阅读说明：本技术 核酸扩增方法和核酸分析用装置 (Nucleic acid amplification method and foranalysis of nucleic acids device ) 是由 横井崇秀 植松千宗 板桥直志 于 2017-04-05 设计创作，主要内容包括：提供一种用于在基板上规则地配置、无扩增偏倚地形成扩增核酸片段簇的方法和装置。本发明涉及的方法中,在具有具备亲水性的多个第一表面、和围绕各个前述多个第一表面且亲水性比前述第一表面低的第二表面的基板上,形成封入有作为模板的核酸的液滴,在基板上的液滴内进行核酸扩增反应后,将液滴除去,进一步在基板上进行核酸扩增反应。(A kind of method and apparatus for regularly configuring on substrate, forming amplification of nucleic acid segment cluster in bias without amplification are provided.In method of the present invention, have hydrophilic multiple first surfaces on the substrate of each aforesaid plurality of first surface and the hydrophily second surface lower than said first surface, form the drop for being sealed with the nucleic acid as template, after carrying out nucleic acid amplification reaction in drop on substrate, drop is removed, nucleic acid amplification reaction is further carried out on substrate.)

1. a kind of nucleic acid amplification method characterized by comprising

The process of prepared substrate, the substrate have hydrophilic multiple first surfaces and around each the multiple the One surface and the hydrophily second surface lower than the first surface, the first surface fix or configured with as template Nucleic acid specificity combine molecule,

The sample solution and contain nucleic acid amplification substrate and nucleic acid that supply contains the nucleic acid as template on the substrate The mixed solution of the reaction solution of synzyme configures the mixed solution, the core as template on the first surface Acid in conjunction with the process on the molecule in conjunction with the nucleic acid specificity as template,

Hydrophobic solvent is supplied on the substrate, is formed and is sealed with the mixed solution of configuration on the first surface The process of drop,

The process that the amplified reaction of the nucleic acid is carried out in the drop,

The process for removing the hydrophobic solvent from the substrate,

The process for supplying the reaction solution containing nucleic acid amplification substrate and nucleic acid synthesizing enzyme on the substrate, and

The process for carrying out the amplified reaction of the nucleic acid.

2. nucleic acid amplification method according to claim 1, the amplified reaction is rolling circle amplification, that is, RCA or polymerase chain Reaction is PCR.

3. nucleic acid amplification method according to claim 1 dilutes the sample solution containing the nucleic acid as template, Form the drop that every 1 drop is sealed with the 1 molecule nucleic acid as described below as template.

4. nucleic acid amplification method according to claim 1, the nucleic acid, which is selected from by mRNA, that is, mRNA, non-coding RNA, is The group of the hybrid nucleic acid of ncRNA, Microrna, genomic DNA and their segment and RNA and DNA composition.

5. nucleic acid amplification method according to claim 1 carries out the work of the amplified reaction of the nucleic acid in the drop After sequence, further comprise:

The sample solution and contain nucleic acid amplification substrate and nucleic acid that supply contains the nucleic acid as template on the substrate The mixed solution of the reaction solution of synzyme, without on the first surface for expanding obtained nucleic acid fragment in the first surface The mixed solution is configured, the nucleic acid as template is incorporated in the molecule in conjunction with the nucleic acid specificity as template On process,

Hydrophobic solvent is supplied on the substrate, is formed and is sealed with the mixed solution of configuration on the first surface The process of drop, and

The process of the amplified reaction of the nucleic acid is carried out in the drop.

6. nucleic acid amplification method according to claim 1, the process for removing the hydrophobic solvent from the substrate It afterwards, further comprise making process dry on the substrate.

7. a kind of foranalysis of nucleic acids device, which is characterized in that

Have the reaction vessel for carrying out nucleic acid amplification reaction, the sample solution for accommodating the sample solution containing the nucleic acid as template The reaction of the reaction solution of slot, the hydrophobic solvent slot for accommodating hydrophobic solvent and receiving at least containing nucleic acid amplification substrate is molten Liquid bath,

Being equipped in the reaction vessel can connect with the sample solution slot, the hydrophobic solvent slot and the reaction solution slot The first opening portion and the second opening portion connect,

Any one of upper surface or bottom surface in the reaction vessel are equipped with substrate, and the substrate has hydrophily Multiple first surfaces with around each the multiple first surface and the hydrophily second surface lower than the first surface,

It is fixed in the first surface or configured with the molecule in conjunction with the nucleic acid specificity as template.

8. the shape of foranalysis of nucleic acids device according to claim 7, the substrate has concave architecture, the concavity knot The most bottom surface of structure is the first surface.

9. foranalysis of nucleic acids device according to claim 7, the size that the first surface is 0.5~2.0 μm of diameter, institute The density for stating the first surface on substrate is 200,000/mm²More than.

10. foranalysis of nucleic acids device according to claim 7, be further equipped with can be to being observed on the substrate The sump pit of the discharge liquor of observation portion and/or the recycling reaction vessel.

11. foranalysis of nucleic acids device according to claim 7, is further equipped at least one temperature control equipment.

12. foranalysis of nucleic acids device according to claim 11, the reaction vessel, described is dredged the sample solution slot Aqueous solvent slot and the reaction solution slot can be adjusted to temperature appropriate using the temperature control equipment.

Technical field

The present invention relates to the nucleic acid amplification methods and foranalysis of nucleic acids device on substrate.

Background technique

In patent document 1, as the nucleic acid amplification method on substrate, describe may include following step production biology The method of the patterned surface of molecule: the step of (a) preparing reagent, the reagent include (i) surface it is discontinuous, have by surface Interval region separate specified point array and (ii) contain there are many different target biological molecules solution；And (b) It the step of reacting reagent, biomolecule is delivered to specified point, each biomolecule is made to be attached respectively to each specified point, should Electric field is applied to interval region in step, obtains biomolecule from interval region.

It is described in patent document 2 and uses the bottom of receiving portion 13 in the method on substrate as enclosing the substances such as nucleic acid Face is that the upper surface of hydrophily and side wall 12 is hydrophobic array.Therefore, it imports in process in pearl using hydrophilic the In the case where one solvent 20, more effectively the first solvent 20 containing pearl 21,21 ' can be imported in receiving portion 13, into one Step can prevent pearl from accommodating hydrophobic second solvent 30 used in process and enter receiving portion 13, it is thus possible to make to accommodate Hydrophilic first solvent 20 in portion 13 is coating and closed by hydrophobic second solvent 30, is formed drop (Droplet).

Summary of the invention

Problems to be solved by the invention

In parallel type sequenator, in order to improve the easiness of analysis, the regularly configuration, single point on substrate is needed to form The technology of the amplification of nucleic acid segment cluster in sub- source.

In patent document 1, the amplification of nucleic acid segment cluster for being formed on substrate regularly configuration, single molecular origin is described Technology.But the amplified reaction of multiple template is to carry out in the solution not being isolated, therefore there are following problems: by In the difference for the speed that template DNA adheres on substrate, previously contributed to form the DNA molecular of cluster become on peripheral substrate into One step forms the template of cluster, generates amplification bias.The skill that therefore, it is necessary to expand each template in the liquid of isolation Art.

On the other hand, it is described in patent document 2 and is regularly configured on substrate, carries out the substances such as nucleic acid according to drop The technology of isolation.But it is not carried out the formation of the amplification of nucleic acid segment cluster on substrate, it is therefore necessary to other device by shape At amplification of nucleic acid segment cluster configure on substrate.

The object of the present invention is to provide regularly configure on substrate, form amplification of nucleic acid segment in bias without amplification The technology of cluster.

The method used for solving the problem

In order to solve the above problems, the present invention provides a kind of nucleic acid amplification method characterized by comprising

The process of prepared substrate, aforesaid base plate have and have hydrophilic multiple first tables with what arbitrary systematicness configured Face with around each aforesaid plurality of first surface and the hydrophily second surface lower than said first surface, in aforementioned first table Face is fixed or configured with the molecule in conjunction with the nucleic acid specificity as template,

On aforesaid base plate sample solution of the supply containing the aforementioned nucleic acid as template with containing nucleic acid amplification substrate and The mixed solution of the reaction solution of nucleic acid synthesizing enzyme configures aforementioned mixed solution on said first surface, aforementioned to be used as template Nucleic acid be incorporated in the process on the molecule in conjunction with the aforementioned nucleic acid specificity as template,

Hydrophobic solvent is supplied on aforesaid base plate, it is molten that formation is sealed with the aforementioned mixing configured on said first surface The process of the drop of liquid,

The process that the amplified reaction of aforementioned nucleic acid is carried out in aforementioned drop,

The process for removing aforementioned hydrophobic solvent from aforesaid base plate,

The process that the reaction solution containing nucleic acid amplification substrate and nucleic acid synthesizing enzyme is supplied on aforesaid base plate, and

The process for carrying out the amplified reaction of aforementioned nucleic acid.

In addition, in order to solve the above problems, the present invention also provides a kind of foranalysis of nucleic acids devices, which is characterized in that has The reaction vessel for carrying out nucleic acid amplification reaction, accommodates the sample solution slot for accommodating the sample solution containing the nucleic acid as template The hydrophobic solvent slot of hydrophobic solvent and the reaction solution slot for accommodating the reaction solution at least containing nucleic acid amplification substrate, Being equipped in previous reaction container can connect with aforementioned specimen solution tank, aforementioned hydrophobic solvent slot and previous reaction solution tank First opening portion and the second opening portion, any one of upper surface or bottom surface in previous reaction container are equipped with substrate, preceding It states substrate and has hydrophilic multiple first surfaces and around each aforesaid plurality of first surface and hydrophily is than aforementioned the The low second surface in one surface is fixed in said first surface or configured with point in conjunction with the nucleic acid specificity as template Son.

The effect of invention

In accordance with the invention it is possible to regularly be configured on substrate, form amplification of nucleic acid segment cluster in bias without amplification.Cause This, can be realized effective and high-precision nucleic acid amplification in parallel type sequenator and foranalysis of nucleic acids later.

Detailed description of the invention

Fig. 1 is the composition figure of the foranalysis of nucleic acids device of embodiment 1.

In Fig. 2A, (a) is the figure for showing the example of the substrate with hydrophily graphics field of embodiment 1.It (b) is to show The figure of the cross section of the example of the substrate with hydrophily graphics field of embodiment 1.

Fig. 2 B is the figure of the specific embodiment of hydrophily graphics field in the foranalysis of nucleic acids device for show embodiment 1.

Fig. 3 shows the synoptic diagram of each process of the nucleic acid amplification method of embodiment 1.

Fig. 4 shows the synoptic diagram of each process of the nucleic acid amplification method of embodiment 2.

Fig. 5 shows the concept map of the result of the nucleic acid amplification method of embodiment 1 or 2.

Fig. 6 shows the synoptic diagram of each process of the nucleic acid amplification method of embodiment 3.

Fig. 7 shows the concept map of the result of the nucleic acid amplification method of embodiment 3.

Fig. 8 shows the synoptic diagram of each process of the nucleic acid amplification method of embodiment 4.

Fig. 9 is the composition figure of the foranalysis of nucleic acids device in embodiment 5.

Figure 10 shows the synoptic diagram of each process of the nucleic acid amplification method of embodiment 5.

Figure 11 shows the flow chart of the nucleic acid amplification method of embodiment 5.

Figure 12 shows the flow chart of the nucleic acid amplification method of embodiment 5.

Figure 13 be carry out embodiment 6 in DNA sequencing in the case where foranalysis of nucleic acids device composition figure.

Specific embodiment

Hereinafter, being illustrated using attached drawing to embodiment, but the present invention is not limited to these embodiments.