ABSTRACT

I. Introduction 112

II. Materials and Methods 114

A. Primers, Molecular Probes, and Target Oligonucleotides 114

B. DNA Templates and PCR Conditions 114

C. SPR-Based BIA 114

1. Protocol A: Determination of the Optimal Conditions for Hybridization, Including Detection of Point Mutations 115 2. Protocol B: Immobilization of Target PCR Products and Injection of Molecular Probes 116 3. Protocol C: Immobilization of Molecular Probes and Injection of Target Asymmetric PCR Products 116

III. Results 116

A. Choice of Primers and Probes 116

B. Characterization of PCR Products Immobilized on Flow Cells 117

C. Use of Peptide Nucleic Acids as Molecular Probes 118

D. Injection of Asymmetric PCR Products to Flow Cells Carrying Specific Probes 119 IV. Discussion 119

A. Advantages of SPR-Based BIA for Characterization of PCR Products 119

B. Problems and Possible Solutions 119

C. Future Perspectives 121

Acknowledgments 121

References 121

I. INTRODUCTION

Surface Plasmon Resonance (SPR) and biosensor technologies for biospecific interaction analysis (BIA)1-3 enable the monitoring of DNA:DNA and DNA:RNA hybridization in real time.3,4 In the BIAcore biosensor system (Figure 14.1A), plane polarized light is reflected from the gold-coated sensor chip (Figure 14.1B), where the molecular interactions take place.3 Surface plasmon resonance in the gold layer results in the extinction of the reflected light at a specific angle. This angle (SPR angle) varies with the refractive index of the solution close to the other side of the sensor chip.