DNA detection method and device therefor
The present invention was made to provide novel methods for detecting DNA and devices therefor. Specifically, the present invention is a method of detecting DNA including the steps of: adding, to an oil, a fluorescently-labeled probe, a DNA intercalator, and a DNA solution containing a target DNA to produce droplets; performing PCR on the droplets; and measuring fluorescence from the fluorescently-labeled probe and fluorescence from the DNA intercalator, wherein the DNA solution has a concentration at which each droplet is produced so as to contain one or less target DNA molecule. In addition, the present invention is a DNA detection device including a droplet production unit, a thermal cycler unit, and a fluorescence detection unit which perform these steps.
1 . A method of determining whether a solution contains a target DNA molecule, the method comprising the steps of:
produce droplets by adding a fluorescently-labeled probe, a DNA intercalator that binds to a double stranded DNA but not to an oil single stranded DNA, and the solution to an oil,
each of the droplets containing the fluorescently-labeled probe, the DNA intercalator and the solution, the fluorescently-labeled probe having a fluorescent dye capable of producing a first fluorescence and a quencher of the fluorescent dye located at its 5′ and 3′ ends respectively, wherein the DNA intercalator emits a second fluorescence only when it intercalates into a double-strand DNA, and wherein each of the droplets has only one or zero of the target DNA molecule when the solution contains the target DNA molecule, and the fluorecently-labeled probe does not emit the first fluorescence before it interacts with the target DNA molecule;
performing a polymerase chain reaction (PCR) in the droplets using a DNA polymerase; and
only after completion of the PCR, measuring the first fluorescence from the fluorescently-labeled probe and the second fluorescence from the DNA intercalator in the droplets by a fluorescence detector, wherein
measuring the first fluorescence in the droplets using a fluorescence detector, the solution contains the target DNA molecule, when both the first fluorescence and the second fluorescence are detected in the droplets;
the solution does not contain the target DNA molecule, when the second fluorescence is not detected in the droplets;
wherein the wavelength of the first fluorescence and the wavelength of the second fluorescence are different; and
wherein the DNA polymerase is a Taq DNA polymerase having 5′ to 3′ exonuclease activity and the fluorescently-labeled probe is a single-stranded hydrolysis probe that anneals to the target DNA molecule and then is hydrolyzed by exonuclease activity of the Taq DNA polymerase during the process of the PCR when the target DNA molecule is present in the droplet such that the fluorescent dye and the quencher located at 5′ and 3′ ends of the fluorexcently-labeled probe respectively are separated from each other and the first fluorescence is emitted in the droplets.
2 . The method according to claim 1 , wherein the oil comprises a fluorine-based oil, a silicone-based oil, or a hydrocarbon-based oil.