Method for constructing RNA library using restriction enzyme

Abstract

The present invention relates to a method for constructing an RNA library, the method comprising the steps of: ligating a first adapter nucleic acid molecule and a second adapter nucleic acid molecule containing restriction enzyme recognition sites to target RNA; and treating the RNA with restriction enzymes. When adapter dimers are formed, the restriction enzymes recognize and cleave the adapter dimers, thereby eliminating noise caused by adapters and increasing the efficiency of RNA library construction.

Description

Method for creating an RNA library using restriction enzymes

[0001] The present invention relates to a method for producing an RNA library, comprising the steps of ligating a first adapter nucleic acid molecule and a second adapter nucleic acid molecule including a restriction enzyme recognition site to a target RNA, and the step of treating with a restriction enzyme.

[0002] A genome refers to all of the genetic information of an organism. Various technologies, including DNA chips, Next Generation Sequencing (NGS), and Next Next Generation Sequencing (NNGS), are being developed to sequence or analyze an individual's genome. NGS is widely used for research and diagnostic purposes. While NGS varies depending on the type of equipment used, it can broadly be divided into three steps: sample collection, library creation, and nucleic acid sequencing. After nucleic acid sequencing, the resulting sequence data can be used to identify various genetic information, including the presence of genetic mutations.

[0003] In particular, as the quality of life improves, interest in early diagnosis of diseases is growing, and molecular diagnostic technology is receiving much attention as a technology that can solve the shortcomings of existing immunodiagnostic technology that detects indirect factors of diseases based on antigen / antibody reactions, as it directly detects the genetic information (DNA / RNA) of pathogens that cause diseases.

[0004] As disease-related genetic information can be explored and secured to build libraries, which can be used to discover candidate substances for new drug development, there is an increasing demand for technology development that enables faster and more accurate library construction.

[0005] In particular, in the production of these libraries, in addition to the library for base sequence analysis, adapter dimers are formed by combining adapters, causing errors in RNA sequence analysis. Therefore, the development of technology to improve these errors is required.

[0006] An object of the present invention is to provide a method for producing an RNA library, comprising the steps of ligating a first adapter nucleic acid molecule and a second adapter nucleic acid molecule including a restriction enzyme recognition site to a target RNA, and the step of treating with a restriction enzyme.

[0007] Another object of the present invention is to provide a composition for producing an RNA library comprising a first adapter nucleic acid molecule and a second adapter nucleic acid molecule including a restriction enzyme recognition site, and a restriction enzyme.

[0008] Another object of the present invention is to provide an RNA library produced by the above method.

[0009] One aspect of the present invention for achieving the above object relates to a method for producing an RNA library, comprising: a) ligating a first adapter nucleic acid molecule to the 3' end of a target RNA; b) ligating a second adapter nucleic acid molecule to the 5' end of the target RNA; c) synthesizing cDNA through a complementary sequence polymerization reaction for the target RNA molecule ligated with the first adapter nucleic acid molecule and the second adapter nucleic acid molecule; d) treating with a restriction enzyme; and e) amplifying the nucleic acid molecule ligated in step c), wherein the first adapter and the second adapter each include a restriction enzyme recognition site.

[0010] Specifically, the target RNA may be a small RNA. The small RNA may collectively refer to RNA consisting of about 50 nucleotides or less, in addition to miRNA and siRNA.

[0011] In the present invention, the “first adapter nucleic acid molecule” is a nucleic acid molecule that is ligated to a target RNA, and a phosphate group at the 5’ end of the first adapter nucleic acid molecule recognizes and binds to the 3’ end of the target RNA.

[0012] Specifically, the first adapter nucleic acid molecule may be in the form of DNA, but is not limited thereto.

[0013] Additionally, specifically, the first adapter nucleic acid molecule may have a 5' end adenylated, but is not limited thereto.

[0014] Additionally, specifically, the first adapter nucleic acid molecule may have a modified 3' end to prevent another adapter molecule from ligating to the 3' end, for example, an amine modification, but is not limited thereto.

[0015] In the present invention, the “second adapter nucleic acid molecule” is a nucleic acid molecule that is ligated to a target RNA and recognizes and binds to the 5’ end of the target RNA.

[0016] Specifically, the second adapter nucleic acid molecule may be in the form of RNA, but is not limited thereto.

[0017] The first adapter nucleic acid molecule and the second adapter nucleic acid molecule may be ligated to the target RNA separately or simultaneously. The order of ligation to the target RNA is not limited, and the first adapter nucleic acid molecule may be ligated first, or the second adapter nucleic acid molecule may be ligated first.

[0018] In the present invention, the "restriction enzyme" may be one that recognizes and cleaves a restriction enzyme recognition sequence within a restriction enzyme recognition site. The restriction enzyme cleavage site may have a sticky end or a blunt end, and is not limited to the shape of the end.

[0019] The above restriction enzyme may be one or more selected from the group consisting of EcoRI, BamHI, HindIII, PstI, XhoI, SalI, KpnI, SmaI, NcoI, ApaI, BglII, ClaI, EcoRV, MluI, NheI, SacI, SpeI, SphI, StuI, XbaI, PvuII, BclI, DAI, NdeI and HpaI, but is not limited thereto.

[0020] In the present invention, a "restriction enzyme recognition site" refers to a site that includes a restriction enzyme recognition sequence that can be recognized by a restriction enzyme. In addition to including the restriction enzyme recognition sequence, additional sequences may be included or bases included in the sequence may be modified as needed. The sequences or modifications that may be additionally included within the restriction enzyme recognition site are not limited.

[0021] Specifically, the above restriction enzyme recognition site may include, but is not limited to, one or more restriction enzyme recognition sequences, and the recognition sequences may be changed or added as needed so that the restriction enzyme can be applied.

[0022] Also, specifically, the restriction enzyme recognition site may include part or all of a restriction enzyme recognition sequence. The first adapter nucleic acid molecule and the second adapter nucleic acid molecule of the present invention each include a restriction enzyme recognition site, and each restriction enzyme recognition sequence included therein may include the entire sequence that the restriction enzyme can recognize, or may include only a part of it.

[0023] More specifically, when the restriction enzyme recognition site included in each of the above adapter nucleic acid molecules includes only a portion of a restriction enzyme recognition sequence, a sequence recognizable by the restriction enzyme may be formed when a portion of the restriction enzyme recognition sequence included in the first adapter nucleic acid molecule and a portion of the restriction enzyme recognition sequence included in the second adapter nucleic acid molecule are connected to each other.

[0024] Additionally, specifically, the fact that a portion of the restriction enzyme recognition sequence included in the first adapter nucleic acid molecule and a portion of the restriction enzyme recognition sequence included in the second adapter nucleic acid molecule are connected to each other may be due to the first adapter nucleic acid molecule and the second adapter nucleic acid molecule being ligated to each other to form an adapter dimer.

[0025] When the first adapter and the second adapter are combined with each other to form an adapter dimer without being ligated to the target RNA, a part of the restriction enzyme recognition sequence included in the first adapter nucleic acid molecule and a part of the restriction enzyme recognition sequence included in the second adapter nucleic acid molecule are connected to each other to form a complete restriction enzyme recognition sequence, and the restriction enzyme can recognize and cut the restriction enzyme recognition sequence of the adapter dimer, thereby cutting the adapter dimer (Fig. 1).

[0026] More specifically, when the first adapter nucleic acid molecule includes a partial restriction enzyme recognition sequence for a type 1 restriction enzyme, the restriction enzyme recognition sequence included in the second adapter nucleic acid molecule may be for the same restriction enzyme as the restriction enzyme that recognizes the restriction enzyme recognition sequence of the first adapter nucleic acid molecule, and may include the recognition sequence of the remaining portion excluding the partial sequence included in the first adapter nucleic acid molecule.

[0027] Also, specifically, the restriction enzyme recognition sequence included in the first adapter nucleic acid molecule may be located at the 5' end of the first adapter nucleic acid molecule, and the restriction enzyme recognition sequence included in the second adapter nucleic acid molecule may be located at the 3' end of the second adapter nucleic acid molecule. This allows the restriction enzyme recognition sequences to be positionally connected when forming a dimer between the first adapter and the second adapter, but is not limited to the above positions.

[0028] In the present invention, the above "complementary strand polymerization" refers to polymerizing a complementary sequence to a molecule that serves as a template, wherein a first adapter nucleic acid molecule is ligated to the 3' end of a target RNA and a second adapter nucleic acid molecule is ligated to the 5' end. One of these complementary strand polymerization methods may include, but is not limited to, reverse transcription, and any sequence that can polymerize a complementary sequence to a molecule serving as a template may be included without limitation.

[0029] Specifically, the complementary sequence polymerization reaction of step b) above may be performed using DNA polymerase, but is not limited thereto, and any reverse transcriptase or polymerase used in a typical reverse transcription reaction may be applied.

[0030] Also, specifically, the restriction enzyme treatment of step d) may be for cleavage of the adapter dimer.

[0031] In one embodiment of the present invention, it was confirmed that when restriction enzymes were treated, the restriction enzyme recognition sequence was recognized and the adapter dimer was cut (Figs. 2 and 3), and it was confirmed that the efficiency of RNA library production was increased by removing obstacles through such restriction enzyme treatment (Figs. 5a to 5c).

[0032] Another aspect of the present invention relates to a composition for producing an RNA library, comprising: a) a first adapter nucleic acid molecule ligated to the 3' end of a target RNA; b) a second adapter nucleic acid molecule ligated to the 5' end of the target RNA; and c) a restriction enzyme, wherein the first adapter and the second adapter each comprise a restriction enzyme recognition site.

[0033] Specifically, the target RNA may be a small RNA.

[0034] The first adapter nucleic acid molecule, the second adapter nucleic acid molecule, and the restriction enzyme recognition site are as described above.

[0035] The composition for producing the above RNA library may further include a buffer, PCR primers, etc., and the additionally included components may be changed and applied as needed.

[0036] Another aspect of the present invention relates to an RNA library constructed using the above-described method. RNAs related to disease, changes in a subject's condition, etc. can be pre-designed into the RNA library constructed as described above. This library can be utilized for the exploration and development of genetic markers and therapeutic candidates for disease treatment or condition improvement.

[0037] The RNA library production method of the present invention includes a step of ligating a first adapter nucleic acid molecule and a second adapter nucleic acid molecule including a restriction enzyme recognition site to a target RNA, and a step of treating with a restriction enzyme, thereby eliminating obstacles and errors caused by adapter dimers that occur during RNA library production, thereby increasing the efficiency of RNA library production.

[0038] In the conventional RNA library production process, adapter dimers formed by adapters binding to each other have caused problems in RNA sequencing analysis. However, by simply cutting and removing adapter dimers through restriction enzyme treatment, the accuracy of the RNA library can be increased in a way that does not affect the RNA library.

[0039] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0040] Figure 1 is a schematic diagram illustrating the production of an RNA library of the present invention. The X mark at the 3' end in the schematic diagram indicates that the amine region has been modified to block additional binding.

[0041] Figure 2 shows the results of confirming adapter dimer cleavage following EcoR1 restriction enzyme treatment.

[0042] Figure 3 shows the results of removing adapter dimers through various types of restriction enzyme treatment and confirming the library produced by the method of the present invention through capillary electrophoresis (Fragment Analyzer).

[0043] Figure 4 is a schematic diagram showing the portions to which the EcoRI_AD, BamHI_AD, and HindIII_AD primers bind when used to confirm the adapter dimer.

[0044] Figure 5 shows the results of confirming the library production efficiency according to restriction enzyme treatment through qPCR. Figure 5a confirms the library production efficiency according to EcoRI restriction enzyme treatment, Figure 5b confirms the library production efficiency according to BamHI restriction enzyme treatment, and Figure 5c confirms the library production efficiency according to HindIII restriction enzyme treatment.

[0045] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited to the following examples.

[0046] Example 1. RNA library production

[0047] As an example, a library was constructed using synthetic miRNA 92a-3p.

[0048] 1-1. Ligation of small RNA and first adapter nucleic acid molecule

[0049] A first adapter nucleic acid molecule was ligated to the 3' end of miR-92a-3p using the T4 RNA Ligase 2, truncated KQ kit (NEB). The first adapter nucleic acid molecule is adenylated at the 5' end and contains a restriction enzyme recognition sequence. 3 μl of miR-92a-3p was mixed with 1 μl of the first adapter nucleic acid molecule (10 pmol / μl), 2 μl of 10X T4 RNA ligase reaction buffer (NEB), 10 μl of 50% PEG8000 (NEB), 1 μl of T4 RNA ligase 2, truncated KQ (NEB), 1 μl of RNase inhibitor (Takara), and 2 μl of RNase / DNase free water, and then reacted using a PCR machine at 28°C for 60 minutes, 65°C for 20 minutes, and 4°C for 5 minutes. The sequence of the first adapter nucleic acid molecule is exemplarily shown in Table 1 below.

[0050] APP-TTCTGG ATA TCT CGG GTG CCA AGGEcoRI2App-TCCTGG ATA TCT CGG GTG CCA AGGBamHI3App-CTTTGG ATA TCT CGG GTG CCA AGGHindIII4App-CAGTGG ATA TCT CGG GTG CCA AGGPstI5App-GAGTGG ATA TCT CGG GTG CCA AGGXhoI6App-GACTGG ATA TCT CGG GTG CCA AGGSalI7App-ACCTGG ATA TCT CGG GTG CCA AGGKpnI8App-GGGTGG ATA TCT CGG GTG CCA AGGSmaI9App-TGGTGG ATA TCT CGG GTG CCA AGGNcoI10App-CCCTGG ATA TCT CGG GTG CCA AGGApaI11App-TCTTGG ATA TCT CGG GTG CCA AGGBglII12App-GATTGG ATA TCT CGG GTG CCA AGGClaI13App-ATCTGG ATA TCT CGG GTG CCA AGGEcoRV14App-CGTTGG ATA TCT CGG GTG CCA AGGMluI15App-AGCTGG ATA TCT CGG GTG CCA AGGNheI16App-CTCTGG ATA TCT CGG GTG CCA AGGSacI17App-AGTTGG ATA TCT CGG GTG CCA AGGSpeI18App-TGCTGG ATA TCT CGG GTG CCA AGGSphI19App-CCTTGG ATA TCT CGG GTG CCA AGGStuI20App-AGATGG ATA TCT CGG GTG CCA AGGXbaI21App-CTGTGG ATA TCT CGG GTG CCA AGGPvuII22App-TCATGG ATA TCT CGG GTG CCA AGGBclI23App-AAATGG ATA TCT CGG GTG CCA AGGDraI24App-ATGTGG ATA TCT CGG GTG CCA AGGNdeI25App-AACTGGATA TCT CGG GTG CCA AGGHpaI

[0051] 1-2. Ligation of the second adapter nucleic acid molecule

[0052] A second adapter nucleic acid molecule was ligated to the 5' end of miR-92a-3p to which the first adapter nucleic acid molecule was ligated through the above 1-1. The second adapter nucleic acid molecule includes a restriction enzyme recognition sequence. T4 RNA Ligase kit (Enzynomics) was used, and 20 μl of the first adapter nucleic acid molecule was ligated to miR-92a-3p, 1 μl of the second adapter nucleic acid molecule (10 pmol / μl), 4 μl of 10X T4 RNA ligase reaction buffer (Enzynomics), 4 μl of 10 mM ATP (Enzynomics), 1 μl of T4 RNA ligase 1 (Enzynomics), 1 μl of RNase inhibitor (Takara), and 9 μl of RNase / DNase free water. The mixture was then mixed and reacted using a PCR machine at 28°C for 30 minutes, 65°C for 20 minutes, and 4°C for 5 minutes. The sequence of the second adapter nucleic acid molecule is exemplarily shown in Table 2 below.

[0053] 서열번호제2 어댑터(RNA) (5'-3')제한효소26GUU CAG UCU ACA GUC CGA CGA UCG AAEcoRI27GUU CAG AGU UCU ACA GUC CGA CGA CGA CGA UCG UCGU CABam UCA AGHindIII29GUU CAG AGU UCU ACA GUC CGA CGA UCC UGPstI30GUU CAG AGU UCU ACA GUC CGA CGA UCC UCXhoI31GUU CAG AGU UCU ACA GUC CGA CGA UCG UCSalI32GUU CAG AGU UCU CGAI ACA UCGGUC CGA GUC CGA CGA UCC CCSmaI34GUU CAG AGU UCU ACA GUC CGA CGA UCC CANcoI35GUU CAG AGU UCU ACA GUC CGA CGA UCG GGApaI36GUU CAG AGU UCU ACA GUC CGA CGA UCA GABglII37GUAGI U CAGU UUCA UUCGA CGA UCU ACA UCU ACA GUC CGA CGA UCG AUEcoRV39GUU CAG AGU UCU ACA GUC CGA CGA UCA CGMluI40GUU CAG AGU UCU ACA GUC CGA CGA UCG CUNheI41GUU CAG AGU UCU ACA GUC CGA CGA UCG AGSac4 U CGU CGUUCA CAGU CAG CAG AGU UCU ACA GUC CGA CGA UCG CASphI44GUU CAG AGU UCU ACA GUC CGA CGA UCA GGStuI45GUU CAG AGU UCU ACA GUC CGA CGA UCU CUXbaI46GUU CAG AGU UCU ACA GUC CGA UCC AGPGA UCC ACUCU47GABclI48GUU CAG AGU UCU ACA GUC CGA CGA UCU UUDraI49GUU CAG AGU UCU ACA GUC CGA CGA UCC AUNdeI50GUU CAG AGU UCU ACA GUC CGA CGA UCG UUHpaI

[0054] 1-3. Complementary strand polymerization

[0055] Using a cDNA Synthesis kit (Xenohelix), cDNA of small RNA ligated with the first and second adapter nucleic acid molecules was synthesized. 12 μl of 5X RTase buffer (Xenohelix), 1 μl of RTase (Xenohelix), 1 μl of RNase inhibitor (Takara), 2 μl of primer containing the index sequence (5 pmol / μl), and 5 μl of RNase / DNase free water were added to miR-92a-3p (40 μl) ligated with the first and second adapter nucleic acid molecules obtained through the above 1-2, and mixed, and then reacted using a PCR machine at 50 °C for 60 minutes, 70 °C for 15 minutes, and 4 °C for 5 minutes.

[0056] 1-4. Purification and restriction enzyme treatment

[0057] Clean-up was performed using a PCR purification kit (Xenohelix), and restriction enzyme treatment was performed to cleave the adapter dimer generated through the above process.

[0058] Specifically, 40 μl of RNase / DNase free water and 500 μl of DB buffer (Xenohelix) were added to 60 μl of the RNA / DNA chimera product (cDNA) generated through the above 1-3, mixed, and passed through a DNA spin column. Then, 2 μl of restriction enzyme (Takara), 4 μl of 10X reaction buffer (Takara), and 34 μl of RNase / DNase free water were added, mixed, placed in a DNA spin column, and allowed to stand at room temperature for 10 minutes. After that, 700 μl of washing buffers P1, P2, and P3 were sequentially passed through the DNA spin column. 40 μl of elution buffer was passed through the DNA spin column, and the purified product was obtained.

[0059] 1-5. Amplification

[0060] Amplification was performed using a HotStart Taq DNA Polymerase kit (Bioneer). 30 μl of the purified product from 1-4 above was mixed with 8 μl of 10X Reaction buffer (Bioneer), 8 μl of 10 nM dNTP (Bioneer), 1 μl of HotStart Taq DNA Polymerase (Bioneer), 3 μl of Forward primer (10 pmol / μl), 3 μl of Reverse primer (10 pmol / μl), and 27 μl of RNase / DNase free water, and then reacted using a PCR machine under the following conditions: 1 cycle of 95°C for 15 minutes, 22 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 15 seconds, 1 cycle of 72°C for 2 minutes, and 1 cycle of 4°C for 5 minutes. After the reaction was completed, cleanup was performed using a PCR purification kit (Xenohelix) according to the product manual.

[0061] A schematic diagram of the production of the above library is shown in Fig. 1.

[0062] Example 2. Verification of adapter dimer cleavage and library creation

[0063] Small nucleic acid fragments were analyzed by capillary electrophoresis using a small fragment kit (Agilent). The kit analyzes small fragments ranging in size from 50 to 1,500 bp, and the analysis was performed according to the product instructions.

[0064] First, steps 1-2, 1-3, and 1-5 were performed using the first and second adapters containing the restriction enzyme EcoR1 recognition sequence, and then step 1-4 was performed. After treating with the restriction enzyme EcoR1, the sample was electrophoresed on a 15% polyacrylamide gel (polyacrylamide gel (19:1)) and confirmed using Gel Doc. The case in which the EcoR1 restriction enzyme was not treated was used as a control. As a result, as shown in Fig. 2, when the EcoR1 restriction enzyme was not treated, the adapter dimer was not cleaved and was maintained, resulting in a 95 bp band, but when the EcoR1 restriction enzyme was treated, it was cleaved into two pieces, resulting in bands of 60 to 80 bp and 20 to 40 bp.

[0065] In addition, steps 1-1 to 1-5 were performed using the first and second adapters containing the restriction enzyme EcoR1 recognition sequence, and small nucleic acid fragments were analyzed using a small fragment kit (Agilent). The case in which EcoR1 restriction enzyme was not treated was used as a control. The experiment was conducted in the same manner for other types of restriction enzymes in addition to EcoR1 restriction enzyme. The sequence recognized by each restriction enzyme was included in the first and second adapters, and steps 1-1 to 1-5 were performed, and small nucleic acid fragments were analyzed using a small fragment kit (Agilent).

[0066] As a result, as shown in Fig. 3, when each restriction enzyme was not treated, a band appeared because the adapter dimer was not cut, but when the restriction enzyme was treated, it was confirmed that the adapter dimer was cut and no band appeared.

[0067] Additionally, qPCR was performed using primers created from the sequences at both ends of the library to confirm the completed library. Specifically, 9 μl of TB Green Premix Ex Taq II (Takara), 2 μl of primer mix (0.5 pmol / μl each), and 12 μl of RNase / DNase free water were added to 2 μl of the library diluted 1 / 500, and mixed, and the reaction was performed using a real-time PCR device under the following conditions: 1 cycle of 95 °C for 3 minutes, 40 cycles of 95 °C for 10 seconds, and 64 °C for 30 seconds.

[0068] As a result, it was confirmed that a library of miRNAs was successfully created regardless of whether restriction enzyme treatment was performed, as shown in Fig. 3.

[0069] Example 3. Confirmation of library production efficiency according to restriction enzyme treatment.

[0070] The efficiency of restriction enzyme treatment during the library production process was confirmed. An RNA library was produced according to the production method of Example 1, but libraries were produced in each case where the restriction enzyme treatment applied in 1-4 was included (restriction enzyme O in the sample) or not included (restriction enzyme X in the sample).

[0071] To confirm the completed library, qPCR was performed using primers made from a partial sequence of the adapter dimer and the 3' end sequence of the library. Specifically, 9 ㎕ of TB Green Premix Ex Taq II (Takara), 2 ㎕ of primer mix (Primer mix, each 0.5 pmol / ㎕), and 12 ㎕ of RNase / DNase free water were added to 1 / 500 diluted library (2 ㎕), and mixed, and the reaction was performed using a real-time PCR device under the following conditions: 1 cycle of 95 ℃ for 3 minutes, 40 cycles of 95 ℃ for 10 seconds, and 64 ℃ for 30 seconds. The reaction was repeated twice, and Cq1 and Cq2 values ​​were obtained for each.

[0072] Figure 4 is a schematic diagram showing the portions to which the EcoRI_AD, BamHI_AD, and HindIII_AD primers bind when used to confirm the adapter dimer.

[0073] Figure 5a shows the efficiency of library production according to the EcoRⅠ restriction enzyme treatment. When the restriction enzyme was not treated, the qPCR result showed a Cq value of approximately 29, which was a significant decrease compared to the Cq value of approximately 36 when the restriction enzyme was treated.

[0074] In addition, Fig. 5b confirms the efficiency of library production according to BamHⅠ restriction enzyme treatment. When the restriction enzyme was not treated, the qPCR result showed a Cq value of approximately 30, which was significantly reduced compared to the Cq value of approximately 36 when the restriction enzyme was treated.

[0075] In addition, Fig. 5c confirms the efficiency of library production according to HindIII restriction enzyme treatment. When the restriction enzyme was not treated, the qPCR result showed a Cq value of approximately 30, which was significantly reduced compared to the Cq value of approximately 36 when the restriction enzyme was treated.

[0076] As described above, in the library production method of the present invention, it was confirmed that when the adapter dimer is cut and removed through restriction enzyme treatment, errors due to the adapter formed as a dimer are resolved, and the accuracy of RNA library production can be increased through the elimination of the amplification reaction for the adapter dimer.

[0077] The respective adapter nucleic acid molecules and primer sequences used in Examples 1 to 3 are summarized in Table 3 below. The primer sequence of the present invention can be synthesized using the nucleic acid sequence of the adapter nucleic acid molecule, regardless of the type of target nucleic acid molecule to be amplified.

[0078] Sequence number distinction sequence (5'-3') Use 51 Complementary sequence polymerization primer CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCCTTGGCACCCGAGATATCCA Complementary sequence polymerization 52 Amp forward primer (primer F) AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA Amplification and library verification 53 Amp reverse primer (primer R) CAAGCAGAAGACGGCATACGAGATA Amplification and library verification 54 EcoRI_ADCCGACGATCGAATTCTGGATATCT Adapter dimer verification 55 BamHI_ADCCGACGATCGGATCCTGGATATCT Adapter dimer verification 56 HindIII_ADCCGACGATCAAGCTTTGGATATCT Adapter dimer verification *AD: Adapter dimer

[0079] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0080] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. a) A step of ligating a first adapter nucleic acid molecule to the 3' end of the target RNA; b) a step of ligating a second adapter nucleic acid molecule to the 5' end of the target RNA; c) a step of synthesizing cDNA through a complementary sequence polymerization reaction for the target RNA molecule ligated with the first adapter nucleic acid molecule and the second adapter nucleic acid molecule; d) a step of treating with restriction enzymes; and e) a step of amplifying the nucleic acid molecule ligated in step c), A method for producing an RNA library, wherein the first adapter and the second adapter each include a restriction enzyme recognition site.

2. In paragraph 1, A method for producing an RNA library, wherein the target RNA is a small RNA.

3. In paragraph 1, A method for producing an RNA library, wherein the first adapter nucleic acid molecule is in the form of DNA.

4. In paragraph 1, A method for producing an RNA library, wherein the first adapter nucleic acid molecule has an adenylated 5' end.

5. In paragraph 1, A method for producing an RNA library, wherein the second adapter nucleic acid molecule is in the form of RNA.

6. In paragraph 1, A method for producing an RNA library, wherein the second adapter nucleic acid molecule is in the form of DNA.

7. In paragraph 1, A method for producing an RNA library, wherein the above restriction enzyme recognition site includes one or more restriction enzyme recognition sequences.

8. In paragraph 1, A method for producing an RNA library, wherein the above restriction enzyme recognition site includes part or all of a restriction enzyme recognition sequence.

9. In paragraph 8, If the above restriction enzyme recognition site includes only a portion of the restriction enzyme recognition sequence, A method for producing an RNA library, wherein a sequence recognizable by a restriction enzyme is formed when a portion of a restriction enzyme recognition sequence included in a first adapter nucleic acid molecule and a portion of a restriction enzyme recognition sequence included in a second adapter nucleic acid molecule are connected to each other.

10. In paragraph 9, A method for producing an RNA library, wherein a portion of a restriction enzyme recognition sequence included in the first adapter nucleic acid molecule and a portion of a restriction enzyme recognition sequence included in the second adapter nucleic acid molecule are connected to each other, resulting in the formation of an adapter dimer by ligation of the first adapter nucleic acid molecule and the second adapter nucleic acid molecule.

11. In paragraph 9, A method for producing an RNA library, wherein, when a first adapter nucleic acid molecule includes a partial restriction enzyme recognition sequence for a type 1 restriction enzyme, the restriction enzyme recognition sequence included in the second adapter nucleic acid molecule is for the same restriction enzyme as the restriction enzyme that recognizes the restriction enzyme recognition sequence of the first adapter nucleic acid molecule, and includes the recognition sequence of the remaining portion excluding the partial sequence included in the first adapter nucleic acid molecule.

12. In paragraph 8, The restriction enzyme recognition sequence included in the first adapter nucleic acid molecule is located at the 5' end of the first adapter nucleic acid molecule, A method for producing an RNA library, wherein a restriction enzyme recognition sequence included in a second adapter nucleic acid molecule is located at the 3' end of the second adapter nucleic acid molecule.

13. In paragraph 1, A method for producing an RNA library, wherein the above restriction enzyme is at least one selected from the group consisting of EcoRI, BamHI, HindIII, PstI, XhoI, SalI, KpnI, SmaI, NcoI, ApaI, BglII, ClaI, EcoRV, MluI, NheI, SacI, SpeI, SphI, StuI, XbaI, PvuII, BclI, DraI, NdeI and HpaI.

14. In paragraph 1, A method for producing an RNA library, wherein the complementary sequence polymerization reaction of step c) above uses DNA polymerase.

15. In paragraph 1, A method for producing an RNA library, wherein the restriction enzyme treatment in step d) above is for cutting an adapter dimer. 16.a) A first adapter nucleic acid molecule ligated to the 3' end of the target RNA; b) a second adapter nucleic acid molecule ligated to the 5' end of the target RNA; and c) Contains restriction enzymes, A composition for producing an RNA library, wherein the first adapter and the second adapter each include a restriction enzyme recognition site.

17. A composition for producing an RNA library, wherein the target RNA is a small RNA in claim 16.

18. RNA library produced by the method of paragraph 1.

Images