................................................................................................................................................. 7 Introduction ........................................................................................................................................... 9 Materials and Methods ....................................................................................................................... 15 1. Determination of the optimal length of probe DNA strands that can be employed for Rth measurements. ................................................................................................................................... 15 1.1. Immobilization of probe ssDNA ............................................................................................ 15 1.2. Evaluation of the functionality of the attached probe ssDNA ............................................... 16 2. PKU mutation analysis in the exons of the gene for PAH ............................................................ 17 2.1 Functionalization of NCD with 3 exons of the PAH gene ...................................................... 17 2.2 Hybridization with synthetically produced fully complementary (wildtype) and mutated target ssDNA ................................................................................................................................. 18 2.3. Rth measurements .................................................................................................................. 18 2.4 Preparation of target ssDNA from real samples ..................................................................... 19 2.5 Hybridization with wildtype target ssDNA from healthy donors ........................................... 21 2.6. Rth measurements with healthy donor material ..................................................................... 22 3. Surface plasmon resonance (SPR) ................................................................................................ 22 Results and Discussions ...................................................................................................................... 23 4. Determination of optimal length of probe DNA strands that can be employed for Rth measurements .................................................................................................................................... 23 5. PKU mutation analysis in the exons of the gene for PAH ............................................................ 24 6. Preparation of target ssDNA from clinical patient samples .......................................................... 27 6.1 Primer design .......................................................................................................................... 27 6.2 Determination of optimal annealing temperature and Mg concentration ............................. 28 6.3 Generation of ssDNA from dsDNA PCR products ................................................................. 29 7. Rth measurements with real samples ............................................................................................ 33 8. Surface Plasmon Resonance ......................................................................................................... 34 Summary and Conclusion .................................................................................................................. 37 References ............................................................................................................................................ 39 Supplemental Information ................................................................................................................. 41