Porous structure and method for manufacturing same

#This Application is a U.S. National Phase Application under 35 U.S.C. § 371 of International Application No. PCT/KR2014/010171, filed Oct. 28, 2014, published as WO 2015/065005 on May 7, 2015, which claims the benefit of Korean Patent Application No. 10-2013-0128696, filed on Oct. 28, 2013, in the Korean Intellectual Property Office, the entire disclosures of which are incorporated herein by reference for all purposes.

#The disclosure described in this specification relates to a porous structure including a primer, a manufacturing method of the porous structure, and a multiplex real-time nucleic acid amplifying method using the porous structure.

#The polymerase chain reaction (PCR) method is a technique to amplify a nucleic acid by cloning the base sequence thereof, and there are an endpoint PCR and a real-time PCR. Hitherto, the endpoint PCR has been often used, but by this method, it is possible to detect or quantify the amplified nucleic acid only after the PCR reaction is completed, and thus there is a problem that a separate result analysis step such as electrophoresis and an apparatus for that are required after the experiment, a long time of 2 hours or longer is required, and it is not possible to detect or quantify the amplified nucleic acid in real time. For example, the first nucleic acid is required to be quantified from the PCR product obtained after the reaction is completed, but it is difficult to accurately quantify the first nucleic acid due to various factors which affect the analysis even if fluorescent staining or the like is employed. The accurate amount of a nucleic acid is measured at the exponential phase, but it is difficult to analyze the accurate amount by the endpoint PCR since a real-time measurement is impossible by it.

#FIG. 1 illustrates the internal structure of the porous structure according to an embodiment of the present invention, and the sphere on the left is a schematic diagram of the porous structure according to an embodiment of the present invention. Specifically, the diagram on the upper-left among the four boxes on the right illustrates a figuration in which a primer in one direction between a forward primer and a reverse primer of a target nucleic acid is fixed as the primer of polymerase chain reaction (PCR), the diagram on the upper-right illustrates a figuration in which the polymerase chain reaction (PCR) of the target nucleic acid bonded with this primer is taking place, the diagram on the lower-left illustrates a figuration in which a primer in one direction between the forward primer and the reverse primer is fixed and this primer is fixed via a linker, and the diagram on the lower-right illustrates a figuration in which both the forward primer and the reverse primer are fixed via a linker.

#FIG. 2 is a schematic diagram illustrating a method for manufacturing a porous structure according to an embodiment of the present invention (upper left), the arrangement of the porous structures that are manufactured by the above method and have different kinds of targets from one another on an array (upper right), the detection of a nucleic acid in real time while amplifying it (lower right), and the analysis of it (lower left).

#FIG. 3 is a schematic diagram illustrating a method for manufacturing a porous structure according to an embodiment of the present invention, specifically it illustrates a step of forming a porous structure in a droplet form by spotting a porous structure-forming solution on an array; a step of curing the porous structures that are manufactured in a droplet form and have different kinds of targets from one another and removing the porogen from the porous structures; and a step of amplifying a nucleic acid and at the same time detecting it in real time.

#Hereinafter, the present invention will be described in detail.

#An embodiment of the present invention provides a porous structure including pores, and the porous structure includes primers in one or more directions between a forward primer and a reverse primer of a target nucleic acid as a primer of polymerase chain reaction (PCR) fixed to the interior of the pores.

#At this time, the primers may be from 10 to 100 base pairs (bp) and more specifically from 20 to 50 base pairs, but the kind and length of the sequence of the primers can be modified depending on the target nucleic acid without limitation.