Label-free characterisation of oligonucleotide hybridisation using reflectometric interference spectroscopy.
The potential of a label-free detection method, reflectometric interference spectroscopy (RIfS), for temperature-dependent DNA hybridisation experiments (for example in single nucleotide polymorphism (SNP) analysis) is investigated. Hybridisations of DNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA) to a single stranded DNA were measured for several temperatures, and the melting curves and temperatures were calculated from the changes in optical thickness obtained. These measurements were performed by hybridising surface-immobilised single stranded oligomers with their complementary ssDNA or with ssDNA containing SNPs at different temperatures. DNA was compared to its analogue oligomers PNA and LNA due to their stability against nuclease. A comparison of melting temperatures demonstrated the higher binding affinities of the DNA analogues. Moreover, a continuous melting curve was obtained by first hybridising the functionalised surface with its complementary DNA at room temperature and then heating up in-flow. Measurement of the continuous melting curve was only possible due to the insensitivity of the RIfS method towards temperature changes. This is an advantage over other label-free detection methods, which are based on determining the refractive index.
Pröll
Florian
F
Institute of Physical and Theoretical Chemistry, Eberhard-Karls-University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany. florian.proell@ipc.uni-tuebingen.de
Möhrle
Bernd
B
Kumpf
Michael
M
Gauglitz
Günter
G
D001483:Base Sequence; D007368:Interferometry / Q000379:methods; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020135:Peptide Nucleic Acids / Q000737:chemistry; D013057:Spectrum Analysis / Q000379:methods*; D044366:Transition Temperature
D020135:Peptide Nucleic Acids