Improved FRET process

摘要

A pair of Fluorescence Resonance Energy Transfer (FRET) hybridization probes comprising nucleotide sequence entity that is complementary to the sequence of the target nucleic acid, fluorescent entity that can be FRET donor entity or FRET acceptor entity, and spacer entity connecting the nucleotide sequence entity and the fluorescent entity, is new. A pair of Fluorescence Resonance Energy Transfer (FRET) hybridization probes comprising nucleotide sequence entity that is complementary to the sequence of the target nucleic acid, fluorescent entity that can be FRET donor entity or FRET acceptor entity, and spacer entity connecting the nucleotide sequence entity and the fluorescent entity, is new. The spacer entity comprises a connecting chain of >=15 atoms, where 2 atoms are carrying negatively charged substituents. Independent claims are also included for: (1) a composition comprising a nucleic acid sample and a pair of hybridization probes; (2) a kit comprising a pair of hybridization probes and nucleic acid amplification primers, template dependent nucleic acid polymerase, deoxynucleoside triphosphates and/or buffer for template dependent nucleic acid amplification reaction; (3) qualitative or quantitative detection of a nucleic acid sequence in a biological sample, where the nucleic acid present in the sample is hybridized with a pair of FRET hybridization probes; (4) determining the melting profile of a hybrid consisting of a target nucleic acid and a pair of FRET hybridization probes, where the fluorescence emission is determined as a function of temperature; (5) chemical solid phase synthesis of multiple oligonucleotides comprising preparing a dye labeled CPG- (N)n, where N is arbitrarily chosen nucleotide residues different from G and n = 1-10, solid phase synthesis of a first oligonucleotide having a first sequence using the CPG, and solid phase synthesis of at least a second oligonucleotide having a second sequence using the CPG.