Development of microscale enzymatic ligation amplification system

The aim of our work was to set up a chemical system that allows linear or exponential amplification of short oligonucleotides under isothermal conditions. This system was designed as a enzymatic test system for replication in a microfluidic structure.


The system consists of a 22mer template A’B’C’ and four oligonucleotides as substrates (two octamers: A, A’ and two 14mers BC, B’C’). The reaction scheme is shown in the following scheme.

Enzymatic_ligation
The first step in this reaction is the enzymatic ligation of the terplex A/A’B’C’/BC. After the Duplex ABC/A’B’C’ has been formed, the recognition sequence which is in part on A and in part on B is complete and the nicking enzyme cleaves the ABC-strand to form the products AB and C. AB serves in the third step as template for the ligation of A’ and B’C’ leading to new template A’B’C’.

The different steps of the reaction cycle were at first investigated by use of Capillary Gel Electrophoresis (CGE) to validate that the reactions work and to find the best conditions (temperature, buffer, concentrations, etc.) for these reactions. It was possible to get qualitative and also some quantitative results for each reaction of the amplification. It was also shown that both enzymes work together to get kinetics of a two-step-process (I. Ligation + II. Nicking) of the scheme shown above. Up to know it was not possible to prove, that the whole amplification cycle works the way it was desired. 
Enzymatic_ligation-microfluidic
The two-step-process (I. Ligation + II. Nicking) was carried out in microfluidic structures (see picture above). For the detection the oligonucleotide BC was internally labelled with an Alexa488-fluorophore in part B and at the 3’-end of C with the quencher Dabcyl. It was proved that it is possible to follow the reaction as the detected fluorescence increases with time. This increase is only possible when both reactions take place as this leads to the Products AB and C where the Alexa-labelled part and the part with the quencher are separated. 



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