KR101785074B1

KR101785074B1 - Microchip for DNA synthesis and DNA synthesis method using the same

Info

Publication number: KR101785074B1
Application number: KR1020160010784A
Authority: KR
Inventors: 천홍구
Original assignee: 고려대학교 산학협력단
Priority date: 2015-01-28
Filing date: 2016-01-28
Publication date: 2017-10-12
Also published as: KR20160092954A

Abstract

The present invention relates to a microchip for DNA synthesis and a DNA synthesis method using the same.
When the DNA is synthesized by using the microchip for DNA synthesis according to the present invention, the error rate of synthesis which occurs in the synthesis of DNA having a long base sequence can be remarkably reduced. This can be achieved by concentrating DNA fragments and DNA ligase at a high concentration in a limited space of the fluid channel included in the microchip for DNA synthesis according to the present invention. In addition, even when a sample with a low concentration is used, it can be concentrated at a high concentration to synthesize DNA, so that the amount of expensive biological / chemical sample can be reduced, which is remarkably superior to the conventional technology.

Description

Microchip for DNA synthesis and method for synthesizing DNA using the same [

The present invention relates to a microchip for DNA synthesis and a DNA synthesis method using the same.

DNA (Deoxyribonucleic acid) is a kind of nucleic acid, which is a substance that stores the genetic information of a living organism in the nucleus of a cell. DNA consists of a backbone chain and a nucleobase, which form a double helix structure by polymerizing four kinds of nucleotides separated by a nucleobase.

In the study of such DNA, the most fundamental and important thing is to efficiently synthesize DNA having necessary genetic information, and currently, a method of synthesizing DNA using oligonucleotide and DNA polymerase is mainly used.

Synthetic genes produced by gene synthesis technology can be used for transformation using synthetic genes and synthetic genomes, transformation of cellulosic feedstocks into biofuels using transformed cells, , Development of new drug molecule production technology through the design and synthesis of gene clusters, optimization of antibody synthesis using gene design algorithm and gene synthesis technology, etc., and can be applied to thousands of base pairs (Kbp ) Is expected to be the base technology for the field of synthetic biology that is currently in the spotlight.

However, in the conventional gene synthesis method, oligonucleotides which are made to overlap only the bases of the ends by using the characteristics of DNA existing in base pair form are synthesized by using DNA polymerase, Therefore, since it is impossible to control the DNA synthesis process, it is known that synthesis of a gene having a size of 1-2 kb at a time makes it impossible to produce a gene without errors.

The most serious problem in conventional gene synthesis is that oligos prepared by chemical synthesis has error rate of about 1 error / 100 bp. Therefore, various studies have been carried out to reduce the error rate of gene synthesis. However, Synthetic methods still take a long time, are laborious and costly, and synthesized genes also have high error rates. In particular, when a long DNA is synthesized using a sample containing a fragment DNA, it is disadvantageous in that a very expensive sample is required to be repeatedly injected repeatedly.

Korean Patent Registration No. 10-0519895 (Patent Document 1) has been disclosed as a prior art related to the present invention, and Patent Document 1 discloses a technique relating to a DNA synthesis method utilizing a DNA synthesis reaction promoter useful in the field of gene engineering .

Patent Document 1. Korean Registration No. 10-0519895

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to solve the problem of high error rate and high cost in synthesis of DNA having a long base sequence, And a DNA synthesis method using the microchip.

In order to solve the above problems, a microchip for DNA synthesis according to one aspect of the present invention comprises

A fluid channel in which the inner surface is negatively charged or negatively charged;

A filtration structure positioned in the middle of the fluid channel and selectively passing only cations therethrough; And

A sample injection unit and a sample outlet at both ends of the fluid channel;

/ RTI >

Characterized in that the filtration structure is a cation selective permeable nanoporous polymer which is a channel portion having a diameter narrower than other channel portions of the fluid channel,

The sample injected through the sample injecting unit may include a first DNA fragment group, a second DNA fragment group including a complementary base sequence to the first DNA fragment, and an DNA ligase.

The DNA synthesis method according to another aspect of the present invention uses the microchip for DNA synthesis according to the present invention.

When the DNA is synthesized by using the microchip for DNA synthesis according to the present invention, the error rate of synthesis which occurs in the synthesis of DNA having a long base sequence can be remarkably reduced. This can be achieved by concentrating DNA fragments and DNA ligase at a high concentration in a limited space of the fluid channel included in the microchip for DNA synthesis according to the present invention. In addition, even when a sample with a low concentration is used, it can be concentrated at a high concentration to synthesize DNA, so that the amount of expensive biological / chemical sample can be reduced, which is remarkably superior to the conventional technology.

1 is a diagram showing a microchip for DNA synthesis according to the present invention.
2 is a photograph showing a filtration structure included in a microchip for DNA synthesis according to the present invention.
FIG. 3 is a diagram showing a process of concentrating DNA fragments and DNA ligases having negative charge in the microchip for DNA synthesis according to the present invention. FIG.
4 is a photograph showing the results of concentration of negative charge samples.
FIG. 5 is a diagram illustrating an example of a process of DNA synthesis by concentrating DNA fragments. FIG.
FIG. 6 is a diagram showing the process of DNA synthesis after concentration. FIG.
7 is an electrophoresis image showing the effect of DNA synthesis after concentration.

Accordingly, the inventors of the present invention have made efforts to develop a method for significantly lowering the error rate in DNA synthesis, and as a result, discovered a microchip for DNA synthesis according to the present invention and a DNA synthesis method using the same, and completed the present invention.

Specifically, the microchip for DNA synthesis according to the present invention comprises

A fluid channel (100) in which the inner surface is negatively charged or negatively charged;

A filtration structure (110) positioned in the middle of the fluid channel for selectively passing only cations therethrough; And

A sample injection unit 120 and a sample outlet 130 at both ends of the fluid channel;

/ RTI >

Characterized in that the filtration structure comprises a cation-selective permeable nanoporous polymer (140)

When the DNA is synthesized using the microchip for DNA synthesis according to the present invention, the error rate occurring in the DNA synthesis can be remarkably lowered by the conventional technique. In addition, even if the sample used in the synthesis of existing DNA is expensive and low in concentration, the conventional method requires only repeated sample injection, resulting in a high production cost for DNA synthesis. Since the DNA is synthesized by concentrating it at a high concentration, the amount of the sample used is remarkably reduced, and the manufacturing cost is also remarkably reduced. Further, since the present invention is a technique of synthesizing DNA fragments and DNA ligase at a high concentration and then synthesizing DNA in a short period of time, the production time of DNA can be drastically shortened.

The reason why the microchip for DNA synthesis according to the present invention can concentrate the first DNA fragment group, the second DNA fragment group and the DNA ligase contained in the sample at a high concentration is that the filtration structure selectively binds only the cation Since it exhibits selective permeability that does not transmit DNA fragments corresponding to anions and DNA ligase.

Meanwhile, in the present invention, the fluid channel may be a fluid channel in which the inner surface is negatively charged or negatively charged, and the charging of the negative charge is not particularly limited, but it can be charged by a method of coating a negative charge. In addition, any fluid channel having a negative charge may be included in the fluid channel, and preferably, a negative charge glass may be used.

On the other hand, the filtration structure may be a channel portion having a diameter narrower than other channel portions of the fluid channel to improve the flow velocity, though there is no particular limitation. The filtration structure may be composed of a cation-selective permeable nanoporous polymer or may include a cation-selective permeable nanoporous polymer as a part. Since the filtration structure is a cation-selective permeable nanoporous polymer or contains it, only the cation is selectively permeated without transmitting the DNA fragment and the DNA ligase, which are anions. In addition, the cationically permeable nanoporous polymer constituting or constituting the filtration structure may be a nanoporous polymer selectively permeable to only cations, but not limited to poly-AMPS (poly- (2-acrylamido-2- methyl-1-propanesulfonic acid)] or poly-SS (poly- (styrene sulfonate)). On the other hand, since the inner surface of the fluid channel is coated with a negative charge as described above, So that negative charges such as DNA fragments or DNA ligase and DNA polymerase can not pass through the filtration structure while only positively charged substances selectively pass through the filtration structure More specifically, the cation (or positively charged) and a portion of the sample are the cation selective permeability of the filtration structure The sample may be discharged through the waste outlet portion 150 through the porous polymer and the sample other than the anion, the cation (or the positive charge) may be discharged through the sample outlet through the fluid channel and the filtration structure. In the case of a structure in which the cationic permeable nanoporous polymer is included in the filtration structure, the top or bottom of the fluid channel in the filtration structure may be made of a cation-selective permeable nanoporous polymer, When the lower end is made of a cation-selective permeable nanoporous polymer, the upper or lower cation-selective permeable nanoporous polymer and the waste outlet may be connected.

The sample injected into the sample injecting unit may include a first DNA fragment group, a second DNA fragment group, and an DNA ligase, and the second DNA fragment group may include a complementary base Sequence. &Lt; / RTI > In particular, the first DNA fragment group may be a set of a plurality of first DNA fragments which are the same or different base sequences. Also, the second DNA fragment group may be a set of a plurality of second DNA fragments having the same or different base sequences including a complementary base sequence to the first DNA fragment. The first DNA fragment or the second DNA fragment may be the same or different base sequence having a length of 10 bp or more, although there is no particular limitation.

On the other hand, DNA synthesis is performed in the fluid channel in the direction of the sample injection part with the filtration structure as the center. More specifically, the filtration structure (particularly the cation-selective permeable nanoporous polymer in the filtration structure) selectively passes cations, and a plurality of first DNA fragments, which are anions in the fluid channel toward the sample injection portion, The plurality of second DNA fragments may be concentrated and the concentrated plurality of first DNA fragments and the plurality of second DNA fragments may be DNA synthesized by DNA ligating with DNA ligase. In addition, the fluid channel induces a flow of the sample in the direction of the sample outlet portion from the sample injecting portion by applying a voltage such that a voltage difference between the direction of the sample injection portion and the direction of the sample outlet portion is formed. Permeable < / RTI > nanoporous polymer). In addition, although there is no particular limitation on the DNA synthesis, DNA synthesis may be performed by DNA ligation using an DNA ligase after phosphorylating the 5 'end of the first DNA fragment or the second DNA fragment .

The filtration structure may further include a waste outlet connected to an upper end or a lower end of the filtration structure. Also, the cation that has passed through the cationic surfactant nanoporous polymer of the filtration structure may be discharged through the waste outlet.

Meanwhile, a DNA synthesis method according to another aspect of the present invention is a DNA synthesis method using the microchip for DNA synthesis according to the present invention. Such a DNA synthesis method significantly improves the error rate of synthesis compared to the prior art.

Specifically, the DNA synthesis method using the microchip for DNA synthesis

1) injecting a sample into a sample injection unit;

2) passing the contained cation or positive charge in the sample through the filtration structure; And

3) a plurality of first DNA fragments in the sample, a second DNA fragment containing a base sequence complementary to the first DNA fragment, and a DNA ligase concentrated in the fluid channel toward the sample injection section, To synthesize DNA by DNA ligase;

.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example

< DNA Fabrication of microchips for synthesis>

First, a microchip for DNA synthesis having the same structure as in FIG. 1 ((a) in cross-section and (b) in stereochemistry) was prepared. The microchip for DNA synthesis is a microfluidic-based glass chip having a depth of 11.6 μm, and the inside of the fluid channel is made of a glass having a negative charge. Particularly, in the microchip for DNA synthesis, the filtration structure is composed of a nanoporous polymer having cation selective permeability above and below the fluid channel, so that only the cation is selectively permeated. The process of forming such a filtration structure will be described in more detail. First, the remaining portion except for the filtration structure is made first, and a polymer having nano-sized holes is made in the upper and lower passages of the intersection. For this purpose, the polymer was first coated with trimethylsilyl metacrylate (TMSMA) so that the polymer and glass could be covalently bonded. Then, the monomer, 2-acrylamido-2-methylpropane sulfonic acid (AMPSA), photoinitiator and cross-linking agent are first filled in the channel and masked to cover the remaining area except the target position using a mask. Light was emitted from the ultraviolet wavelength band using an exposure apparatus to form a filtration structure (hereinafter referred to as " poly-AMPS ").

< DNA Sample injection into synthetic microchip>

A second DNA fragment group composed of a first DNA fragment group composed of first DNA fragments as a sample, a second DNA fragment as a complementary base sequence thereof, and a second DNA fragment group composed of DNA ligase ( ligase) was injected into the wells. Here, M0202S of New England Biolabs was used as the DNA ligase, and B0202S of the same company was used as the buffer. In addition, a voltage difference is formed in the fluid channel to induce flow of the sample.

< DNA Sample Concentration in Synthesis Microchip>

FIG. 3 is a diagram showing a process of concentrating DNA fragments and DNA ligase that are negatively charged in the DNA synthesis microchip. 3, in FIG. 3 (a), 60 V is applied to the left sample injection port and 45 V is applied to the right sample discharge portion. No voltage is applied to the top and bottom waste outlet. At this time, the fluid flows to the right by the electroosmosis effect. In FIG. 3 (b), 0 V is applied to the reservoir at the upper and lower waste outlet portions. Since poly-AMPS (filtration structure) is a polymer with negative charge, it selectively passes only cations. Only cations and positively charged samples enter the waste outlet. In FIG. 3 (c), as the positive ions are sucked into the upper and lower poly-AMPS (filtration structure), an electric field is generated and an ion exhaustion region is generated. 3 (d), the negative charge samples are subjected to the electrophoretic force toward the sample injection part, and the negative charge samples (the first DNA fragment, the second DNA fragment and the DNA ligase ligase) accumulates at a high concentration.

4 is a photograph showing the results of concentration of the negative charge samples (10 mM phosphase buffer, 100 μM flurescein, 100 V applied to the sample injection part, and 63 V applied to the drainage outlet part).

&Lt; Concentration of DNA Synthesis>

The first DNA fragment group (S1, S2 ,,,, Sn-1, Sn) and the complementary second DNA fragment group (NS1, NS2 ,,, NSn-1, NSn) , The long - chain DNA was synthesized by concentrating it at a sufficiently high concentration on the left side of the fluid channel. First, the DNA fragment was phosphorylated at the 5 'end of the DNA fragment. When all of the DNA fragments were mixed, the fragments became sticky to each other and became the original double helix DNA form. After that, double-stranded DNA was prepared by covalently bonding DNAs located on both sides with DNA ligase.

To further examine the phosphorylation and ligation process, T4 Polynucleotide Kinase (T4 PNK buffer) buffer, 10 mM ATP, and Nuclease free water were used as a reagent for DNA fragmentation phosphorylation. The enzyme was T4 PNK. 100 μM of DNA was added to each well and reacted separately. The phosphorylated DNA fragments were collected. To ligate the merged DNA fragments, phosphorylated DNA fragments were mixed with a mixture of T4 ligase buffer, T4 ligase, and Nuclease free water.

FIG. 5 is a schematic diagram illustrating a process of synthesizing DNA by concentrating such DNA fragments. (When S1, S2, S3, NS1, and NS2 phosphorylated are mixed, half of complementary DNA fragments are used. DNA synthesis is completed by ligation with ligase. 6 is a diagram showing a process of DNA synthesis after concentration in a microchip for DNA synthesis.

Experimental Example

< DNA Using microchip for synthesis DNA Measurement of synthesis efficiency>

In order to confirm the improvement of ligation efficiency by using the concentrated chip, the phosphorylated DNA fragments were ligation-enriched at the same time as the experimental group, and as the control group, the non-enrichment reaction pulled out from the reservoir located at the sample- A sample was used. Polymerase chain reaction (PCR) was performed to amplify template DNA with S1, S2, and S3, and a total of 16 cycles were carried out. As a sample used in the PCR, AccuPower® PCR PreMix was used. This PreMix contains Top DNA Polymerase, dNTP, Tris-HCl, KCl, MgCl ₂ , trace amounts of stabilizers and trace dyes used in electrophoresis. 7 is a photograph showing the result thereof. In FIG. 7, lane 1 is a DNA ladder (shown on the left of the picture, 200 and 400 base). In FIG. 7, the second lane is Control (S (1), S (2), S (3) are connected). Also in FIG. 7, the lane No. 3 starts at a concentration of 0.1X, is concentrated, and is extracted by pulling it out. Also in FIG. 7, the lane 4 is a sample which was made into IN at a concentration of 0.1X. As shown in FIG. 7, S (1), S (2) and S (3) were connected in the lane 3, which is the result of concentration, but not in lane 4. Therefore, it can be seen that the DNA synthesis can also be performed in the case of the concentrated sample according to the present invention (lane 3 in FIG. 7).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. It is natural.

100: fluid channel
110: filtration structure
120:
130: sample outlet
140: cation-selective permeable nanoporous polymer
150: waste outlet

Claims

A fluid channel in which the inner surface is negatively charged or negatively charged;
A filtration structure positioned in the middle of the fluid channel and selectively passing only cations therethrough; And
A sample injection unit and a sample outlet at both ends of the fluid channel;
/ RTI >
Characterized in that the filtration structure comprises a cation-selective permeable nanoporous polymer,
The sample injected through the sample injecting unit may include a first DNA fragment group, a second DNA fragment group including a complementary base sequence to the first DNA fragment, and an DNA ligase.
Further comprising a waste outlet connected to the top or bottom of the filtration structure,
The cationically permselective nanoporous polymer is poly- (2-acrylamido-2-methyl-1-propanesulfonic acid) or poly-SS (poly-
DNA synthesis is carried out in the fluid channel in the direction of the sample injection part centering on the filtration structure,
Wherein the first DNA fragment group is a set of a plurality of first DNA fragments which are the same or different base sequences,
Wherein the second DNA fragment group is a set of second DNA fragments including a complementary base sequence to a first DNA fragment and a plurality of identical or different base sequences,
The fluid channel is configured to apply a voltage to form a voltage difference between the direction of the sample injection part and the direction of the sample outlet part to induce the flow of the sample in the direction of the sample outlet part from the sample injection part,
The cations passing through the cation-selective permeable nanoporous polymer of the filtration structure are discharged through the waste outlet,
Wherein a plurality of first DNA fragments and a plurality of second DNA fragments which are anions are concentrated in the fluid channel in the direction of the sample injecting portion and the plurality of second DNA fragments are concentrated around the filtration structure, 1 < / RTI > DNA fragment and a plurality of second DNA fragments are synthesized by DNA ligation by DNA ligase.

delete

A DNA synthesis method using the microchip for DNA synthesis according to claim 1
1) injecting a sample into a sample injection unit;
2) passing the contained cation or positive charge in the sample through the filtration structure; And
3) a plurality of first DNA fragments in the sample, a second DNA fragment containing a base sequence complementary to the first DNA fragment, and a DNA ligase concentrated in the fluid channel toward the sample injection section, To synthesize DNA by DNA ligase;
A DNA synthesis method using a microchip for DNA synthesis.