Use of Real-Time Quantitative PCR to Compare DNA Isolation Methods

Abstract

PCR-based diagnostics of genomic DNA and DNA from viruses, microorganisms, or tumor cells are rapidly becoming routine practice in the clinical laboratory. DNA isolation is usually the first step toward detection. Because of expanding numbers of DNA isolation methods, the choice between methods is becoming increasingly difficult. Especially when a low amount of target DNA is present, when only a limited amount of clinical sample is available, or when PCR inhibitors are expected to be present, isolation efficiency, repeatability, and removal of PCR inhibitors by the isolation method become critical factors, seriously affecting the final outcome of the experiment. Recently, a real-time quantitative PCR system [ABI PrismTM 7700 Sequence Detection System, Perkin-Elmer Applied Biosystems (PE)] was developed. Quantification is automatically performed in the exponential phase of the PCR reaction, where there is a linear relationship between the log of input target DNA quantity and the number of PCR cycles to reach an arbitrary fluorescence threshold (1)(2). The objective of this study was to evaluate this PCR system for standardization of DNA isolation methods. Therefore, we added a known amount of marker DNA copies, close to the detection limit of the PCR, to plasma, which we chose because of recent developments in diagnostic molecular oncology (3)(4). After isolating the DNA with four different methods, we used the PCR system to determine isolation efficiency and repeatability of each method. Blood from five healthy donors was collected in 10-mL EDTA tubes. Plasma was pooled and stored at −80 °C. A 12.8-kb plasmid containing a cDNA sequence construct of the bcr-abl gene translocation was used as marker DNA. The plasmid was linearized with a restriction enzyme, cleaned using a QIAquick column (QIAGEN), and quantified using GeneQuant II RNA/DNA calculator (Pharmacia). A 10-fold serial dilution of the marker DNA …