Method development and evaluation

Quantitative real time PCR

Amplification plot: Relative fluorescence vs. cycle number (Graphic: C.Wolf)

Real-time polymerase-chain-reaction, also known as qPCR, is widely used in environmental analysis to obtain reliable and accurate qualitative and quantitative information on specific phytoplankton species in field samples. Quantification of nucleic acids is performed during the exponential phase of the PCR, avoiding carryover problems of standard end-point PCR analysis. Amplicon formation is detected by measuring a fluorescence signal that increases proportional to the starting quantity of the target molecule. Fluorescence is generated either by intercalating dyes or sequence-specific fluorescence labeled probes. The amount of target molecules is calculated by using standard reference curves. Real-time PCR is an effective and sensitive alternative or complement to e.g. traditional microscopy cell counts, as they reduce person to person variability, time and ultimately cost.

AutoFiM - Automatic filtration system for marine microbes

AutoFiM connected to the ships pump system on RV Polarstern. (Image: J.Hessel)

The newly developed automated and remote-controlled filtration system for marine microbes (AutoFiM) allows high resolution collection and filtration of marine or freshwater samples at defined time intervals or at defined stations for further molecular analysis of e.g. microalgae. Up to 5 Liters could be sampled and filtrated. In total 12 filters could be stored for about one week in e.g. lysis buffer until the sample plate has to be replaced. To date AutoFiM is operated on RV Polarstern to examine its applicability on expeditions and its long-term performance. AutoFiM works autonomous without persistent attendance of a scientist and can therefore reduce cost and time and render the whole microalgae treatment economically feasible.  In the future AutoFiM should be expand by an ultrasound module for generating cell extracts and free nucleic acids subsequent to the filtration procedure.

 

 

Next Generation Sequencing

Next Generation Sequencing: 454-Pyrosequencing (Graphic: C. Wolf)
Next Generation Sequencing: 454-Pyrosequencing (Graphic: C. Wolf)

In the past decade, the so called next-generation sequencing (NGS), such as Illumina sequencing, has revolutionized our understanding and interpretation of the unseen diversity of unicellular eukaryotic organisms. Especially the identification of pico-eukaryotes and very rare abundant species is now feasible which are hardly detectable with conventional methods such as light microscopy. The new sequencing techniques allows a relatively cost and time saving, massive-parallel analysis of DNA samples. During one Illumina sequencing cycle, millions of short and mostly error-free DNA fragments are produced. This flood of information has to be compressed and further quality checked before the sequences can be assigned to protist taxa. Our group is specialized on sequence processing strategies and analysis of 18S rRNA gene sequences from samples taken in the North Sea, Fram Strait and Central Arctic Ocean.

Nucleic acid biosensor

Detection principle of the nucleic acid biosensor: Sandwich hybridization (Graphic: J.Hessel)

The nucleic acid biosensor module ALGADEC is used for autonomous, highly sensitive and quantitative detection of microalgae in marine water samples including relevant toxic algae species. The detection principle is based on sandwich hybridization involving two labeled, molecular species-specific probes binding to the ribosomal RNA of the target organism of interest. The hybridization reaction takes place on a sensor chip with 8 carbon electrodes. If hybridization is successful a redox-reaction of an antibody enzyme complex is followed that can be measured as an electrochemical signal. This signal can further be used to quantify the cell numbers of the species based on calibration curves. The supply of the different solutions into the hybridization chamber as well as the single detection steps are conducted automatically through the software controlled microfluidic system. For the development and optimization of further biosensor devices, evaluation is done concerning the general sensor design including the construction of the hybridization chamber and the microfluidic system and concerning the optimal chip design.