Abstract Assay for detecting nucleic acid sequence, which comprises the steps: [A] contacting (1) a target single-stranded nucleic acid, (2) a pair of nucleic acid primers having nucleotide sequence complementary to the target nucleic acid and its complementary chain respectively, (3) a fluorescent-labeled nucleic acid probe which has a nucleotide sequence designed so as to be hybridized to the target nucleic acid downstream (at the side of 3'-terminus) from where the nucleic acid primer is hybridized and has a modified 3'-end so that the nucleotide chain is not elongated by a nucleic acid polymerase, and (4) a nucleic acid polymerase having exonuclease activity specific to a double-stranded nucleic acid in the presence of substrates for the enzyme (four kinds of nucleoside triphosphate), and thereby elongating the said nucleic acid primer chain and simultaneously hydrolyzing only the fluorescent-labeled nucleic acid probe hybridized to the target nucleic acid; [B] denaturating the chain-elongated product of primer to a single-stranded form; [C] amplifying the target nucleic acid by repeating the steps A and B; and [D] determining the change in fluorescence polarization resulted by the hydrolysis of the probe. This method is effective to detect the target nucleic acid sequence more simply with higher sensitivity and higher reliability in comparison with known methods, and further has advantages that because of no carryover or contamination of the amplified nucleic acids into subsequent reactions, undesirable false positive result can be avoided.
An assay method for detecting nucleic acid sequence, which comprises the steps: [A] contacting (1) a target single-stranded nucleic acid, (2) a pair of nucleic acid primers having nucleotide sequence complementary to the target nucleic acid and its complementary chain respectively, (3) a fluorescent-labeled nucleic acid probe which has a nucleotide sequence designed so as to be hybridized to the target nucleic acid downstream (at the side of 3'-terminus) from where the nucleic acid primer is hybridized and has a modified 3'-end so that the nucleotide chain is not elongated by a nucleic acid polymerase, and (4) a nucleic acid polymerase having also an exonuclease activity specific to a double-stranded nucleic acid in the presence of four kinds of nucleoside triphosphate which are a substrate for the enzyme, and thereby elongating the said nucleic acid primer chain, and simultaneously hydrolyzing only the fluorescent-labeled nucleic acid probe hybridized to the target nucleic acid by the exonuclease activity of said nucleic acid polymerase, [B] denaturating the chain-elongated product of primer to a single-stranded nucleic acid, [C] amplifying the target nucleic acid by repeating the above steps [A] and [B] the desired times, [D] determining the change in fluorescence polarization resulted by the hydrolysis of the fluorescent-labeled nucleic acid probe.
Claim 2
Dependent Claim
27 words
The assay method according to claim 1, wherein the target nucleic acid is DNA, and the nucleic acid moiety of the fluorescent-labeled nucleic acid probe is DNA.
Claim 3
Dependent Claim
37 words
The assay method according to claim 1, wherein the target nucleic acid is a cDNA which is derived from RNA by a reverse transcriptase, and the nucleic acid moiety of the fluorescent-labeled nucleic acid probe is DNA.
Claim 4
Dependent Claim
24 words
The assay method according to claim 1, wherein the fluorescent-labeled nucleic acid probe is a DNA probe which is labeled with a fluorescein derivative.
Claim 5
Dependent Claim
14 words
The assay method according to claim 4, wherein the fluorescein derivative is fluorescein isothiocyanate.
Claim 6
Dependent Claim
15 words
The assay method according to claim 1, wherein the nucleic acid polymerase is DNA polymerase.
Claim 7
Dependent Claim
20 words
The assay method according to claim 6, wherein the DNA polymerase is a DNA polymerase originated from Thermus aquaticus .
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