A PCR amplification product is detected, in quantitative determination of nucleic acid and measurement of the number of bacterial cells or specific genes, by addition of a dye compound which does not fluoresce in the free state but fluoresces in the bonded state to a double-stranded nucleic acid.
대표청구항▼
A method for quantifying a target nucleic acid, comprising the steps of: conducting PCR of a nucleic acid sample with a primer set required for amplification of a specific base sequence region of the target nucleic acid to provide a double stranded nucleic acid; reacting the double-stranded nucleic
A method for quantifying a target nucleic acid, comprising the steps of: conducting PCR of a nucleic acid sample with a primer set required for amplification of a specific base sequence region of the target nucleic acid to provide a double stranded nucleic acid; reacting the double-stranded nucleic acid with a dye compound represented by the following formula to bind the dye compound to the double stranded nucleic acid; and measuring intensity of the fluorescence from the dye compound bound to the double-stranded nucleic acid to quantitatively determine the target nucleic acid in the sample, [Figure] [I] wherein [Figure] is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te; R1 and R2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl; R3 is a group of －A or －L－A wherein L is －L1－, L2－L3－, or －L4－L5－L6－, L1 to L6 being independently －(CH〓CH)－, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or －CH〓R4－, wherein R4 is a cyclic structure having an oxo group; A is a substituted or unsubstituted aryl group, or －CH〓R5, wherein R5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring; in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R1, R2, or R3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group; and Y-is an anion.
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