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Capillary electrophoresis with laser-induced fluorescent detection (CE-LIF) as a novel tool in molecular genetic analyses of human disease

Objective

- To construct an ultrasensitive, compact laser-induced fluorescence (ucLIF) detector for capillary electrophoresis (CE)
- To optimise the instrument for detection of mutations, and validate the new technology by comparison with conventional techniques based on gel electrophoresis
- To reengineer the ucLIF to be integrated with the modular CE apparatus from PRINCE Technologies.

The small sample volumes (nanoL range) analysed by the powerful technique capillary electrophoresis put great demands on the performance of the detector system, and the most sensitive detection is afforded by laser-induced fluorescence (LIF). A ultrasensitive compact LIF-detector will be constructed, based on sheath flow technology and confocal optics. The detector is specifically adapted to the modular CE instrument (PRINCE) from PRINCE Technologies. The instrument will be optimised for the quantitative detection of a)PCR amplification products, b) precolumn hybridisation products c) and for the identification of mutations by primer extension or SSCP. These techniques will in turn will be used for the detection of mutations in enzymes methylenetetrahydrofolate reductase (MTHFR) and cystathionine B-synthase (CBS), which have recently been demonstrated to occur with high frequency in patients with early onset cardiovascular disease and hyperhomocysteinemia. This will form a platform for the validation and demonstration of the potential of the CE-ucLIF technology for the analyses of mutations in the human genom. Finally, the prototype (version 2) will be reengineered to be integrated and interfaced with the PRINCE modular CE apparatus, and the performance of this reconstructed unit will be validated by comparison with the prototype upon which this demonstration was carried out.

Funding Scheme

DEM - Demonstration contracts

Coordinator

Universitetet i Bergen
Address
Armauer Hansens Hus
5021 Bergen
Norway

Participants (3)

Katholieke Universiteit Nijmegen - Stichting Katholieke Universiteit
Netherlands
Address
18,Geert Grooteplein Zuid 18
6500 HB Nijmegen
Prince Technologies BV
Netherlands
Address
7 B,handelsweg 7 B
7826 TC Emmen
THE PROVOST, FELLOWS AND SCHOLARS OF THE COLLEGE OF THE HOLY AND UNDIVIDED TRINITY OF QUEEN ELIZABETH NEAR DUBLIN HEREINAFTER TRINITY COLLEGE DUBLIN
Ireland
Address
Smurfit Institute,trinity College
2 Dublin