LightCycler qPCR optimisation for low copy number target DNA

Abstract The LightCycler is a rapid air-heated thermal cycler which incorporates a fluorimeter for the detection and quantification of Polymerase Chain Reaction (PCR) amplified products. It provides real-time cycle-by-cycle analysis of product generation. Amplification occurs in glass capillary tubes. The products are detected using a fluorescent double stranded DNA binding dye or fluorescent probes. However, conditions that work well in conventional PCR reactions do not readily translate to the LightCycler. Whilst using this new technology to study an infectious pathogen in human tissue samples, several parameters were identified which can have an adverse effect on the reliable and reproducible quantification of low copy number target DNA. They included abstraction of PCR reagents on glass, primer–dimer formation, non-specific product generation, and a failure to amplify low copy number target when it is present in a high background of human chromosomal DNA. For each problem identified, several solutions are described. Novel approaches are also described to ensure that amplification of target DNA and of the quantification standards occurs with the same efficiency. With appropriate changes to the protocols currently in use, LightCycler quantitative Polymerase Chain Reaction (LC-qPCR) can be used to achieve a level of accuracy that exceeds that of an enzyme immunoassay. The LC-qPCR optimisation strategies described are of particular relevance when applying this technology to the study of pathogens in tissue samples. The technique offers the enormous potential for reliable and reproducible quantitative PCR of low copy number target DNA.