Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers.
Surface plasmon field-enhanced fluorescence spectroscopy was employed to extensively investigate the hybridization behaviors of polymerase chain reaction (PCR) amplicons on a peptide nucleic acid (PNA) or DNA probe layer that was previously attached on a streptavidin-modified gold surface via biotin/streptavidin interaction. Despite the neutral backbone of PNA, the hybridization reactions were strongly influenced by the variation of ionic strength. The association rates exhibited a monotonic decrease with ionic strength increase and the maximum hybridization signal was achieved at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA/DNA electrostatic barrier, particularly in lower ionic strength range (e.g., 10 approximately 150 mM Na(+)). The electrostatic cross talk was shown to be largely reduced if the PNA probe layer was sufficiently diluted by following a strategic templated immobilization method. As a consequence, a pseudo-first-order kinetic model was applicable to describe the hybridization kinetics, and affinity constants were derived for evaluating the influence of single nucleotide polymorphisms (SNPs).
Yao
Danfeng
D
Max-Planck-Institute for Polymer Research, 55128 Mainz, Germany.
Kim
Junyoung
J
Yu
Fang
F
Nielsen
Peter E
PE
Sinner
Eva-Kathrin
EK
Knoll
Wolfgang
W
D001703:Biophysics / Q000379:methods; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D015342:DNA Probes / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D007477:Ions; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012964:Sodium / Q000737:chemistry; D055672:Static Electricity; D019809:Streptavidin / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*; D013499:Surface Properties; D013696:Temperature; D013997:Time Factors
D015342:DNA Probes; D007477:Ions; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D010455:Peptides; D001710:Biotin; D019809:Streptavidin; D012964:Sodium