NUCLEIC ACID AMPLIFICATION METHOD AND NUCLEIC ACID ANALYZER
Provided are a method and apparatus for forming clusters of amplified nucleic acid fragments without amplification bias, in a regular arrangement on a substrate. In the method according to the present invention, droplets enclosing the template nucleic acid are formed on a substrate that has a plurality of first surfaces having hydrophilicity and a second surface surrounding each of the plurality of first surfaces and being less hydrophilic than the first surfaces. Then, after a nucleic acid amplification reaction is performed in the droplets on the substrate, the droplets are removed and a nucleic acid amplification reaction is further performed on the substrate.
1 . A nucleic acid amplification method comprising the steps of:
preparing a substrate having a plurality of first surfaces having hydrophilicity and a second surface surrounding each of the plurality of first surfaces, the second surface being less hydrophilic than the first surfaces, wherein a molecule that specifically binds to a template nucleic acid is fixed or arranged on the first surfaces;
supplying a mixed solution of a sample solution comprising the template nucleic acid and a reaction solution comprising a nucleic acid amplification substrate and a nucleic acid synthetic enzyme on the substrate to arrange the mixed solution on the first surfaces, and bind the template nucleic acid to the molecule that specifically binds to the template nucleic acid;
supplying a hydrophobic solvent on the substrate to form droplets in which the mixed solution arranged on the first surfaces is enclosed;
performing an amplification reaction of the nucleic acid in the droplets;
removing the hydrophobic solvent from the substrate;
supplying a reaction solution comprising a nucleic acid amplification substrate and a nucleic acid synthetic enzyme on the substrate; and
performing an amplification reaction of the nucleic acid.
2 . The nucleic acid amplification method according to claim 1 , wherein the amplification reaction is rolling circle amplification (RCA) or polymerase chain reaction (PCR).
3 . The nucleic acid amplification method according to claim 1 , wherein the sample solution comprising the template nucleic acid is diluted to form droplets in which one molecule or less of the template nucleic acid is enclosed per droplet.
4 . The nucleic acid amplification method according to claim 1 , wherein the nucleic acid is selected from the group consisting of messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, genomic DNA, fragments thereof, and hybrid nucleic acids of RNA and DNA.
5 . The nucleic acid amplification method according to claim 1 , further comprising, after the step of performing an amplification reaction of the nucleic acid in the droplets, the steps of:
supplying a mixed solution of a sample solution comprising the template nucleic acid and a reaction solution comprising a nucleic acid amplification substrate and a nucleic acid synthetic enzyme on the substrate to arrange the mixed solution on first surfaces that do not comprise an amplified nucleic acid fragment among the first surfaces, and bind the template nucleic acid to the molecule that specifically binds to the template nucleic acid;
supplying a hydrophobic solvent on the substrate to form droplets in which the mixed solution arranged on the first surfaces is enclosed; and
performing an amplification reaction of the nucleic acid in the droplets.
6 . The nucleic acid amplification method according to claim 1 , further comprising the step of drying the substrate, after the step of removing the hydrophobic solvent from the substrate.
7 . An apparatus for nucleic acid analysis comprising:
a reaction vessel in which a nucleic acid amplification reaction is performed;
a sample solution tank for storing a sample solution comprising a template nucleic acid;
a hydrophobic solvent tank for storing a hydrophobic solvent; and
a reaction solution tank for storing a reaction solution comprising at least a nucleic acid amplification substrate,
wherein the reaction vessel is provided with a first opening and a second opening that are connectable to the sample solution tank, the hydrophobic solvent tank, and the reaction solution tank;
a substrate that has a plurality of first surfaces having hydrophilicity and a second surface surrounding each of the plurality of first surfaces is provided on either a top surface or a bottom surface in the reaction vessel, the second surface being less hydrophilic than the first surfaces; and
a molecule that specifically binds to a template nucleic acid is fixed or arranged on the first surfaces.
8 . The apparatus for nucleic acid analysis according to claim 7 , wherein the shape of the substrate has a concave structure, and the bottommost surface of the concave structure is the first surface.
9 . The apparatus for nucleic acid analysis according to claim 7 , wherein the first surfaces have a diameter of 0.5 to 2.0 μm, and the density of the first surfaces on the substrate is 200,000/mm 2 or more.
10 . The apparatus for nucleic acid analysis according to claim 7 , further comprising:
an observation unit capable of observing the substrate, and/or
a drainage tank for collecting drainage from the reaction vessel.
11 . The apparatus for nucleic acid analysis according to claim 7 , further comprising at least one temperature control device.
12 . The apparatus for nucleic acid analysis according to claim 11 , wherein the temperature of the reaction vessel, the sample solution tank, the hydrophobic solvent tank, and the reaction solution tank can be adjusted to an appropriate temperature by the temperature control device.