KR20140006369A

KR20140006369A - Method for preparing rnai library

Info

Publication number: KR20140006369A
Application number: KR1020120073003A
Authority: KR
Inventors: 황병준; 김민정; 박성균
Original assignee: (재) 스크립스코리아항체연구원
Priority date: 2012-07-04
Filing date: 2012-07-04
Publication date: 2014-01-16

Abstract

The present invention relates to a method for manufacturing an RNAi library using molecular biology methods including several restriction enzymes, wherein, compared with existing technologies, the method for manufacturing the RNAi library can reduce time and costs consumed in manufacturing the RNAi library and also manufacture the RNAi library about genes of various animals and plants whose information within genomes are not well-known as well as about genomes of humans, rats and the like. The present invention is expected to greatly contribute to studies using the domestic RNAi library, which are absolutely in poor situations compared with overseas countries, by solving problems of expensive development and maintenance of RNAi libraries used in many areas such as biology, pharmacy, medicine and the like, including new medicine development and mechanism research for causes of diseases.

Description

ＲＮＡｉ library production method {Method for preparing RNAi library}

The present invention relates to a method for preparing an RNAi library, and more particularly, to a method for preparing an RNAi library, which is an improvement of the prior art for preparing an RNAi library through PCR amplification using an oligonucleotide as a template.

Specifically, the present invention relates to a RNAi library fabrication method using molecular biological methods including various restriction enzymes, while reducing the time and cost required for RNAi library fabrication compared to the prior art, as well as in genomes such as humans and mice The present invention relates to a method for producing RNAi libraries for genes of various plants and animals whose information on genes is not well known.

siRNA is a small RNA fragment of 21 to 25 nucleotides in size produced by cleavage of double-stranded RNA by Dicer and specifically binds to mRNA with complementary sequences to inhibit protein expression. It is known. The presence of siRNA was first confirmed by an experiment by Andrew Fire of Carnegie Laboratories and Craig Mello of the University of Massachusetts Medical School in 1998. When the double-stranded RNA was injected into the nematode, it was complementary to mRNA and sequence specific cells. It was confirmed that by inducing gene silencing (gene silencing). This phenomenon is called RNA interference (RNA interference).

After the first report of siRNA successful knock-down of a target gene in human somatic cells in 2001, it has been applied to functional genome research, drug target discovery, and siRNA drug development. siRNA is expected to be able to greatly alleviate the problem of gene delivery efficiency, which is a drawback of the existing gene therapy methods, because it can be delivered intracellularly with much higher efficiency than the nucleic acid used in conventional gene therapy.

Therefore, by controlling the expression of target genes in specific disease cells by using siRNA as a gene therapy agent, it is possible to treat refractory diseases such as cancer, ischemic diseases, viral diseases (HIV, HCV, HBV, etc.). With this in mind, siRNAs are being actively developed as therapeutic agents for various diseases.

In particular, therapeutics using the RNAi concept can destroy specific mRNAs in much lower amounts than conventional antisense methods, and RNAi requires only double-stranded RNA information because the mechanisms present in the cell There is an advantage that can induce specific mRNA destruction even at low concentrations.

In addition, high-throughput screens using RNAi libraries composed of siRNAs / shRNAs for many genes have been widely performed at home and abroad for functional dielectric studies and new drug targets targeting various phenomena in cells. Nematode and Drosophila researchers have already built RNAi libraries for 10,000 and 15,000 genes, and have built human disease-related model systems for screening genes. Based on these results, various important disease-related genes and useful genes have been discovered in the model system, and the role of human disease-related genes is being visualized.

On the other hand, RNAi libraries targeting genomes of various higher organisms, including humans and mice, are also used, but are not widely used in Korea because they require a lot of costs for the production, maintenance, and use of the library. And new drug target derivation studies are also being hampered.

Accordingly, in the technical field of the present invention, while providing RNAi library for genes of humans and mice, as well as genes of various plants and animals whose information on genes in the genome is not well known, the time and cost required for RNAi library production are provided. There is a need for new technologies that can be saved.

Republic of Korea Patent Publication No. 10-2004-0072643 (2004.08.18)

SUMMARY OF THE INVENTION An object of the present invention is to improve a conventional technique for preparing RNAi libraries through PCR amplification using oligonucleotides as a template, and to provide a method for preparing RNAi libraries, which is shorter in production time and more cost effective than RNAi libraries. .

Specifically, an object of the present invention is to provide a method for preparing RNAi library using molecular biological methods including various restriction enzymes, while reducing the time and cost of RNAi library production compared to the prior art, such as human, mouse, and the like Of course, it provides a way to create RNAi libraries for genes of several plants and animals whose information on genes in the genome is not well known.

The present invention provides a method for producing RNAi library to achieve the object as described above.

RNAi library production method of an embodiment of the present invention,

(a) cleaving and fragmenting genomic DNA to obtain a DNA fragment;

(b) binding a first adapter comprising a first restriction enzyme recognition site to the ends of the DNA fragments,

(c) amplifying the DNA fragment to which the first adapter is bound using a primer set labeled with a first label,

(d) treating the DNA amplification product obtained in step (c) with a first restriction enzyme and generating a DNA fragment by cleaving the DNA amplification product by recognizing the first restriction enzyme recognition site. Steps,

(e) separating the DNA fragment with the first label from the cut DNA fragment using a medium that specifically binds to the first label,

(f) a second adapter corresponding to each of the plurality of library types to be produced is coupled to the ends of the separated DNA fragments, each having a different sequence for each of the plurality of library types and including a second restriction enzyme recognition site; Combining the two adapters,

(g) amplifying a plurality of DNA fragments each having a second adapter coupled to each of a plurality of library types using a primer set labeled with a second label,

(h) generating a DNA fragment by treating the DNA amplification product obtained in step (g) with a second restriction enzyme and cleaving the DNA amplification product by recognizing the second restriction enzyme recognition site. Steps,

(i) separating the DNA fragments with the second label from the cleaved DNA fragments by a plurality of library types using a medium that specifically binds the second label;

(j) constructing a library by cloning a DNA fragment separated by a plurality of library types into a vector in step (i).

RNAi library production method of an embodiment of the present invention, the DNA fragment separated by a plurality of library types in the step (i), each having a different sequence for each of the plurality of library types, including a third restriction enzyme recognition site Binding a third adapter of the topography; and processing a third restriction enzyme to recognize the third restriction enzyme recognition site, thereby cleaving the DNA amplification product to cut the DNA amplification product. The method may further include coupling the hairpin structure to the generated DNA fragment.

In the RNAi library preparation method of an embodiment of the present invention, the first label may be biotin, and the medium specifically binding to the first label may be streptavidin.

In the RNAi library preparation method of an embodiment of the present invention, the second label may be Dioxygenin, and the medium specifically binding to the second label may be an anti-dioxygenin antibody.

In the RNAi library production method of an embodiment of the present invention, the first restriction enzyme may be MmeI restriction enzyme, and the second restriction enzyme may be BpmI restriction enzyme. In addition, the third restriction enzyme may be a BsmI restriction enzyme.

In the RNAi library production method of an embodiment of the present invention, the first adapter may be an oligonucleotide pair of SEQ ID NO: 1 and 2, and / or an oligonucleotide pair of SEQ ID NO: 3 and 4.

In the RNAi library manufacturing method of an embodiment of the present invention, the second adapter may be an oligonucleotide pair of SEQ ID NOs: 9 and 10, or an oligonucleotide pair of SEQ ID NOs: 11 and 12.

In an RNAi library manufacturing method of an embodiment of the present invention, the branched third adapter is an oligonucleotide pair of SEQ ID NOs: 17 and 18, an oligonucleotide pair of SEQ ID NOs: 19 and 20, or an oligonucleotide of SEQ ID NOs: 21 and 22 May be a pair.

In the RNAi library manufacturing method of an embodiment of the present invention, the hairpin structure is a group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 23 to SEQ ID NO: 27 and oligonucleotides having a base sequence complementary to these oligonucleotides Can be selected from.

According to the RNAi library manufacturing method of the present invention, the RNAi library is shorter production time and cost-effective than the prior art.

In particular, the present invention is to prepare RNAi library using molecular biological methods including a number of restriction enzymes, while reducing the time and cost required to produce RNAi library, information on genes in the genome as well as genomes such as humans and mice RNAi libraries can be made for genes of several unknown species.

Therefore, the present invention solves the problem of high development and maintenance cost of RNAi library used in many fields such as biology, pharmacy, medicine, including new drug development, disease cause mechanism research, etc. It is expected to contribute greatly to the research using the RNAi library.

1 is a photograph of electrophoresis on 1X TBE polyacrylamide gel after DNA fragments were prepared by treating fragmented enzymes on extracted genomic DNA. In Figure 1 it can be seen that the size and amount of the fragments change as the enzyme treatment time elapses.
Figure 2 is a photograph of an electrophoresis on 1X TBE polyacrylamide gel after attaching the blunt terminal adapter to the genomic DNA fragment prepared in Example 1 and performing PCR amplification. Lane 1 shows the results of electrophoresis of PCR amplification products, and lane 2 shows the results of electrophoresis after MmeI restriction enzyme treatment for PCR amplification products (about 60bp can be confirmed).
Figure 3 is a photograph of the electrophoresis on 1X TBE polyacrylamide gel after binding of the adapter according to the library type to the enzyme amplified DNA amplification products and PCR amplification reaction (polymerase chain reaction II). The results of electrophoresis of amplification products for libraries of type 1, type 2 and type 3 in the order of lanes 1, 2 and 3 are shown, which are the same size and form nonspecific products in addition to the target DNA. Can be observed.
Figure 4 is a photograph of electrophoresis on 1X TBE polyacrylamide gel after performing the re-amplification reaction (polymerase chain reaction III) after separating only the target DNA. It can be seen that only products specific to the target DNA were produced.
Figure 5 is a photograph of the electrophoresis on 1X TBE polyacrylamide gel after the final amplification reaction (polymerase chain reaction V) for the target DNA for type 1, type 2 and type 3 libraries. Lanes 1 and 2 are type 2, lanes 3 and 4 are type 3 and lanes 5 and 6 are type 1 electrophoresis results.

Hereinafter, the present invention will be described in more detail based on the embodiments of the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are incorporated herein by reference.

Example

Example 1: Cell Line and gDNA Extraction

A549 and SK-OV-3 cell lines were used from the laboratory of Professor Dong-Wan Seo of Kangwon National University. After culturing this cell line in a 5% CO ² , 37 ° C. incubator, gDNA was extracted using a QIAamp DNA Mini Kit (51304) sold by Qiagen. In preparation for the possibility of RNA extraction, Roche's RNase (10109142001) was treated, and the quality of gDNA was confirmed through an absorber (Nano-drop machine, Thermo).

Example 2: RNAi Library Preparation Sequence

(1) cleavage of gDNA

DNA fragments were prepared by cutting the genomic DNA extracted in Example 1 for 60 minutes (or time adjustable according to the desired size) using NEB's double stranded DNA fragmentation enzyme (dsDNA Fragmentase) (M0348S). (See FIG. 1), which is used as the base material for RNAi library preparation.

(2) Blunt end formation and adapter attachment

Since the material obtained in step (1) is not large enough, a blunt end adapter (a DNA fragment in which restriction enzymes and library related sequences are located) is amplified and linked to DNA ligase. Heat the DNA fragments that are not double-stranded to bond the adapters of set A) and the adapters of set B) as shown below, and then connect them to the original DNA fragment material [the DNA fragment obtained in step (1)]. .

A) Set Adapter: HwaSkai8, HwaSkai9

HwaSkai8 (SEQ ID NO: 1):

5'- TAA TAC GAC TCA CTA TAG GG A TGA AGC TTG GTG CAG GGT GAG ACC GCT GGC C-3 '

HwaSkai9 (SEQ ID NO: 2):

5'-GGC CAG CGG TCT C-3 '

B) Set Adapters: HwaMme-3, HwaMme-4

HwaMme-3 (SEQ ID NO: 3):

5'- TAC GAT TTA GGT GAC ACT ATA G GG CGA CAG TTG GAA TG C TGG AG G CTA CAG CAG TCC AAC -3 '

HwaMme-4 (SEQ ID NO: 4):

5'-GTT GGA CTG CTG TAG-3 '

(3) polymerase chain reaction I (PCR amplification I)

The DNA fragment obtained in step (2) was subjected to polymerase chain reaction using Clontech's Advantage 2 PCR kit (639206) (see lane 1 of FIG. 2). As primers for PCR amplification, the following primer sets are used.

Primer set: pair of HwaSkai10 and HwaSkai13 (biotinylated)

HwaSkai10 (SEQ ID NO: 5):

5'-ATG AAG CTT GGT GCA GGG TGA GAC CGC-3 '

Biotinylated HwaSkai13 (SEQ ID NO: 6):

Biotin-TEG-5'- ATA G GG CGA CAG TTG GAA TG C TGG AGG -3 '

(4) cleavage of MmeI restriction enzyme

PCR amplification reaction of step (3) is purified using a Qiagen PCR purification kit, and then cleaved with MmeI (NEB R0637) restriction enzymes located in HwaMme-3 and HwaMme-4 adapters (see lane 2 in FIG. 2). ). Approximately 60 bp of amplification products (HwaMme-3, HwaMme-4 adapter + gDNA fragment 20bp = 60bp) can be identified, and the size of the gDNA is changed to about 20bp during cleavage.

This process is performed the same regardless of the library type, but in the subsequent process, the adapter to be attached depends on the library type.

(5) Streptavidin tube attachment and PCR amplification adapter connection

A fragment of DNA obtained after cleavage of the MmeI enzyme for the PCR amplification product in step (4) has a protein called Biotin attached thereto to remove the remaining adapter and impurities not necessary for the subsequent process. Biotin protein specifically binds to a protein called streptavidin, which is obtained in step (4) in a polymerase chain reaction vessel (PCR tube: mRNA capture kit, Roche) to which streptavidin protein is attached. Attached DNA fragments. PCR amplification adapters connected according to the library type (type 1, type 2 and type 3) are as follows.

Type 1: HwaMmeDuap-1, HwaMmeDuap-2

HwaMmeDuap-1 (SEQ ID NO: 7):

Phospho-5'-TTT TCT GTC TTC ACT GCG TTG ATA CCA ATG GTT TGT TCC GTA-3 '

HwaMmeDuap-2 (SEQ ID NO: 8):

5'-AGT GAA GAC AGA AAA NN-3 '

Type 2: HwaSBIBsm-1, HwaSBIBsm-2

HwaSBIBsm-1 (SEQ ID NO: 9):

Phosphate-5'-CTg CAt TcC TAC TCT GCG TTG ATA CCA ATG GTT TGT TCC GTA-3 '

HwaSBIBsm-2 (SEQ ID NO: 10):

5'-AGA GTA GgA aTG cAG NN-3 '

Type 3: HwaMmeBsm-1, HwaMmeBsm-2

HwaMmeBsm-1 (SEQ ID NO: 11):

Phosphate-5'-TAG CAt TcC TAC TCT GCG TTG ATA CCA ATG GTT TGT TCC GTA-3 '

HwaMmeBsm-2 (SEQ ID NO: 12):

5'-AGA GTA GgA aTG CTA NN-3 '

(6) polymerase chain reaction II (PCR amplification II)

The DNA fragment obtained in step (5) was subjected to polymerase chain reaction using Clontech's Advantage 2 PCR kit (639206) (see FIG. 3). As primers for PCR amplification, the following primer sets are used.

Primer set: pair of HwaSkai13 (biotinylated) and HwaMme-23

Biotinylated HwaSkai13 (SEQ ID NO: 6):

Biotin-5'-TEG- ATA G GG CGA CAG TTG GAA TG C TGG AGG -3 '

HwaMme-23 (SEQ ID NO: 13):

5'-TAC GGA ACA AAC CAT TGG TAT CAA CGC-3 '

(7) isolation of target DNA fragments

TBE 8% polyacrylamide gels are used to isolate only the desired DNA fragment and attach it back to the streptavidin tube. Biotin is attached to one end of the DNA fragment obtained through amplification of step (6) and separated through a polyacrylamide gel. The binding of biotin and streptavidin tubes attached to the target DNA fragment is used to remove impurities (eg, enzymes and buffers) other than the target DNA fragment.

(8) Polymerase Chain Reaction III (PCR Amplification III)

The same procedure as in (6), and the primer set below is used as a primer for PCR amplification (see FIG. 4). Figure 4 shows the results of electrophoresis of amplification products for libraries of type 1, type 2 and type 3 in the order of lane 1, lane 2 and lane 3, showing that only products specific to each target DNA were produced. You can check it. On the other hand, the target DNA produced as a result of amplification has a protein called Dioxygenin (Dioxygenin) attached to one end.

Primer set: pair of mHwaSkai13 (no modification) and HwaMme-24 (deoxygeninization)

HwaSkai13 (SEQ ID NO: 6):

5'- ATA G GG CGA CAG TTG GAA TG C TGG AGG -3 '

Deoxygeninated HwaMme-24 (SEQ ID NO: 14):

DIG-5'-TAC GGA ACA AAC CAT TGG TAT CAA CGC-3 '

(9) BpmI restriction enzyme cleavage

The amplified product amplified in step (8) was cut for 2 hours at 37 ° C using BpmI restriction enzyme from NEB.

(10) Bead Binding Using Dioxygenin Antibodies

One end of the DNA cleaved with BpmI in step (9) has a protein called deoxygenin attached. Thus, the DNA (BpmI cleavage) in which the deoxygenin is bound to one end is connected to the protein A / G magnetic beads (100-03D, 100-01D of Invitrogen) using anti-oxygenin (Roche, 11333062910). do. As a result, when the BpmI cleavage-anti-deoxygenin-A / G magnetic bead conjugate is produced and attached to the magnet, all impurities except the linked magnetic bead complex can be removed.

This process is performed in the same manner except that different adapters are connected according to three types of libraries, but different processes are performed for Type 1, Type 2, and 3 in subsequent processes. In other words, the adapter to be attached depends on the library type.

(11) Brachy adapter (Biotinylated) or with adapter

For type 1 libraries:

DNA ends for the type 1 library obtained in step (10) were made blunt ends, and the following Hwang2011CJ and Hwang2011CK adapters were attached thereto to make a structure that can be inserted into a viral vector.

Hwang2011CJ (SEQ ID NO: 15):

5'-gg tag gcc gAA TAT Ttc ttg gct GGa tCC atc ttg tgg aaa gga cga aaa aaa g-3 '

Hwang2011CK (SEQ ID NO: 16):

5'-c ttt ttt tcg tcc ttt cca c-3 '

For type 2 libraries:

A branched adapter, HwanoHair-32, Hwang2011BK or HwangAAA, HwangBBB, is connected to the DNA terminus for the type 2 library in the manner described above without blunt termination.

Biotinylated HwanoHair-32 (SEQ ID NO: 17):

Biotin-TEG-5'-CAG ATA GAG AGT ACG TGC TCC TGC TGA AGG AGG gGc AGT AGG CAC-3 '

Hwang2011BK (SEQ ID NO: 18):

phosphate-5'-G CCT ACT GCA TAG GAA TTC TGC CCT CAC GAG TCG AAG GCA CAG-3 '

Biotinylated HwangAAA (SEQ ID NO: 19):

Biotin-TEG-5'-CAG ATA GAG AGT ACG TGC TCC TGC TGA AGG Acc tGc AGT AGG CAC-3 '

HwangBBB (SEQ ID NO: 20):

phosphate-5'-G CCT ACT gCc TcG GAA TTC TGC CCT CAC GAG TCG AAG GCA CAG-3 '

For type 3 libraries:

A branched adapter, BiotinHwaSBI-6, HwaSkai15, is linked to the DNA terminus for the type 3 library in the same manner as described above without blunt termination.

BiotinHwaSBI-6 (SEQ ID NO: 21):

Biotin-TEG-5'-CAG ATA GAG AGT ACG TGC TAC ACT GGA GGG CTA GAG CAG GAG CAC-3 '

HwaSkai15 (SEQ ID NO: 22):

Phospho-5'-GCTCCTGCTAGCTACTTCAGAATGCCACATGTAGACGCCACGT-3 '

(12) BsmI cleavage generation for attaching hairpin structures

Only the products for type 2 and type 3 libraries of the products obtained in step (11) are performed. BsmI (NEB, R0134) restriction enzyme treatment is performed to remove the adapter for the polymerase chain reaction in the BpmI cleavage-anti-Dig-A / G magnetic bead-specific branched adapter.

(13) Streptavidin tube attachment and pre hairpin structure attachment

Biotin-attached BsmI fragments (types 2 and 3) obtained in step (12) were attached to a polymerase chain reaction vessel (PCR tube: mRNA capture kit, Roche) to which streptavidin protein was attached. Connect the Pre Hairpin structure as shown below. Amplify this structure because not enough is available.

Free hairpin constructs for type 2: HwaSBIBseBsm-19E, HwaSBIBseBsm-19F, HwaSBIBseBsm-19G, HwaSBIBseBsm-19H

HwaSBIBseBsm-19E (SEQ ID NO: 23):

Phosphate-5'- AAG AGA T GGAG TC gGA TGCAT g tcc ga ggag A TCT CTT cA-3 '

HwaSBIBseBsm-19F (SEQ ID NO: 24):

Phosphate-5'- AAG AGA T GGAG TC gG A TGCAT ga cc ga ggag A TCT CTT cA-3 '

HwaSBIBseBsm-19G (SEQ ID NO: 25):

Phosphate-5'- TCC GA CT CTC C CT C CCTGCAG acaa ga ggag AG TC GGA cA-3 '

HwaSBIBseBsm-19H (SEQ ID NO: 26):

Phosphate-5'- TCC GA CT CTC TCT C CCTGCAG acaa gag gag AG TC GGA cA-3 '

Free hairpin structure for type 3: Hwang2011CF

Hwang2011CF (SEQ ID NO: 27):

Phosphate-5'-TGAAG CCAC AGATGA GCTC agaa gaggag gtgg cttCA CT-3 '

(14) polymerase chain reaction IV (PCR amplification IV)

PCR amplification of the target DNA fragment for the type 1 library obtained through step (11), and the target DNA fragment for the type 2 and type 3 libraries obtained through step (13). In the PCR process, the reaction is performed using a hot start tag (Finzyme's 540S polymerase). As primers for PCR amplification, a primer set in which only one primer is attached to biotin is used as follows.

Primer pair of type 1: primer pair of Hwang2011CM (biotinylated) and HwaMme23

Biotinylated Hwang2011CM (SEQ ID NO: 28):

Biotin-TEG-5'-gg tag gcc gAA TAT Ttc ttg gct GGa tCC-3 '

HwaMme-23 (SEQ ID NO: 13):

5'-TAC GGA ACA AAC CAT TGG TAT CAA CGC-3 '

Primer pairs of type 2: primer pairs of HwaSBI-7 and HwaSkai17 (biotinylated)

HwaSBI-7 (SEQ ID NO: 29):

5'-CAG ATA GAG AGT ACG TGC TAC ACT GG-3 '

Biotinylated HwaSkai17 (SEQ ID NO: 30):

Biotin-TEG-5'-ACG TGG CGT CTA CAT GTG GCA TTC TGA-3 '

Primer pairs of type 3: primer pairs of Hwang2011BM and HwangCCC (biotinylated)

Hwang2011BM (SEQ ID NO: 31):

5'-CTG TGC CTT CGA CTC GTG AGG GCA G-3 '

Biotinylated HwangCCC (SEQ ID NO: 32):

Biotin-TEG-5'-ATA GAG AGT ACG TGC TCC TGC TGA AGG Acc-3 '

(15) Target DNA Fragment Isolation

Repeat the above procedure (7) to isolate the target DNA as well as the target product, as well as the target product.

(16) polymerase chain reaction V (PCR amplification V)

Same as step (14), but use primer set with biotin attached to both primers as below. The amplification result is as shown in FIG.

Primer pair of type 1: a pair of biotinylated Hwang2011CM (SEQ ID NO: 28) and biotinylated HwaMme-23 (SEQ ID NO: 13)

Primer pair of type 2: a pair of biotinylated HwaSBI-7 (SEQ ID NO: 29) and biotinylated HwaSkai17 (SEQ ID NO: 30)

Primer pair of type 3: a pair of biotinylated Hwang2011BS (SEQ ID NO: 33) and biotinylated HwangCCC (SEQ ID NO: 32)

Biotinylated Hwang2011BS (SEQ ID NO: 33):

Biotin-TEG-5'-CTG TGC CTT CGA CTC GTG AGG GCA G-3 '

(17) The amplification products obtained in step (16) are inserted into a carrier vector suitable for the target DNA for the type 1, type 2 and type 3 libraries, respectively. The vector to be used is well known in the art and readily available, and the insertion into the vector is also performed according to methods known in the art.

Example 3: RNAi Library Construction

In this example, RNAi libraries were prepared from genomic DNA according to the RNAi library preparation procedure described in Example 2 under the conditions described below. The same or similar contents as in Example 2 are omitted for convenience.

(1) cleavage of gDNA or desired DNA

Fragmentation conditions in the present embodiment was 60 minutes and genomic DNA or the desired DNA was prepared and proceeded as follows. First, except for fragmentation enzyme (dsDNA Fragmentase enzyme) was mixed with gDNA and the remaining components as shown in the composition of Table 1 below. Fragmentation enzyme was added later. The reaction was allowed to stand on ice for 5 minutes and soaked in a 37 ° C. water bath until the desired size was obtained.

Reaction components Starting DNA Volume (μg) DNA (μg) One 2 3 4 5 10X fragmentation reaction buffer (μl) 2 4 6 8 10 100X BSA (μl) 0.2 0.4 0.6 0.8 1.0 Fragmentase (μl) 2 4 6 8 10 Final volume (μl) 20 40 60 80 100

Once the desired fragment size was produced, 5 μl of 0.5 M EDTA was added to stop the reaction. Integrin for fragment identification was electrophoresed on 1.0% agarose gel.

(2) Blunt end formation and adapter attachment

1) Blunt end formation

At the end of step (1), put it on ice and cool it completely. Polymerase Chain Reaction Container (PCR Tube) On ice, the cold machine was also pre-set to 12 ° C. 0.1 mM of dNTP and 5 μl of T4 DNA polymerase were added thereto, followed by vortexing and transfer to a cold PCR tube. And this was kept at 12 ° C. for 30 minutes. After the reaction was completed, the impurities were removed in addition to the DNA using a PCR purification kit, and then blunt terminal adapters were attached.

2) With blunt end adapter

Blunt end adapter formation

The fragments described in step (2) of Example 2 were made into 10 pmol and held at 80 ° C. for 5 minutes and then slowly cooled to room temperature. After reaching room temperature it was kept at 4 ° C. for 20 minutes and stored at −20 ° C.

Adapter adaptation (20µl)

2.0 μl of 10 × T4 DNA ligase buffer, 14.0 μl of the DNA, 2.0 μl of T4 DNA ligase and 2.0 μl of blunt end adapters were mixed and left at 4 ° C. overnight or for 6 hours.

(3) polymerase chain reaction I (PCR amplification I)

DNA obtained in step (2) was amplified under the composition of Table 2 below using the biotinylated primer set described in step (3) of Example 2.

Advantage 2 buffer Remarks dNTP (5mM each) Primers: 10 pmol HwaSkai10 and HwaSkai13 (biotinylated) Mold (Ligation Mixture) Phusion DNA pol (not hot start) 50X advantage 2 pol DMSO DW

On the other hand, PCR amplification conditions were 55 ℃, 10 minutes-[95 ℃, 1 minute-64 ℃, 0.5 minutes-68 ℃, 5 minutes] X cycle-68 ℃, 7 minutes-4 ℃, 8 after completion of the amplification process Amplification products were identified on the% TBE polyacrylamide gel.

(4) cleavage and dephosphorylation of MmeI restriction enzyme

Mme I restriction enzyme digestion (40 μl) DNA (1ug) Buffer (4) enzyme BSA (10x) SAM DW 4 4 0 2

Incubate at 37 ° C. for 4 hours under the composition of Table 3. The DNA of this state was cleaved with MmeI and subjected to electrophoresis on 8% TBE polyacrylamide gel with 25 bp and 100 bp ladders (DNA standard) to identify 62 bp fragments.

Dephosphorylation (Shirimp Akaline Phosphatase, Roche) treatment was performed by standing at 37 ℃ for 20 minutes.

(5) Streptavidin tube attachment and PCR amplification adapter connection

Process 1) Only about 100 ng of the DNA fragment obtained in step (4) was attached to the streptavidin tube.

① PCR tubes were washed with 250 μl of wash buffer.

② Biotin-labeled DNA (MmeI digested) was mixed with 2X binding buffer (Binding Buffer).

③ It was allowed to stand at 37 ° C. for 5 minutes.

④ after rotating at a high speed at room temperature.

⑤ After washing three times with a washing buffer.

⑥ Dilute the adhesion buffer with 1x and wash.

Course 2) Adapter Ligation

① A NIN ₂ mixed adapter was prepared. The following fragments were made into 10 pmol and held at 80 ° C. for 5 minutes and then slowly cooled to room temperature. After reaching room temperature it was kept at 4 ° C. for 20 minutes and stored at −20 ° C.

Type 1: HwaMmeDuap-1, HwaMmeDuap-2

Type 2: HwaSBIBsm-1, HwaSBIBsm-2

Type 3: HwaMmeBsm-1, HwaMmeBsm-2

② Adapter ligation was performed on the tube prepared in step 1). 2.0 μl 10 × T4 DNA ligase buffer, DW 14.0 μl, T4 DNA ligase 2.0 μl and blunt end adapter 2.0 μl were mixed and left overnight at 4 ° C. or for 6 hours.

(6) polymerase chain reaction II (PCR amplification II)

① Remove the supernatant of the tube (5) is completed using a pipette.

② was cleared with Roche cleaning solution, and in this process, the pipette was used to remove impurities.

③ After that, three clearings were performed with the same washing solution.

④ PCR amplification is performed using a new primer set because separation from polyacrylamide gel is required, using the biotinylated primer set described in step (6) of Example 2 under the composition of Table 4 below. Amplified.

10 × Advantage 2 buffer Remarks dNTP (10mM each) Primers: HwaSkai13 (Biotinylated) and HwaMme-23 Mold (Ligation Mixture) Attached 50X advantage 2 pol DMSO DW

PCR amplification conditions were 95 ° C, 0.5min-[95 ° C, 1min-64 ° C, 0.5min-68 ° C, 1min] X cycle-68 ° C, 5min-4 ° C. found.

(7) isolation of target DNA fragments

1) Electrophoresis after gel formation

① Big gel needs 45ml of polyacrylamide gel mix and hardened by adding aps and temed to prevent the gel from counting at the bottom.

② Se 600 Ruby (GE) Big gel system was assembled and the gel prepared above was poured for 2 hours and used.

③ Put the above system into the buffer tank and fill it with 1X TBE buffer and electrophoresis for 2 hours and 40 minutes at 200V and 500mA.

④ Et-Br (2ug / ml) staining was performed for 10 minutes.

⑤ Destaining was performed three times for 2 minutes.

2) Gel Extraction

① The product of about 104bp (which can vary in size depending on the purpose) was cut out finely so that there was no contamination of other products.

② Centrifuge for 5 minutes at maximum speed using the prepared tube system.

③ Repeat the process of ② was centrifuged for about 5 minutes and if the gel remained in the upper tube, centrifugation time was increased.

④ 150μl of 1X Adhesion Buffer (Binding buffer) was added and kept at 50 ° C for 30 minutes.

⑤ The supernatant except for the gel pieces was transferred to the prepared tube.

⑥ Again, 150 μl of the binding buffer (Binding buffer) was added and the process ④ and ⑤ were repeated.

3) Streptavidin tube binding: 20 μl of DNA extracted in step 2) was bound.

① PCR tubes were washed with 250 μl of wash buffer.

② Up to 20 μl of biotin-labeled DNA was incubated at 37 ° C. for 5 minutes.

③ After the rotation at room temperature at a high speed was stationary.

④ it was washed three times with a washing buffer.

(8) Polymerase Chain Reaction III (PCR Amplification III)

10 × Advantage 2 buffer Remarks dNTP (5mM each) mmHwaSkai13 (no deformation) and HwaMme-24 (deoxygeninated) 10 pmol Mold (Ligation Mixture) Attached 50X advantage 2 pol DMSO DW

PCR amplification conditions were 95 ° C, 0.5min-[95 ° C, 1min-68 ° C, 0.5min-68 ° C, 1min] X cycle-68 ° C, 5min-4 ° C. found.

(9) BpmI restriction enzyme cleavage

BpmI restriction enzyme digestion (40 μl) DNA (0.2 ug) Buffer (3) enzyme BSA (10x) SAM DW 4 4 4 0

Incubated for 2 hours at 37 ℃ under the composition of Table 6 was digested with restriction enzymes.

(10) Bead Binding Using Dioxygenin Antibodies

① Dynabead was completely suspended by pipetting or rotating.

② 50 μl of dynabe was added to the prepared 200 μl tube.

③ The tube was placed over the magnetic magnet to remove the solution from the beads.

④ The supernatant was removed and the tube was removed from the magnet.

⑤ 20 μl (0.1 mg / ml) of anti-deoxygenin antibody was placed in the tube after step ④ and spun at room temperature for 10 minutes.

⑥ Remove the supernatant after placing the tube on the magnet.

⑦ The tube was removed from the magnet.

⑧ About 0.01 pmol (diluted in 20 μl) of the BpmI cleavage obtained in step (9) was attached to the anti-dioxygenin antibody and then homogenized completely by pipetting or rotation.

⑨ Rotate at room temperature for 10 minutes.

⑩ The tube was placed back on the magnet and the supernatant was removed.

세척 washed three times with distilled water.

(11) Brachy adapter (Biotinylated) or with adapter

For type 1 libraries: DNA ends for the type 1 libraries obtained in step (10) were blunt ends in the same manner as step (2), and then Hwang2011CJ and Hwang2011CK adapters were attached.

For type 2 libraries: A branched adapter HwanoHair-32, Hwang2011BK or HwangAAA, HwangBBB was attached to the DNA terminus for the type 2 library obtained in step (10) without blunt termination.

For type 3 libraries: The branched adapters BiotinHwaSBI-6, HwaSkai15 were attached to the DNA ends for the type 3 libraries obtained in step (10) without blunt termination.

(12) BsmI cleavage generation for attaching hairpin structures

Of the DNA products obtained in step (11), the DNA products for the type 2 and type 3 libraries were attached to a magnet, washed, and digested with restriction enzymes under the composition of Table 7 below.

Bsm I restriction enzyme digestion (20 μl) DNA Buffer (3) enzyme BSA (10x) SAM DW 17 2 One 0 0

Then, after placing on the magnet, the supernatant was obtained and the rest was stored. This procedure was performed to remove the dioxingenin portion cut off with restriction enzymes.

(13) Streptavidin tube attachment and pre hairpin structure attachment

Procedure 1) Attaching Streptavidin Tube

① After the procedure (12), the DNA fragments were eluted with 40 μl by adding 2X binding buffer.

② Streptavidin PCR tube was washed with 250 μl of washing buffer.

③ 2 μl of type 2 and 0.4 μl of type 3 were contained in the biotin-labeled DNA, which was adjusted to 50 μl with 1 × adhesion buffer and incubated at 37 ° C. for 5 minutes.

(4) Rotate to stand at room temperature for 5 minutes.

⑤ After washing three times with 200μl wash buffer, the last wash using a Tip (Tip) was removed completely to leave no residue.

⑥ washed with 1 × ligase buffer and cooled.

Step 2) Attaching a Free Hairpin Adapter

The adapter preparation process was performed in the same manner as the process (2), and the adapter ligation was performed using the adapters as described in the process (13) of the second embodiment. 5.0 μl of 10 × T4 DNA ligase buffer, DW 41.0 μl, 2.0 μl of T4 DNA ligase and 2.0 μl of adapter were mixed and left overnight at 4 ° C. or for 6 hours.

(14) polymerase chain reaction IV (PCR amplification IV)

The tube was cleared and then amplified under the composition of Table 8 below.

5X Hot start buffer Remarks dNTP (10mM each) Corresponding primer sets of type 1, type 2 and type 3 Mold (Ligation Mixture) Attached Hot start taq polymerase DMSO 0 DW

PCR amplification conditions were 98 ° C, 1 minute-[98 ° C, 0.5 minutes-68 ° C, 1 minute] X cycle-68 ° C, 5 minutes-4 ° C.

(15) Target DNA Fragment Isolation

Repeat step (7).

(16) polymerase chain reaction V (PCR amplification V)

The procedure (14) was repeated with the primer set as described in the procedure (16) of Example 2.

(17) The amplification products obtained in step (16) were inserted into a carrier vector suitable for the target DNA for libraries of type 1, type 2 and type 3, respectively. The vector to be used is well known in the art and readily available, and the insertion into the vector was also performed according to methods known in the art.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

<110> Scripps Korea Antibody Institute <120> Method for preparing RNAi library <130> P12-0293KR <160> 33 <170> Kopatentin 1.71 <210> 1 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> HwaSkai8 <400> 1 taatacgact cactataggg atgaagcttg gtgcagggtg agaccgctgg cc 52 <210> 2 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> HwaSkai9 <400> 2 ggccagcggt ctc 13 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> HwaMme-3 <400> 3 tacgatttag gtgacactat agggcgacag ttggaatgct ggaggctaca gcagtccaac 60 60 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HwaMme-4 <400> 4 gttggactgc tgtag 15 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HwaSkai10 <400> 5 atgaagcttg gtgcagggtg agaccgc 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HwaSkai13 <400> 6 atagggcgac agttggaatg ctggagg 27 <210> 7 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> HwaMmeDuap-1 <400> 7 ttttctgtct tcactgcgtt gataccaatg gtttgttccg ta 42 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> HwaMmeDuap-2 <400> 8 agtgaagaca gaaaann 17 <210> 9 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> HwaSBIBsm-1 <400> 9 ctgcattcct actctgcgtt gataccaatg gtttgttccg ta 42 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> HwaSBIBsm-2 <400> 10 agagtaggaa tgcagnn 17 <210> 11 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> HwaMmeBsm-1 <400> 11 tagcattcct actctgcgtt gataccaatg gtttgttccg ta 42 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> HwaMmeBsm-2 <400> 12 agagtaggaa tgctann 17 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HwaMme-23 <400> 13 tacggaacaa accattggta tcaacgc 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HwaMme-24 <400> 14 tacggaacaa accattggta tcaacgc 27 <210> 15 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Hwang2011CJ <400> 15 ggtaggccga atatttcttg gctggatcca tcttgtggaa aggacgaaaa aaag 54 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hwang2011CK <400> 16 ctttttttcg tcctttccac 20 <210> 17 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> HwanoHair-32 <400> 17 cagatagaga gtacgtgctc ctgctgaagg aggggcagta ggcac 45 <210> 18 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Hwang2011BK <400> 18 gcctactgca taggaattct gccctcacga gtcgaaggca cag 43 <210> 19 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> HwangAAA <400> 19 cagatagaga gtacgtgctc ctgctgaagg acctgcagta ggcac 45 <210> 20 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> HwangBBB <400> 20 gcctactgcc tcggaattct gccctcacga gtcgaaggca cag 43 <210> 21 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> BiotinHwaSBI-6 <400> 21 cagatagaga gtacgtgcta cactggaggg ctagagcagg agcac 45 <210> 22 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> HwaSkai15 <400> 22 gctcctgcta gctacttcag aatgccacat gtagacgcca cgt 43 <210> 23 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> HwaSBIBseBsm-19E <400> 23 aagagatgga gtcggatgca tgtccgagga gatctcttca 40 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> HwaSBIBseBsm-19F <400> 24 aagagatgga gtcggatgca tgaccgagga gatctcttca 40 <210> 25 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> HwaSBIBseBsm-19G <400> 25 tccgactctc cctccctgca gacaagagga gagtcggaca 40 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> HwaSBIBseBsm-19H <400> 26 tccgactctc tctccctgca gacaagagga gagtcggaca 40 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Hwang 2011CF <400> 27 tgaagccaca gatgagctca gaagaggagg tggcttcact 40 <210> 28 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Hwang2011CM <400> 28 ggtaggccga atatttcttg gctggatcc 29 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> HwaSBI-7 <400> 29 cagatagaga gtacgtgcta cactgg 26 <210> 30 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HwaSkai17 <400> 30 acgtggcgtc tacatgtggc attctga 27 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Hwang 2011 BM <400> 31 ctgtgccttc gactcgtgag ggcag 25 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HwangCCC <400> 32 atagagagta cgtgctcctg ctgaaggacc 30 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Hwang2011BS <400> 33 ctgtgccttc gactcgtgag ggcag 25

Claims

(a) cleaving and fragmenting genomic DNA to obtain a DNA fragment;
(b) binding a first adapter comprising a first restriction enzyme recognition site to the ends of the DNA fragments,
(c) amplifying the DNA fragment to which the first adapter is bound using a primer set labeled with a first label,
(d) treating the DNA amplification product obtained in step (c) with a first restriction enzyme and generating a DNA fragment by cleaving the DNA amplification product by recognizing the first restriction enzyme recognition site. Steps,
(e) separating the DNA fragment with the first label from the cut DNA fragment using a medium that specifically binds to the first label,
(f) a second adapter corresponding to each of the plurality of library types to be produced is coupled to the ends of the separated DNA fragments, each having a different sequence for each of the plurality of library types and including a second restriction enzyme recognition site; Combining the two adapters,
(g) amplifying a plurality of DNA fragments each having a second adapter coupled to each of a plurality of library types using a primer set labeled with a second label,
(h) generating a DNA fragment by treating the DNA amplification product obtained in step (g) with a second restriction enzyme and cleaving the DNA amplification product by recognizing the second restriction enzyme recognition site. Steps,
(i) separating the DNA fragments with the second label from the cleaved DNA fragments by a plurality of library types using a medium that specifically binds the second label;
(j) RNAi library manufacturing method comprising the step of constructing a library by cloning a DNA fragment separated by a plurality of library types in a vector (i) to a vector.

The method of claim 1,
Coupling a branched third adapter to a DNA fragment separated by a plurality of library types in step (i), each having a different sequence for each of the plurality of library types and including a third restriction enzyme recognition site;
Treating the third restriction enzyme to generate a DNA fragment for binding the hairpin structure by cutting the DNA amplification product by recognizing the third restriction enzyme recognition site;
RNAi library manufacturing method further comprising the step of coupling the hairpin structure to the generated DNA fragment.

3. The method according to claim 1 or 2,
Wherein said first label is biotin and the medium that specifically binds said first label is streptavidin.

3. The method according to claim 1 or 2,
The second label is deoxygenin, and the medium specifically binding to the second label is an RNAi library manufacturing method, characterized in that the anti-deoxygenin antibody.

The method of claim 1,
The first restriction enzyme is MmeI restriction enzyme, the second restriction enzyme RNAi library production method, characterized in that the BpmI restriction enzyme.

3. The method of claim 2,
The third restriction enzyme is a RNAi library production method, characterized in that the BsmI restriction enzyme.

3. The method according to claim 1 or 2,
Wherein said first adapter comprises oligonucleotide pairs of SEQ ID NOs: 1 and 2, or oligonucleotide pairs of SEQ ID NOs: 3 and 4, or both.

3. The method according to claim 1 or 2,
Wherein said second adapter is an oligonucleotide pair of SEQ ID NOs: 9 and 10, or an oligonucleotide pair of SEQ ID NOs: 11 and 12.

3. The method according to claim 1 or 2,
The branched third adapter is an oligonucleotide pair of SEQ ID NOs: 17 and 18, an oligonucleotide pair of SEQ ID NOs: 19 and 20, or an oligonucleotide pair of SEQ ID NOs: 21 and 22.

3. The method of claim 2,
The hairpin structure is an RNAi library manufacturing method, characterized in that selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 23 to SEQ ID NO: 27 and oligonucleotides having a base sequence complementary to these oligonucleotides.