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Validation of an innovative method for the DNA isolation for quantitative PCR applications

01.06.2010

Polymerase chain reaction (PCR) is a well established technique to amplify specific sequences of a DNA strand in vitro. It enables researchers to produce millions of copies of a particular fragment of DNA within a short time for the subsequent detection, analysis and application by usual laboratory methods. Theoretically, a single molecule is sufficient for this approach, making PCR one of the most sensitive analytical methods.

The principle of PCR is based on repeated thermal cycles, consisting of an  enzymatic DNA replication and leading to an exponential amplification (“chain reaction”). Compared to the qualitative detection of DNA, the quantitative or real-time PCR permits additionally the quantification of formed DNA molecules during the amplification process. For the detection of products in real-time PCR either non-specific fluorescent dyes incorporating in any double-stranded DNA or sequence-specific DNA probes labelled with a fluorescent reporter are applied.

Before amplification by means of PCR, DNA has to be isolated from the respective biological sample and purified. However, the recommended DNA isolation method is time-consuming and work-intensive and requires a multitude of extraction and centrifugation steps.

The aim of the research project, performed in collaboration with Dr. Lerche KG, is the proof of an enhanced specificity and sensitivity of polymerase chain reactions by applying an innovative method for the isolation and purification of DNA from human whole blood. This procedure is based on the separation principle using size exclusion chromatography with new porous substrates. Perturbing impurities introduced by other DNA isolation procedures are expected to be eliminated and influences on the specificity of the PCR by cross reactivities are avoided. For the first time, sensitivity and specificity of the new procedure to usual preparations will be compared quantitatively by real-time PCR. To validate this innovative technology, the recovery rate of a defined number of DNA copies will be determined by employing in-house developed flow cytometric reference procedures for the determination of cell concentrations and cell sorting.

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