Nucleic acid probes and methods to detect and/or quantify nucleic acid analytes

A method for the detection or quantification of a nucleic acid analyte comprising the steps of:

• a. providing a nucleic acid probe, wherein said nucleic acid probe is comprised of a nucleic acid that is derivatized with two non-identical covalently attached dyes, of which at least one dye is fluorescent, and of which one dye is a pH-sensitive dye, wherein the dyes are attached at either the same or at adjacent nucleotides of the nucleic acid probe;
• b. contacting said nucleic acid probe with a nucleic acid analyte so as to allow for the hybridization of the nucleic acid probe with the nucleic acid analyte;
• c. removing the unhybridized nucleic acid probe from the mixture;
• and
• d. measuring the fluorescence of the hybridized nucleic acid probe under one or more conditions where the pH of the medium is defined.

A method of preparing nucleic acid probes, said probes comprised of a nucleic acid that is derivatized with two or more non-identical covalently attached dyes comprising the steps of:

• a. providing an oligonucleotide that is derivatized with a diene-moiety and an amino group;
• b. reacting the oligonucleotide with a first dye that is derivatized with a dienophile-moiety;
• and
• c. reacting the oligonucleotide with a second dye that is derivatized with an amine-reactive moiety.

A method of preparing an oligonucleotide that is derivatized with a diene moiety and an amino group comprising the steps of:

• a. synthesizing an oligonucleotide sequence using solid phase phosphoramidite synthesis cycles;
• b. conducting a last phosphoramidite synthesis cycle wherein a phosphoramidite is applied in the coupling step of the synthesis cycle that carries a diene-moiety and a protected amino group;
• and
• c. deprotecting the oligonucleotide.