title: Single-tube two-round polymerase chain reaction using the LightCycler instrument.
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.
  
  
  METHODS
  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.
authors.affiliation: Institute for Laboratory Medicine, General Hospital Linz, Krankenhausstr. 9, A-4020 Linz, Austria. joerg.berg@akh.linz.at
authors.forename: J
authors.initials: J
authors.lastname: Berg
authors.forename: V
authors.initials: V
authors.lastname: Nagl
authors.forename: G
authors.initials: G
authors.lastname: Mühlbauer
authors.forename: H
authors.initials: H
authors.lastname: Stekel
mesh_terms: D004247:DNA / Q000032:analysis; D017931:DNA Primers; D006801:Humans; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D012680:Sensitivity and Specificity; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D004247:DNA