ABSTRACT
It has been very difficult to detect and quantify multiple somatic mutations simultaneously in single-tube qPCR. Here, a novel method for simultaneous detection of multiple mutations and a melting curve analysis was developed by using clamping PNA and detection PNA probes. Each PNA probe was designed to have a specific melting temperature by the introduction of γ-PNA monomer. This technique was successfully applied to the detection of six genotypes in two different mutations of K-RAS at the same time. Such simultaneous analysis of an amplified curve and a melting curve in qPCR can be widely used for the early diagnosis of cancer and determining the prognosis of drug treatments.
Subject(s)
Genes, ras/genetics , Mutation , Peptide Nucleic Acids/genetics , Polymerase Chain Reaction/methods , Alanine/chemistry , Cell Line , Dimerization , Genotyping Techniques/methods , HeLa Cells , Humans , Lysine/chemistry , Molecular Probes , TemperatureABSTRACT
Peptide nucleic acids (PNAs) are DNA analogues in which the nucleic acid backbone is replaced by a pseudopeptide backbone and nucleobases are attached to the backbone by methylene carbonyl linkers. γ-Carbon modification of the PNA structure allows monomers, and subsequently oligomers, with improved properties to be obtained. In this study, we report the convenient synthesis of γ-lysine-modified PNA monomers for pyrimidine bases (thymine and cytosine) with high optical purity (> 99.5%) and direct enantiomer separation of γ-lysine-modified PNA analogs, using chiral HPLC to determine the optical purity.
Subject(s)
Lysine/chemical synthesis , Peptide Nucleic Acids/chemical synthesis , Chemical Fractionation/methods , Chromatography, High Pressure Liquid , Isomerism , Lysine/analogs & derivatives , Molecular StructureABSTRACT
Synthesis of self-activated peptide nucleic acid (PNA) monomers and an efficient method for PNA synthesis using a benzothiazole-2-sulfonyl (Bts) group as an amine-protecting group as well as an acid-activating group are reported. Couplings were complete within 120 min, and the deprotection was performed in 10 min. This Bts strategy provides a high purity PNA oligomer and is appropriate for large-scale synthesis. The results of the 15-mer PNA oligomer are described.
Subject(s)
Peptide Nucleic Acids/chemical synthesis , Amides/chemistry , Combinatorial Chemistry TechniquesABSTRACT
We present a way to improve the dispersion tolerance of an electrical-binary-signal-based duobinary transmitter, implemented by using a dual-arm Mach-Zehnder modulator driven with two complementary binary signals. Successful transmission over 200 km of single- mode fiber is achieved by optimizing the relative time delay between the binary signals and the driving voltage.
