Microfluidic integration of spatially confined replication

An important issue in PACE was to perform spatial resolved on-chip replication. Therefore two kinds of gels were tested. The first is the so-called Pluronic® (BASF), a triblock copolymer of the type (ExPyEx) with E, P, and subscript denoting oxyethylene, oxypropylene and segment length respectively. We chose two different Pluronic® block copolymer surfactants for our tests: F127 (E99P69E99) and the F87 (E61P40E61). The advantage in terms of microfluidic integration is, that these kinds of hydrogel monomers are reversible thermo-responsive gels. At low temperatures the dissolved materials acts as a free flowing solution while at elevated temperatures self assembled micelles form a quasi-lattice with a face-centered cubic structure leading to an increase in viscosity and hence to the formation of a gel. Furthermore, Pluronic® F127 is known to be a good separation medium for electrophoresis. Both the autocatalytic amplification and the thioDNA replication were tested in F127 as well as F87 using different concentrations (10%, 15%, 20%, 25%, 30% w/v). The use of Pluronic® as a reaction matrix to establish spatially confined replication, leads to a strong bleaching of the fluorophores attached to the oligonucleotides concerned. 


To solve this problem we chose another gelling agent agarose (Super LM, Fa. Roth) with a gelation temperature ≤ 20°C and a melting-temperature ≤ 62°C. Pre-experiments in capillaries showed that the ligation process in thioDNA replication works well in 1.5% w/v agarose gel.


Thiol_cycle_spatially

The opposite curve plot represents the ligation-step of the thioDNA replication. All components (3 Oligonucleotides: Alexa488Dabcyl-9mer, Template 18mer, Thiooligonucleotide-9mer) are mixed with the agarose-gel (1.5 % w/v in Hepes-buffer 0.2 mM) at 60°C. At this elevated temperature the melted agarose is liquid and the reaction cannot take place as no hybridisation occurs. Cooling down the sample to 20°C leads to the start of the reaction. The increase in fluorescence intensity can be traced back on the ligation as well as on the temperature-dependency of the fluorophores. A repeated increase of temperature to 60°C shows the increase in intensity just for the ligation.


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