For many years we have been developing software for medical and biological research and can look back on many successfully completed projects, for example in the fields of cell biology, laboratory device development, molecular biology, medical research and bioinformatics.
Project examples
One of our most recent projects in this field is a three-year research project that started on 1 February 2021 and is funded by the Federal Ministry of Education and Research. The research consortium consists of five partners: a research organisation for applied research, commercial companies and a research institute.
The overall aim of the project is to research a novel platform for digital multiplex assays. The new device is intended to make it possible to analyse a large number of DNA segments (DNA sequences) in a sample particularly quickly, cost-effectively and with unprecedented complexity (degree of multiplexing). To determine which DNA segments are present in the sample and in what quantity, fluorescent molecules (fluorophores) are used to distinguish the DNA. The laboratory device will use digital PCR (dPCR) as its basic method.
Polymerase chain reaction (PCR)
The polymerase chain reaction is a standard method in molecular biology for amplifying DNA. In many successive cycles, the desired DNA segment is amplified in a thermocycler using the enzyme DNA polymerase. The reaction is called a chain reaction because the DNA that has already been amplified always serves as the starting material for the new, exponential amplification cycles (see figure).
Since the biochemist Kary Mullis invented PCR in 1983, it has been used as one of the most important standard methods in basic research in molecular biology as well as in everyday laboratory work. Examples include cloning, the identification of hereditary diseases and the detection of viral infections. It is no coincidence that Mullis received the Nobel Prize in Chemistry in 1993 for developing PCR.
Quantitative real-time polymerase chain reaction (qRT-PCR): analysis method for detecting COVID-19 infections
Especially since the start of the worldwide coronavirus pandemic in 2020, the polymerase chain reaction has become a familiar term to the general public around the world. Since the start of the pandemic, qRT-PCR (quantitative real-time PCR), a special form of PCR, has been used to test for SARS-CoV-2. At present, it is the most precise and least expensive known method for testing patients for a CoV-2 infection.
An extracted DNA sample is placed in a reaction tube together with other reaction solutions and put into a qRT-PCR device. In real-time PCR, the device measures fluorescence during the polymerase chain reaction whenever fluorescence-labelled DNA probes detect the presence of a specific target DNA sequence during the reactions.
Since the target DNA, such as a DNA sequence of SARS-CoV-2, is continuously amplified in a reaction tube during a qPCR, the intensity of the emitted fluorescence also increases over time as the amount of DNA rises.
By comparing the fluorescence intensity of reference samples with that of the sample under examination, the presence and the amount (quantity) of a target DNA sequence in the sample can be calculated once the measurements are complete.
Since this method does not measure the amount of DNA directly but only determines it via reference samples, it is not an absolute determination of quantity (quantification) but a relative one.
Digital polymerase chain reaction (dPCR)
Like qRT-PCR, the digital polymerase chain reaction (dPCR) is used to quantify a target DNA sample. Before the analysis, the reaction mix is partitioned into thousands of droplets or compartments, which are read out individually after the analysis. This enables a very precise analysis and an absolute quantification of DNA sequences. In dPCR, too, the DNA polymerase amplifies the DNA under examination, but in this case individually in each droplet or compartment of the reaction mix. Whether the target DNA sequence is present, and in what quantity, is determined by the presence of fluorescence.
Since the amplification of the DNA is assumed to follow a Poisson distribution, i.e. the amplification of the DNA proceeds uniformly over time, the amount of DNA in the entire initial sample can be determined by counting the fluorescent and non-fluorescent droplets and the total number of droplets.
Unlike qRT-PCR, dPCR is much less sensitive to factors that inhibit the PCR reaction (PCR inhibitors), does not depend on reference samples for quantification and can achieve a considerably higher detection limit. The detection limit of a measurement method indicates the extreme value at which a measured quantity can still just be detected. Although dPCR can deliver much more accurate measurements of DNA quantities, its major drawback so far has been that dPCR instruments are considerably more expensive than qRT-PCR devices, which is why the latter are mostly used for measurements. This is exactly where our research project FREEDOM comes in (see above).
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