DNA Primers -
- Humans -
- Polymerase Chain Reaction - instrumentation Polymerase Chain Reaction - methods
- Sensitivity and Specificity -
- Translocation, Genetic -
- Tumor Cells, Cultured -
- Find related publications in this database (Keywords)
- polymerase chain reaction
- two-round PCR
- nested PCR
- LightCycler (TM)
- rapid-cycle PCR