Thesis presented November 05, 2009

Abstract:
This Ph.D. thesis studies phenomena concerning DNA duplex stability. Exploiting a real-time detection system based on Surface Plasmon Resonance imaging (SPRi), we use a temperature scan method to analyze DNA interactions on functionalized biochips. First, a detailed study about a comparison of two DNA immobilization methods and the influence of different buffer components on surface hybridized DNA duplexes is presented. Especially the influence of salt and denaturing agents in the buffer are discussed. Second, we apply the temperature scan method to point mutation detection, as well for oligonucleotides as for longer DNA or RNA targets produced by Polymerase Chain Reaction (PCR) or Nucleic Acid Sequence Based Amplification (NASBA) amplification protocols, respectively. Competition between targets containing point mutations and surface hybridization is addressed. Also, secondary structures in solution may alter target capture on probes. For the isothermal NASBA amplification, we show that it is possible to make an integrated system with amplification and detection on the SPRi chip. Finally, a study of DNA lesions and repair enzymes is presented in collaboration with the 'Laboratory of Nucleic Acids Lesions' (LAN) at the CEA Grenoble profiting once again from the flexible temperature regulation to characterize enzyme activity on surface grafted DNA.

Keywords:
Surface Plasmon Resonance imaging (SPRi), temperature scans, point mutation, thiol self-assembling, DNA lesions, Base Excision Repair (BER)

On-line thesis.