KR101576709B1 - Method of collecting sequence-verified nucleic acid fragments and the equipment for amplifying sequence-verified nucleic acid fragments - Google Patents

Abstract

(a) preparing a sequencing plate having a DNA library; (b) providing a scaled down nucleic acid fragment library sequenced through hyperparallel sequencing of said DNA library; (c) positioning the sequencing plate with all of the sequence-identified nucleic acid fragments in an amplifier; (d) amplifying the entire nucleic acid fragments located in the amplifier; And (e) recovering the amplified nucleic acid fragments.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for recovering sequence-confirmed nucleic acid fragments and an apparatus for amplifying the sequence-verified nucleic acid fragments,

The techniques disclosed herein relate to methods for recovering nucleic acid fragments whose sequences have been identified after sequence sequencing analysis, and also to a sealing chamber for performing such a method.

As biotechnology advances, the importance of high-speed, superparallel DNA synthesis and analysis technology is also growing. In the 20th century, the development of next-generation sequencing has led to ultra-fast, superparallel DNA analysis. Along with the development of new analytical methods, we have made remarkable progress in reducing the time required for analysis and increasing the amount of data that can be analyzed. Currently, next-generation base analysis methods such as Illumina, Roche-454, and Ion-Torrent analyze the base sequence based on the chemical reactions occurring at each site by connecting the DNA libraries to be analyzed on the solid phase. Recently, as gene synthesis technology has been developed and its application range has been increased, the development of super fast and super parallel gene synthesis technology is also increasing in importance. A prerequisite for super-parallel gene synthesis is the synthesis of gene libraries. Conventional gene synthesis methods have limitations on the synthesis of ultra-fast and superparallel sequences of gene libraries, but using the recently developed 'shotgun DNA synthesis' method, it has become possible to synthesize gene libraries.

Previously, nucleic acid fragments analyzed by the next-generation base analysis method were only able to acquire the nucleotide sequence information, and it was very difficult to recover the nucleic acid fragments themselves. Recently, after the next-generation base analysis, Was developed. The first method is to map the information of each well of the assay plate and the analyzed base sequence information after the next generation base analysis and to pick up the bead to which the desired nucleic acid fragment remaining on the plate is connected, (High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing, 2010 Nature Biotechnology, Mark Matzas et al). The second method involves linking the barcode sequence to a DNA library obtained from an organism or artificially synthesized, and analyzing a portion of the DNA pool using next-generation sequencing ('Shotgun DNA synthesis' for the high-throughput construction of large DNA molecules, 2012 Nucleic Acids Research, Kim et al). Then, the desired nucleic acid fragment is selectively amplified from the remaining DNA pool using a primer containing a bar code sequence. These methods have the advantage of being able to selectively recover the nucleic acid fragments for which the nucleotide sequence has been confirmed using the next generation sequencing method.

However, the method of picking the bead has the disadvantage of requiring expensive equipment for bead picking. In addition, the method of recovering a nucleic acid fragment using a barcode sequence has a limitation in recovering a desired nucleic acid fragment because of the size of the large DNA library when a population of artificially synthesized DNA library obtained from an organism is very large . For example, if hundreds of genes are synthesized at the same time, the pool will contain hundreds of millions to tens of millions of kinds of nucleic acid fragments, regardless of whether they are errors or not. Selective amplification of only one type of nucleic acid fragment desired by the experimenter from this pool is considerably difficult due to the large size of the library and the recovery rate is also low.

Michael L. Metzker, Nature Reviews, Vol. January 2010.

It is an object of the present invention to provide a method for easily recovering nucleic acid fragments that have been identified from a whole DNA library.

It is another object of the present invention to provide a sealing chamber for recovering sequence-recognizing nucleic acid fragments.

According to one aspect of the present invention, there is provided a method of detecting nucleic acid, comprising: (a) positioning nucleic acid fragments identified by sequencing in an amplifier; (b) amplifying the sequence-confirmed nucleic acid fragments; And (c) recovering the amplified nucleic acid fragments.

According to another aspect of the present invention there is provided a method of amplifying nucleic acid comprising: (a) connecting an adapter sequence to nucleic acid fragments before sequencing; (b) sequencing nucleic acid fragments to which said adapter is attached; (c) positioning said sequence-identified nucleic acid fragments in an amplifier; (d) amplifying the sequence-confirmed nucleic acid fragments; And (e) recovering the amplified nucleic acid fragments.

According to another aspect of the present invention, there is provided a method for preparing a DNA library, comprising: (a) preparing a sequencing plate having a DNA library; (b) providing a scaled down nucleic acid fragment library sequenced through hyperparallel sequencing of said DNA library; (c) positioning the sequencing plate with all of the sequence-identified nucleic acid fragments in an amplifier; (d) amplifying the entire nucleic acid fragments located in the amplifier; And (e) recovering the amplified nucleic acid fragments.

According to another aspect of the present invention, there is provided a sealing chamber for amplifying nucleic acid fragments, comprising: a base chamber capable of receiving a sequencing plate having nucleic acid fragments; An upper chamber which is engageable with and detachable from the base chamber and has a sealing structure as a whole when engaged with the base chamber; And an elastic gasket disposed between the base chamber and the upper chamber for sealing, wherein an inner space for accommodating the PCR solution for amplifying the nucleic acid fragments is formed by the coupling of the base chamber and the upper chamber , At least one of the base chamber and the upper chamber is provided with a sealing chamber made of a material having thermal conductivity.

According to another aspect of the present invention, there is provided a sealing chamber for amplifying nucleic acid fragments, comprising: a base chamber having a storage chamber; An upper chamber disposed on the base chamber; A sample accommodating portion accommodated in the accommodating chamber and having a receiving space for accommodating a sample for amplifying the nucleic acid fragments; A gasket disposed between the base chamber and the upper chamber and having elasticity; A jig having a threaded hole connected to the base chamber and extending to an upper portion of the upper chamber, the threaded hole having an internally threaded portion; And a pressing portion that is screwed to the screw hole, wherein the pressing portion includes a screw portion having a male screw portion, and a head disposed on the screw portion, wherein a displacement of the pressing portion with respect to the jig A sealing chamber is provided in which pressing of the upper chamber by the pressing portion is achieved.

In the method of amplifying nucleic acid fragments after sequencing of the present invention, the DNA library to be amplified can be shrunk to remarkably increase the recovery rate of a desired nucleic acid fragment. That is, if the size of the DNA library is very large, the recovery rate of the desired nucleic acid fragment is low because of the complexity of the DNA library. Accordingly, shrinking the DNA library using the present invention can reduce the complexity of the DNA library and increase the recovery rate of the desired nucleic acid fragment. Further, although a method of utilizing conventional sequence-confirmed nucleic acid fragments has been limited, it has been efficiently improved to increase utilization of nucleic acid fragments that have been identified.

In addition, according to the sealing chamber of the present invention, a base chamber capable of accommodating a sequencing plate having sequence-identified nucleic acid fragments can be combined with and detachable from the base chamber, and has an overall sealing structure when the base chamber is combined with the base chamber. An inner space in which a PCR solution for amplifying the nucleic acid fragments is accommodated is formed by the combination of the base chamber and the upper chamber, and an elastic gasket disposed between the base chamber and the upper chamber for sealing, It is possible to effectively prevent foreign substances or the like from penetrating into the sample. Further, as the pressurizing piece is provided in the upper chamber, damage to the upper chamber can be prevented, and maintenance, maintenance, and management can be easily performed only by replacing the pressure piece. In addition, since the gasket, the upper chamber, and the base chamber are formed with predetermined concavities and convexities, the infiltration path of the foreign matter is extended, so that foreign matter penetration can be more effectively blocked.

Figure 1 shows a flow chart of a method for recovering sequenced nucleic acid fragments through sequencing.
Figure 2 shows the recovered product of nucleic acid fragments sequenced through sequencing.
Figure 3 shows individual nucleic acid fragments amplified using selective amplification primers using the recovered products of nucleic acid fragments identified by sequencing.
FIG. 4 shows the nucleotide sequence analysis results of Sanger sequencing of selectively amplified individual nucleic acid fragments.
5 is a perspective view of a sealing chamber according to an embodiment of the present invention.
6 shows an exploded view of a sealing chamber according to an embodiment of the present invention.
7 shows a top view of a sealing chamber according to an embodiment of the invention.
Figure 8 shows a cross-sectional view along AA of Figure 7;
9 is a cross-sectional view taken along the line BB of Fig.

Hereinafter, various embodiments of the present invention will be described in detail.

In order to overcome the limitations of the bead picking method or the method of recovering nucleic acid fragments using the bar code sequence as described above, the present inventors have effectively reduced the DNA library to alleviate the restriction by the size of the DNA library, A method and an apparatus capable of recovering the entire nucleic acid fragments analyzed without loss have been developed and the present invention has been completed.

According to an embodiment of the present invention, there is provided a method of amplifying nucleic acid comprising: (a) locating nucleic acid fragments identified by sequencing in an amplifier; (b) amplifying the sequence-confirmed nucleic acid fragments; And (c) recovering the amplified nucleic acid fragments.

In one embodiment, prior to sequencing the nucleic acid fragments to be sequenced, an adapter sequence may be coupled to the nucleic acid fragments to be sequenced prior to sequencing, and sequencing of the nucleic acid fragments to which the adapter is coupled.

Specifically, first, the sequence-identified nucleic acid fragments are placed in an amplifier.

The sequencing may be performed by a method of synthesizing nucleic acid fragments. In this case, the nucleic acid fragment may be synthesized by a Sanger method or a hyperparallel method (Michael L. Metzker, Nature Reviews, Vol. II, 2010 January, 'Seqeuencing technologies-the next generation'). Here, the hyperparallel method may be a method such as pyrosequencing chemistry, bridge amplification, next generation sequencing, third generation sequencing, next generation sequencing, or semiconductor sequencing, but is not limited thereto.

If the length of the nucleic acid fragment to be sequenced is longer than the length of the nucleic acid fragment proposed in the next generation sequencing method to be used, it can be fragmented using a restriction enzyme method or a physical shearing method. When the length of the nucleic acid fragment is short, it is possible to extend the length using a method such as assemble or ligation.

In addition, the amplifier may be a sealed chamber in which a predetermined sample is received and sealed. Said sealing chamber for amplifying nucleic acid fragments comprises: a base chamber capable of containing a sequencing plate with said nucleic acid fragments; An upper chamber which is engageable with and detachable from the base chamber and has a sealing structure as a whole when engaged with the base chamber; And an elastic gasket disposed between the base chamber and the upper chamber for sealing. At this time, the inner space in which the PCR solution for amplifying the nucleic acid fragments is accommodated is formed by the coupling of the base chamber and the upper chamber. At least one of the base chamber and the upper chamber is made of a thermally conductive material so that the temperature of the internal PCR solution can be easily adjusted according to temperature control of the water tank when the sealing chamber is immersed in the water tank.

An embodiment of the sealing chamber is shown in Fig. 5, the sealing chamber 1 includes a base chamber 100, an upper chamber 200, a sample receiving portion 300, a gasket 400, a jig 500, and a pressing portion 600 But is not limited thereto.

In addition, when the sequence-confirmed nucleic acid fragments are placed in the amplifier, the sequencing plate in which the sequence-confirmed nucleic acid fragments are present can be placed in the amplifier after the completion of the sequence check. That is, the recovered plate for next-generation nucleotide sequencing can be mounted and sealed in the sealed chamber. When placing the sequence-identified nucleic acid fragments in the amplifier, the whole plate is placed in the amplifier in the sequenced state without cutting off the filler portion of the sequencing plate or separately separating the nucleic acid fragments identified in the plate.

Next, the nucleic acid fragments that have been identified are amplified.

Injecting a DNA polymerase, a dNTP, a primer, a buffer, and / or a PCR solution into the sequence-identified nucleic acid fragments for amplification of the sequence-identified nucleic acid fragments. The DNA polymerase may be, but is not limited to, Tag polymerase, Pfu polymerase, and the like.

The next amplified nucleic acid fragments are recovered. For example, a PCR solution and amplified nucleic acid fragments can be recovered using a pipette. With this method, the entire nucleic acid fragment whose nucleotide sequence has been identified can be recovered without loss.

Next, desired nucleic acid fragments of the recovered nucleic acid fragments are recovered. As a method of recovering the desired nucleic acid fragments, a conventional method for recovering nucleic acid fragments can be used. A kit for recovering nucleic acid fragments sold on the market or a method using the kit may be applied. In addition, if the amplified nucleic acid fragments are bound to beads or have a specific bar code sequence connected thereto, the nucleic acid fragments can be recovered using a bead recovery or a sequence homologous to the bar code sequence.

Amplification of sequence-identified nucleic acid fragments can be amplified or extended using a temperature-controllable instrument such as an oven or water bath or PCR instrument.

It is further contemplated that prior to performing the step, prior to sequencing the nucleic acid fragments identified in the sequence, connecting the adapter sequence to the nucleic acid fragments to be sequenced and sequencing the nucleic acid fragments to which the adapter is attached can do.

The step of connecting the adapter sequence may be a method using PCR assemble or a method using ligation. When connecting the adapter, a 15 to 30 bp bar code sequence can be added as needed.

In addition, after the base sequence analysis, the step of recovering the base sequence analysis plate may further include the step of recovering the base sequence analysis plate. At this time, do not perform the washing and bleaching steps as the last step of next-generation sequencing.

According to another embodiment of the present invention, there is provided a method for recovering a nucleic acid fragment using a method of shrinking a nucleic acid fragment library through super parallel sequencing. The method comprises the steps of: (a) preparing a sequencing plate with a DNA library; (b) providing a scaled down nucleic acid fragment library sequenced through hyperparallel sequencing of said DNA library; (c) positioning the sequencing plate with all of the sequence-identified nucleic acid fragments in an amplifier; (d) amplifying the entire nucleic acid fragments located in the amplifier; And (e) recovering the amplified nucleic acid fragments.

In a preferred embodiment, if necessary, (f) recovering the desired nucleic acid fragments from the recovered nucleic acid fragments may be further included.

The process of shrinking the DNA library through the superparallel sequencing can be described as follows. For example, a DNA library containing nucleic acid fragments of several hundreds of millions to several hundreds of millions is analyzed using Roche-454 GS Junior. First, during the sequencing preparation, the concentration of the first DNA library is measured, and the number of molecules is calculated and diluted. At this time, about 5 to 20 x 10 ⁶ nucleic acid fragments are reacted with about 10 ⁷ sequencing beads to carry out emulsion PCR. Then, sequencing beads with nucleic acid fragments amplified by immersion PCR are collected, washed and bead enrichment is carried out, and sequencing is performed by injecting into a sequencing plate. Among these, the number of sequencing readings obtained by successful sequencing is about 50,000 to 100,000, so that the number of nucleic acid fragments becomes smaller than that of the original DNA library. For example, when analyzing a DNA library containing hundreds of millions to several tens of millions of nucleic acid fragments using Ion-Torrent Proton, the nucleic acid fragments of about 7 to 10 × 10 ¹¹ , After mixing with the beads for sequencing, emulsion PCR allows only one kind of nucleic acid fragments to form double stranded nucleic acid in one kind of beads. In order to enable sequencing through the enrichment process, only about 3 to 5 x 10 ¹¹ (half of the total) nucleic acid fragments remain when the nucleic acid fragments having different adapter sequences are separated from each other. Among these, the number of sequencing readings obtained by successful sequencing is about 60 to 80 million, so that the number of nucleic acid fragments becomes smaller than that of the original DNA library.

According to the present inventors' method, since the DNA library of the entire DNA library is analyzed by the next-generation sequencing method, the size of the DNA library is effectively reduced by the number of the next generation sequencing analysis readings . The analyzed sequencing plate can be placed in a sealed chamber and recovered from the sequencing plate by amplifying the reduced DNA library. In addition, when the DNA library contains a bar code sequence, the desired nucleic acid fragment can be obtained from the reduced DNA library at a higher recovery rate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of a sealing chamber according to the present invention will be described with reference to the accompanying drawings. The present embodiments are not intended to be limiting.

Spatially relative terms such as "bottom "," top ", and the like can be used to easily describe one member or components and other members or components as shown in the figures. Spatially relative terms should be understood to include, in addition to the orientation shown in the drawings, terms that include different orientations of the device during use or operation. For example, when changing the viewpoint by changing the orientation of a member shown in the drawing, a portion described as "side" can constitute a "front portion ". Thus, the exemplary term "side" may include both forward and backward directions outside the side. The members can also be oriented in different directions, so that spatially relative terms can be interpreted according to orientation.

The thickness and size of each member in the drawings are exaggerated, omitted, or schematically shown for convenience and clarity of explanation. Also, the size and area of each component do not entirely reflect actual size or area.

FIG. 5 is a perspective view of a sealing chamber according to an embodiment of the present invention, and FIG. 6 is an exploded view of a sealing chamber according to an embodiment of the present invention. FIG. 7 is a plan view of a sealing chamber according to an embodiment of the present invention, FIG. 8 is a sectional view taken along line A-A of FIG. 7, and FIG. 9 is a sectional view taken along line B-B of FIG.

5 and 6, a sealing chamber 1 according to an embodiment of the present invention includes a base chamber 100, an upper chamber 200, a sample receiving portion 300, a gasket 400, a jig 500, and a pressurizing portion 600.

A predetermined accommodating chamber 110 is formed in the base chamber 100 so that the sample accommodating unit 300, which will be described later, can be accommodated. For example, the accommodating chamber 110 may include a predetermined depression formed on the upper surface of the base chamber 100. 6, the sample accommodating part 300 and the accommodating chamber 110 (see FIG. 6) may have a shape corresponding to the outer shape of the sample accommodating part 300, which will be described later. May have a rectangular shape having an inclined surface at one corner, but the present invention is not limited thereto.

The base chamber 100 may be made of a metal such as stainless steel to prevent deformation and damage even in an atmosphere of high pressure, high temperature, etc., but is not limited thereto. Preferably, the upper surface of the base chamber 100 may be provided with a predetermined guide portion 120 so that the gasket 400 and the upper chamber 200 to be described later are appropriately disposed and fixed. For example, the guide part 120 may be a predetermined guide protrusion provided at each corner of the base chamber 100.

The upper chamber 200 is disposed on the upper portion of the base chamber 100 and is disposed on the base chamber 100. The upper chamber 200 includes a sample receiving portion 300 and a sample receiving portion 300, Cover. The upper chamber 200 may be made of a metal such as stainless steel to prevent deformation and breakage even in an atmosphere of high pressure, high temperature, etc., but is not limited thereto.

The sample accommodating part 300 may be formed of a plate-like member having a predetermined accommodation space 310, for example. The receiving space 310 may be formed of a predetermined depression into which the sample can be inserted, or may include a predetermined receiving hole into which a separate member such as a glass containing the sample can be received, but is not limited thereto. As described above, the outer shape of the sample accommodating portion 300 may preferably have a shape corresponding to the shape of the accommodating chamber 110, but is not limited thereto.

On the other hand, preferably, the accommodation space 310 formed in the sample accommodating portion 300 may have various volumes and shapes as required. For example, when the amount of the sample required for the experiment is very small, the sample accommodating part 300 having a small accommodation space 310 can be used. Various samples can be tested under the same conditions, A plurality of receiving spaces 310 may be formed in the sample accommodating unit 300, but the present invention is not limited thereto. Accordingly, even when various types of experiments are carried out by accommodating the sample, appropriate experiments are performed by appropriately replacing the sample accommodating portion 300 without performing replacement of other members or the entire sealing chamber 1 according to the present invention The convenience of the user can be improved.

The housing chamber 110 is formed in the base chamber 100 and the sample accommodating portion 300 is accommodated in the accommodating chamber 110 so that the sealing chamber 1 according to the present invention can have a double- have. That is, a dual storage structure in which the sample is received in the sample storage part 300 and the sample storage part 300 is housed in the storage chamber 110 formed in the base chamber 100 can be achieved.

The gasket 400 may be disposed between the base chamber 100 and the upper chamber 200 and may be made of a material having heat resistance and elasticity. For example, the gasket 400 is made of rubber, plastic, or synthetic resin having heat resistance and elasticity, but is not limited thereto. Preferably, the gasket 400 may be formed of a plate-like member so as to be in close contact with the base chamber 100 and the upper chamber 200, and may be mounted on the upper portion of the sample accommodating portion 300. That is, after the sample accommodating portion 300 is housed in the accommodating chamber 110 formed in the base chamber 100, the gasket 400 is mounted on the sample accommodating portion 300, The chamber 200 may be disposed. 6, the gasket 400 may be formed with a space similar to the predetermined hole 410 corresponding to the accommodation space 310 formed in the sample accommodating portion 300, but the present invention is not limited thereto No.

A gasket 400 having elasticity is disposed between the base chamber 100 and the upper chamber 200. Accordingly, when the upper chamber 200 is pressed, sealing with the gasket 400 is achieved, The foreign object or the like contained in the sample accommodating portion 300 and the sample accommodated in the sample accommodating portion 300 can be prevented from penetrating. Details will be described later.

The jig 500 may be connected to the base chamber 100 and may be configured to have a threaded hole 530 formed on the upper portion of the upper chamber 200 and having a female thread. The jig 500 may be fixedly connected to the base chamber 100, but may preferably be composed of a separate member from the base chamber 100 so as to be removed as required, as shown in Fig. 6 , But is not limited thereto.

For example, the jig 500 may have a configuration in which the support portions 510 on the both sides are bent in a direction facing each other, and the support portions 510 on both sides are connected to each other through the upper extension portion 520 have. The base chamber 100 and the side portion of the upper chamber 200 are mounted on the supporter 510 and a screw hole 530 is formed in the extension 520. In order to mount the base chamber 100 and the side portions of the upper chamber 200 on the support portion 510, predetermined mounting portions 130 and 260 are formed on the side portions of the base chamber 100 and the upper chamber 200, respectively. , But is not limited thereto.

5 and 6 illustrate that one jig 500 is provided but the present invention is not limited thereto and a plurality of jigs 500 and pressing units 600 corresponding to the jigs 500 may be provided, But not limited thereto.

The pressurizing unit 600 is provided to press the upper chamber 200 to achieve close contact between the upper chamber 200, the gasket 400 and the base chamber 100.

The pressing portion 600 may include a predetermined screw portion 610 connected to the screw hole 530 formed in the jig 500 and a head 620 provided on the screw portion 610. The head 620 is provided on the upper portion of the screw portion 610 and the screw portion 610 is screwed to the screw hole 530 formed in the jig 500, Displacement of the pressing portion 600 relative to the jig 500 can be achieved. That is, when the head 620 is turned in one direction, the pressing portion 600 is downwardly displaced to press the upper chamber 200 by the pressing portion 600, and the head 620 is moved in the other direction The pressing portion 600 is displaced upward so that the pressing can be released.

6, the upper chamber 200 includes a main body 210, a pressing piece 220 which is pressed to be in contact with the pressing portion 600, And a receiving groove 230 formed in the receiving groove 220 and accommodating the pressing piece 220 therein.

The main body 210 constitutes a main body of the upper chamber 200. A predetermined receiving groove 230 is formed in a part of the main body 210 and the pressing portion 600 The pressing piece 220 can be received. That is, when the pressing portion 600 is moved downwardly in accordance with the rotation of the head 620 to press the upper chamber 200, the pressing portion 600 directly contacts the main body portion 210 constituting the upper chamber 200 The upper chamber 200 may be pressurized as the pressing piece 220 provided in the receiving groove 230 of the upper chamber 200 is pressed. Accordingly, direct contact and pressurization of the pressurizing unit 600 and the entire upper chamber 200 are avoided, and the entire upper chamber 200 can be prevented from being damaged despite a plurality of operations. That is, since the member directly in contact with the pressing portion 600 is limited to the pressing piece 220, when the pressing piece 220 is damaged by a plurality of operations, Since the problem can be solved by replacing only the part 220, the economics of management and maintenance can be achieved.

A predetermined connection hole 240 is formed at the side of the main body 210 and connected to the receiving groove 230. A predetermined fixing member 250 is inserted through the connection hole 240 . At this time, a predetermined fixing groove 222 connected to the fixing member 250 may be formed on the side of the pressing piece 220. The pressing member 220 is disposed in the receiving groove 230 and the fixing member 250 is inserted into the fixing groove 222 through the connecting hole 240 so that the pressing member 220 Fixation can be achieved. Accordingly, it is possible to prevent the pressing piece 220 from being unnecessarily displaced or disengaged in spite of the contact between the pressing portion 600 and the pressing piece 220 and the screw rotation of the pressing portion 600. [

According to a preferred embodiment of the present invention, a predetermined first concave-convex portion is formed on the upper surface and the lower surface of the gasket 400 contacting with the base chamber 100 and the upper chamber 200, The upper surface of the base chamber 100 and the lower surface of the upper chamber 200 may have second uneven portions corresponding to the uneven portions. That is, as the first concave-convex portion and the second concave-convex portion are provided, the adhesion between the gasket 400 and the base chamber 100 and the upper chamber 200 is more reliably achieved, and the gasket 400 and the base chamber 100 and the upper chamber 200, it is possible to more effectively prevent the penetration of foreign matter by the protrusions and the penetration of foreign substances and reaching the sample.

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, It should be understood that various modifications may be made by those skilled in the art without departing from the spirit and scope of the invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples and test examples, but the present invention is not limited to the following examples and test examples.

Production Example 1. Production of Sealing Chamber

A sealing chamber for mounting a plate for next-generation nucleotide sequence analysis was prepared. The sealing chamber was made of metal with a good thermal conductivity and was designed to use a rubber cover so that the PCR solution between the sealing chamber and the plate was sealed when the plate for the next generation sequencing was sealed without leakage. Also make sure that the side of the cover is exposed on the side of the gasket.

A sealing chamber (not shown) including the base chamber 100, the upper chamber 200, the sample receiving portion 300, the gasket 400, the jig 500, and the pressing portion 600 according to an embodiment of the present invention 1) (see Figs. 5 and 6).

Example 1. Sequencing of a gene

Prior to sequencing the acquired DNA library, the process involved linking the adapter sequences proposed in the next generation sequencing. At this time, as a method of connecting the adapter sequence, a method using PCR assemble or a method using ligation was used. In addition, a 20 bp bar code sequence was added, and the length of the bar code sequence was adjustable.

When the adapter sequence is ligated using PCR assemble, the composition of the PCR solution is as follows. 10 μl of 2X Pfu polymerase master mix, 1 μl of 10 μM adapter for primer, 1 μl of 10 μM adapter rev primer, 5 μl of dw. The Pfu polymerase master mix used in the experiments was a 2X Pfu polymerase premix product sold by Solgent. Also, the primers used in the experiment include the 454 adapter sequence - the bar code sequence - the complementary sequence to the end sequence of the DNA library, and the actual sequence of the primer is as follows (adaptation forward primer (SEQ ID NO: 1): CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNNNNNNNNNNNNNNACGTACGACAGAGTACTCGT, adapter reverse primer (SEQ ID NO: 2): CCTATCCCCTGTGTGCCTTGGCAGTCTCAGNNNNNNNNNNNNNNNNNNNNTCGAACTAATCGGATTGCG). At this time, the DNA denaturation was maintained at 95 ° C. for 10 minutes, and then 95 ° C., 30 seconds, 58 ° C., 30 seconds, 72 ° C. and 45 seconds were performed 15 times to 20 times, , And maintained for 10 minutes. The DNA library to which the adapter was connected was sequenced by Sanger sequencing method to confirm the connection.

F1_1
(SEQ ID NO: 3) CTCTTCTCCGCCGCAAAATCTGGCAACTGAAAGAGCTGGGTTATGCAGCCGTGGATGATGAAACCACGCAACAGACAATGCGTGAGTTAAAAGAACTGGGCTACACTTCGGAGCCGCACGCTGCCGTAGCTTATCGTGCGCTGCGTGATCAGTTGAATCCAGGCGAATATGGCTTGTTCCTCGGCACCGCGCATCCGGCGAAATTTAAAGAGAGCGTGGAAGCGATTCTCGGTGAAACGTTGGATCTGCCAAAAGAGCTGGCAGAACGTGCTGATTTACCCTTGCTTTCACATAATCTGCCCGCCGATTTTGAGAGACC F1_2
(SEQ ID NO: 4) CTCTTCTCTTGAAGGCACCGATACGCTGGCGTATACCGATGCGCAGTATCAACAGCTTGCGGCGGTTACGCGCGCACTGATTGATTGCTATCCGGATATCGCTAAAAACATGACGGGCCATTGTGATATTGCGCCGGATCGCAAAACCGATCCCGGTCCTGCATTTGATTGGGCACGCTTTCGTGTGCTGGTCAGCAAGGAGACAACATGACGCTATTTACAACCTTACTGGTGTTAATTTTCGAGCGCCTGTTTAAGTTGGGCGAGCACTGGCAGCTTGATCATCGTCTTGAAGCGTTCTTTCGCCGCGTGAGAGACC F1_3
(SEQ ID NO: 5) CTCTTCTGCCGCCGACTCAAACACCTCGTCCGTCACCTCCATCCCGCCGTGCAGATCGAACTCCTTCGCCATCTGCTTGCCGAGCGTAGTCTGGTCGTCATGGAACGCCGGCAGACAGTGCAGGAACTTCACGTTCGGGTTGTCGGTCAGCGCCATCATCTGCGCGTTCACCTGATACCCGCGCAGCAGCGCAATGCGCTCTGCCCACTTCTCTTTGGCCTCGCCCATCGACACCCACACGTCGGTATAGATAAAGTCCGCGCCCTTAACGCCTGCCGCCACGTCTTCCGTCAGAGTAATTTTCCCGCCGTGAGAGACC F2_1
(SEQ ID NO: 6) CTCTTCTGCGGGTAACCACGCCCTGGCGAATGTGTTCTACCAGCGGCGCATGGCAATCACTCAGCGAGCTCGACGCCAGGGTCAGGTTTTTAAAGCCCATCTTCGCGATGACGTCCATCACCATATTGACGGTCAGGTCACCGCCACGGAAAGCGTGATGGAACGAAACCGTCATGCCGTCCTGTAAACCTGAGCGACGAATCGCTTCTTCCAGGTTGGCGCACAGTTTGCGATCGCGCGCTTTTTCAGCCTGGTAGGTTTGCTTTGGCGAGTTCTGGAAAGCGGCAAGATCGCATTCAGCGCGACGATTCCAGAGACC F2_2
(SEQ ID NO: 7) CTCTTCTAGCTGGATAACTTCCGTCAGGAAGTTCACGGCAATGGCCTCTCATCGTATCCGCACCCGAAACTGATGCCGGAATTCTGGCAGTTCCCGACCGTATCAATGGGTCTGGGTCCGATTGGTGCTATTTACCAGGCTAAATTCCTGAAATATCTGGAACACCGTGGCCTGAAAGATACCTCAAAACAAACCGTTTACGCGTTCCTCGGTGACGGTGAAATGGACGAACCGGAATCGAAAGGTGCGATCACCATCGCTACCCGTGAAAAACTGGATAACCTGGTCTTCGTTATCAACTGTAACCTGCAGAGAGACC F3_1
(SEQ ID NO: 8) CTCTTCTCCCGGCGTTGATGGCGTGAACAAAACAATGCTACAGGCCCGTCTGGCTGTTGAGCTGCAAATCCTCCGTGATGAATTACTCTCAGGCCACTACCAGCCCTTGCCCGCCCGTCGCGTTTACATCCCTAAAAGCAACGGCAAACTGCGCCCACTGGGTATCCCCGCGTTGCGCGATCGTATTGTTCAGCGCGCCATGCTGATGGCGATGGAGCCGATTTGGGAGAGTGATTTTCATACGCTCTCATATGGCTTCCGCCCTGAGCGCAGTGTCCACCACGCGATCCGCACGGTGAAATTACAGCTCACAGAGACC F3_2
(SEQ ID NO: 9) CTCTTCTAACGCCTGCCGCCACGTCTTCCGTCAGAGTAATTTTCCCGCCGTGCTTCTCCGCCAGCGCGCTGCACTCCGCCACCAGGCTCTCTTCCGGCCAGCAGGCTTTCGGGGCCAACAGGCGCAGATCCAGCCCGGTCAGCGCCGCCGCTTCCAGCATCGAGTTGCCCATGTTGTTGCGCGCATCGCCCGCGTAGACCAGCGTCATCTCGTTAAACGCCTTGCCCGGCAGGTGCTCCTGCATGGTCATCAGGTCCGCCAGCAGCTGGGTCGGGTGGAACTCGTTGGTCAGCCCGTTCCACACCGGCACGCAGAGACC F4_1
(SEQ ID NO: 10) CTCTTCTCAGTGCGAGTTCCGGCTGACCAGGAATGTAACGTTGCGCATGCAGCCACCAGTAGAGGGCATTGCTTCCCAGTCCACGTTTTTCTGCCTGTTGTAGCCAGCGATCGCGAGCCGCACCATTTCCTGCCGCCTGGGCGGTATTGGCAGCAGCAAGCAGATCCTCATTGCTCATGTCGTGAAGACTGATTTTCTGCCAGGCCGCCAGTGCGGTGGCGTAGTCCTCAACCTGATACGCCTGATAGGCTACCGCACGATGTTGCCAGGCGCTCGGTTGGCGTTGTTCGGCCTGAAGCCATGCATACAACGAGAGACC

Example 2. Increase of sequenced product

A sequencing plate, which was sequenced in the biofilm system in Example 1, was mounted and sealed in the sealing chamber produced in Production Example 1. The PCR solution was then injected using a syringe through the side of the rubber lid exposed on the side of the encapsulation chamber. At this time, the PCR solution contained DNA polymerase, dNTP, a primer containing the adapter sequence proposed in the next generation sequencing, a buffer or a corresponding PCR solution master mix.

The composition of the PCR solution is as follows. 600 μl of 2X Pfu polymerase master mix, 15 μl of 100 μM primer, 585 μl of dw. Amplification and extension of the nucleic acid fragments were performed by injecting the sealed chamber into which the PCR solution was injected into a water bath. The Pfu polymerase master mix used in the experiments was a 2X Pfu polymerase premix product sold by Solgent. The primer sequence used in the experiment was the 454 For sequence of the 454 adapter sequence (CCATCTCATCCCTGCGTGTCTCCGACTCAG: SEQ ID NO: 11). For the initial DNA denaturation, the cells were maintained at 95 ° C. for 10 minutes, followed by 95 ° C., 5 minutes, 60 ° C., 5 minutes and 72 ° C. for 5 minutes for 3 to 10 times, Lt; 0 > C for 10 minutes.

Example 3. Identification of amplified products

The sealed chamber was removed from the water bath and the PCR solution and amplified and extended nucleic acid fragments were recovered through the side of the rubber lid exposed on the side of the sealing chamber using a syringe. In this way, a DNA library having undergone the sequencing was recovered. This means that the entire DNA library has been reduced to the number of readings of next-generation sequencing.

The recovered and reduced DNA library was selectively amplified by a primer containing the bar code sequence to which it was linked. The amplified individual nucleic acid fragments completed the analysis of the nucleotide sequence through Sanger sequencing and showed a much improved recovery rate compared to the conventional method of collecting individual nucleic acid fragments ('Shotgun DNA synthesis' for the high- According to Kim et al., the efficiency of recovering individual nucleic acid fragments from the entire DNA library is about 77%, but 100% recovery is possible with the present invention, according to the throughput construction of large DNA molecules, 2012 Nucleic Acids Research, Kim et al.

The present invention reduces overall DNA library through sequencing to increase recovery of final target nucleic acid fragments. In addition, it provides a method that can more effectively utilize nucleic acid fragments that have not been used differently after sequencing. In addition, an amplifier capable of amplifying the nucleic acid fragments identified by the parallel synthesis is provided, and the amplified nucleic acid fragments can be rapidly amplified when amplified using such an amplifier, It can be effectively processed and thus it is considered to be an industrially useful invention.

1: sealing chamber 100: base chamber
110: storage chamber 120: guide part
130: mounting part 200: upper chamber
210: main body 220: pressing piece
222: fixing groove 230: storage groove
240: connection hole 250: fixing member
260: mounting part 300: sample receiving part
310: accommodation space 400: gasket
410: Hall 500: Jig
510: Support part 520: Extension part
530: screw hole 600: pressing portion
610: threaded portion 620: head

<110> University-Industry Foundation <120> Method of collecting sequence-verified nucleic acid fragments and          the equipment for amplifying sequence-verified nucleic acid          fragments <130> P0530STN <160> 11 <170> Kopatentin 2.0 <210> 1 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> adapter forward primer <400> 1 ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn nnnnnnnnnn acgtacgaca 60 gagtactcgt 70 <210> 2 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> adapter reverse primer <400> 2 cctatcccct gtgtgccttg gcagtctcag nnnnnnnnnn nnnnnnnnnn tcgaactaat 60 cggattgcg 69 <210> 3 <211> 319 <212> DNA <213> Artificial Sequence <220> <223> sequencing product F1_1 <400> 3 ctcttctccg ccgcaaaatc tggcaactga aagagctggg ttatgcagcc gtggatgatg 60 aaaccacgca acagacaatg cgtgagttaa aagaactggg ctacacttcg gagccgcacg 120 ctgccgtagc ttatcgtgcg ctgcgtgatc agttgaatcc aggcgaatat ggcttgttcc 180 tcggcaccgc gcatccggcg aaatttaaag agagcgtgga agcgattctc ggtgaaacgt 240 tggatctgcc aaaagagctg gcagaacgtg ctgatttacc cttgctttca cataatctgc 300 ccgccgattt tgagagacc 319 <210> 4 <211> 319 <212> DNA <213> Artificial Sequence <220> <223> sequencing product F1_2 <400> 4 ctcttctctt gaaggcaccg atacgctggc gtataccgat gcgcagtatc aacagcttgc 60 ggcggttacg cgcgcactga ttgattgcta tccggatatc gctaaaaaca tgacgggcca 120 ttgtgatatt gcgccggatc gcaaaaccga tcccggtcct gcatttgatt gggcacgctt 180 tcgtgtgctg gtcagcaagg agacaacatg acgctattta caaccttact ggtgttaatt 240 ttcgagcgcc tgtttaagtt gggcgagcac tggcagcttg atcatcgtct tgaagcgttc 300 tttcgccgcg tgagagacc 319 <210> 5 <211> 319 <212> DNA <213> Artificial Sequence <220> <223> sequencing product F1_3 <400> 5 ctcttctgcc gccgactcaa acacctcgtc cgtcacctcc atcccgccgt gcagatcgaa 60 ctccttcgcc atctgcttgc cgagcgtagt ctggtcgtca tggaacgccg gcagacagtg 120 caggaacttc acgttcgggt tgtcggtcag cgccatcatc tgcgcgttca cctgataccc 180 gcgcagcagc gcaatgcgct ctgcccactt ctctttggcc tcgcccatcg acacccacac 240 gtcggtatag ataaagtccg cgcccttaac gcctgccgcc acgtcttccg tcagagtaat 300 tttcccgccg tgagagacc 319 <210> 6 <211> 319 <212> DNA <213> Artificial Sequence <220> <223> sequencing product F2_1 <400> 6 ctcttctgcg ggtaaccacg ccctggcgaa tgtgttctac cagcggcgca tggcaatcac 60 tcagcgagct cgacgccagg gtcaggtttt taaagcccat cttcgcgatg acgtccatca 120 ccatattgac ggtcaggtca ccgccacgga aagcgtgatg gaacgaaacc gtcatgccgt 180 cctgtaaacc tgagcgacga atcgcttctt ccaggttggc gcacagtttg cgatcgcgcg 240 ctttttcagc ctggtaggtt tgctttggcg agttctggaa agcggcaaga tcgcattcag 300 cgcgacgatt ccagagacc 319 <210> 7 <211> 319 <212> DNA <213> Artificial Sequence <220> <223> sequencing product F2_2 <400> 7 ctcttctagc tggataactt ccgtcaggaa gttcacggca atggcctctc atcgtatccg 60 cacccgaaac tgatgccgga attctggcag ttcccgaccg tatcaatggg tctgggtccg 120 attggtgcta tttaccaggc taaattcctg aaatatctgg aacaccgtgg cctgaaagat 180 acctcaaaac aaaccgttta cgcgttcctc ggtgacggtg aaatggacga accggaatcg 240 aaaggtgcga tcaccatcgc tacccgtgaa aaactggata acctggtctt cgttatcaac 300 tgtaacctgc agagagacc 319 <210> 8 <211> 319 <212> DNA <213> Artificial Sequence <220> <223> sequencing product F3_1 <400> 8 ctcttctccc ggcgttgatg gcgtgaacaa aacaatgcta caggcccgtc tggctgttga 60 gctgcaaatc ctccgtgatg aattactctc aggccactac cagcccttgc ccgcccgtcg 120 cgtttacatc cctaaaagca acggcaaact gcgcccactg ggtatccccg cgttgcgcga 180 tcgtattgtt cagcgcgcca tgctgatggc gatggagccg atttgggaga gtgattttca 240 tacgctctca tatggcttcc gccctgagcg cagtgtccac cacgcgatcc gcacggtgaa 300 attacagctc acagagacc 319 <210> 9 <211> 319 <212> DNA <213> Artificial Sequence <220> <223> sequencing product F3_2 <400> 9 ctcttctaac gcctgccgcc acgtcttccg tcagagtaat tttcccgccg tgcttctccg 60 ccagcgcgct gcactccgcc accaggctct cttccggcca gcaggctttc ggggccaaca 120 ggcgcagatc cagcccggtc agcgccgccg cttccagcat cgagttgccc atgttgttgc 180 gcgcatcgcc cgcgtagacc agcgtcatct cgttaaacgc cttgcccggc aggtgctcct 240 gcatggtcat caggtccgcc agcagctggg tcgggtggaa ctcgttggtc agcccgttcc 300 acaccggcac gcagagacc 319 <210> 10 <211> 319 <212> DNA <213> Artificial Sequence <220> <223> sequencing product F4_1 <400> 10 ctcttctcag tgcgagttcc ggctgaccag gaatgtaacg ttgcgcatgc agccaccagt 60 agagggcatt gcttcccagt ccacgttttt ctgcctgttg tagccagcga tcgcgagccg 120 caccatttcc tgccgcctgg gcggtattgg cagcagcaag cagatcctca ttgctcatgt 180 cgtgaagact gattttctgc caggccgcca gtgcggtggc gtagtcctca acctgatacg 240 cctgataggc taccgcacga tgttgccagg cgctcggttg gcgttgttcg gcctgaagcc 300 atgcatacaa cgagagacc 319 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 454 Forward sequence <400> 11 ccatctcatc cctgcgtgtc tccgactcag 30

Claims (22)

(a) positioning nucleic acid fragments identified by sequencing in an amplifier;
(b) amplifying the sequence-confirmed nucleic acid fragments; And
(c) recovering said amplified nucleic acid fragments,
Wherein the step (a) is performed by placing the sequencing plate in which the sequence-confirmed nucleic acid fragments are present in the amplifier in the state after completion of the sequence confirmation. The method according to claim 1,
(d) recovering the desired nucleic acid fragments from the recovered nucleic acid fragments. The method according to claim 1,
Wherein the sequencing is performed by a method of synthesizing nucleic acid fragments. The method of claim 3,
Wherein the synthetic method is a Sanger method or a super parallel method. 5. The method of claim 4,
Wherein the hyperparallel method is selected from the group consisting of pyrosequencing chemistry, bridge amplification, next generation sequencing, third generation sequencing, next generation sequencing, and semiconductor sequencing. The method according to claim 1,
Wherein the amplifier is a sealed chamber in which a predetermined sample is received and sealed. The method according to claim 1,
Further comprising injecting a polymerase, dNTP, or DNA polymerase solution into the amplifier for amplification of the nucleic acid fragment. delete 3. The method of claim 2,
Wherein the recovery of the desired nucleic acid fragments is carried out using the beads when the amplified nucleic acid fragments are bound to the beads. 3. The method of claim 2,
Wherein the recovery of the desired nucleic acid fragments is recovered using a sequence homologous to the barcode sequence when a specific barcode sequence is linked to the amplified nucleic acid fragments. (a) linking adapter sequences to nucleic acid fragments prior to sequencing;
(b) sequencing nucleic acid fragments to which said adapter is attached;
(c) positioning said sequence-identified nucleic acid fragments in an amplifier;
(d) amplifying the sequence-confirmed nucleic acid fragments; And
(e) recovering the amplified nucleic acid fragments. 12. The method of claim 11,
Wherein the step of connecting the adapter sequence is a method using PCR assemble or a method using ligation. (a) preparing a sequencing plate having a DNA library;
(b) providing a scaled down nucleic acid fragment library sequenced through hyperparallel sequencing of said DNA library;
(c) positioning the sequencing plate with the sequence-identified reduced nucleic acid fragment library in an amplifier;
(d) amplifying the entire nucleic acid fragments in the library located in the amplifier; And
(e) recovering the amplified nucleic acid fragments. 14. The method of claim 13,
(f) recovering the desired nucleic acid fragments from the recovered nucleic acid fragments. A sealing chamber for amplifying nucleic acid fragments,
A base chamber capable of receiving a sequencing plate having the nucleic acid fragments;
An upper chamber which is engageable with and detachable from the base chamber and has a sealing structure as a whole when engaged with the base chamber; And
And an elastic gasket disposed between the base chamber and the upper chamber for sealing,
An inner space in which the PCR solution for amplifying the nucleic acid fragments is accommodated is formed by the coupling of the base chamber and the upper chamber,
Wherein at least one of the base chamber and the upper chamber is made of a material having thermal conductivity. A sealing chamber for amplifying nucleic acid fragments,
A base chamber in which a storage chamber is formed;
An upper chamber disposed on the base chamber;
A sample accommodating portion accommodated in the accommodating chamber and having a receiving space for accommodating a sample for amplifying the nucleic acid fragments;
A gasket disposed between the base chamber and the upper chamber and having elasticity;
A jig having a threaded hole connected to the base chamber and extending to an upper portion of the upper chamber, the threaded hole having an internally threaded portion; And
And a pressing part screwed to the screw hole,
Wherein the pressing portion includes a screw portion having a male screw portion and a head disposed on the screw portion,
Wherein the pressing of the upper chamber by the pressing portion is achieved as the head is pivoted to displace the pressing portion with respect to the jig. 17. The method of claim 16,
The jig,
A U-shaped support portion bent on both sides facing each other, and
And a predetermined extension portion connecting the upper portion of the support portion,
The screw hole is formed in the extended portion,
And the base chamber and the upper chamber are supported by the support portion. 17. The method of claim 16,
Wherein the base chamber comprises:
And a guide portion for allowing the gasket and the upper chamber to be fixed in position. The method of claim 16, wherein
The upper chamber includes:
The body portion,
A pressing piece which is pressed in contact with the pressing portion, and
And a housing groove formed in the main body portion and accommodating the pressing piece. The method of claim 19, wherein
The upper chamber includes:
A connection hole formed at a side of the main body and connected to the receiving groove,
A fixing groove formed on a side of the pressing piece,
And a fixing member connected to the connection groove through the connection hole. The method of claim 16, wherein
A predetermined first irregular portion is formed on an upper surface and a lower surface of the gasket in contact with the base chamber and the upper chamber,
And a second concavo-convex portion corresponding to the first concavo-convex portion is formed on an upper surface of the base chamber and a lower surface of the upper chamber, which contact the gasket. The method of claim 6, wherein the sealing chamber is the sealing chamber of any one of claims 15 to 21.

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