The invention relates to 5apos; rACE technology optimization method and application

Abstract

The invention relates to the technical field of cDNA terminal amplification, and provides an optimization method and application of a 5 'RACE technology. The optimization method of the 5 'RACE technology comprises the following steps: S1, primer design: predicting an RG4 structure and position of RNA according to a known partial cDNA sequence of a target gene, and designing a reverse transcription primer crossing the RG4 at the upstream of the RG4 structure; s2, performing reverse transcription based on the primer in the step S1 to obtain a cDNA first chain; and S3, carrying out PCR amplification by taking the cDNA first chain in the step S2 as a template to obtain a target gene segment. The invention provides an innovative primer design strategy on the basis of the existing 5 'RACE technology. According to the primer design method, Oligo (dT) 20 or a general specific reverse transcription primer commonly used in a traditional 5 'RACE test is abandoned, an RG4 complex structure on mRNA is fully considered and avoided during primer design, the probability of early termination of reverse transcription is effectively reduced, and acquisition of a real sequence at the mRNA 5'end is facilitated. Therefore, the success rate of the 5 'RACE can be greatly improved, so that the requirements of molecular biology experiments are met.

Description

Technical Field

[0001] The present invention relates to the technical field of cDNA end amplification, and particularly to an optimized method and application of 5'RACE technology. Background Art

[0002] Race technology is a technique for rapidly amplifying the 5′-end and 3′-end of cDNA from a sample based on reverse transcription, invented by Frohman et al. in 1988. This technique obtains the 5′ and 3′ ends of the complete cDNA through the known partial cDNA sequence. RACE technology has been applied in multiple fields with its unique advantages, such as the establishment of cDNA libraries, cloning of target genes, research on the functions of viral genomes, and development of new expressed sequence tags.

[0003] With the in-depth study of gene functions and regulatory mechanisms, scientists are increasingly facing the challenge of dealing with the complex problems of RACE technology. These challenges include, but are not limited to, reverse transcription problems: the 5’-end of mRNA usually has a high GC content and a complex secondary structure, which may hinder the extension of reverse transcriptase, resulting in incomplete reverse transcription and the inability to obtain the complete 5’-end cDNA sequence. Among them, the RNA secondary structure - RNA G-quadruplex (RG4) has a direct impact on RNA reverse transcription. RNA G-quadruplex (RG4) is an atypical secondary nucleic acid structure widely present in the transcriptome, folded from RNA sequences rich in guanine (G). RG4 also widely exists in non-coding RNA (ncRNA) and plays a key role in gene regulation, stress granule formation, and neurodegenerative diseases. The presence of the RG4 structure has a significant impact on the RNA reverse transcription process. Due to the stability and complex secondary structure of RG4, it may hinder the extension of reverse transcriptase, leading to premature termination of reverse transcription, thereby reducing the efficiency of cDNA synthesis. This phenomenon is particularly obvious when dealing with mRNA with a complex structure.

[0004] Existing Race technologies usually use methods such as SMARTer Race and nested Race. These methods still have the problem of low success rate in actual experiments. Due to the unknown or difficult-to-predict secondary and tertiary structures of mRNA, the complexity of the mRNA sequence increases, thus affecting the success rate of reverse transcription; on the other hand, if the primer specificity is improved, this will effectively solve the problem of premature termination of reverse transcription. Therefore, there is an urgent need for an optimized method that can effectively deal with complex structures and improve the success rate of its reverse transcription. This will be beneficial to promoting the progress of downstream gene-related experiments and improving the efficiency of molecular biology experiments. Summary of the Invention

[0005] In view of this, the present invention provides an optimized method and application of 5'RACE technology that can improve the success rate of reverse transcription.

[0006] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides an optimized method of 5'RACE technology, including the following steps:

[0007] S1, primer design: Predict the RG4 structure and position of RNA based on the known partial cDNA sequence of the target gene, and design a reverse transcription primer spanning the RG4 upstream of the RG4 structure;

[0008] S2, perform reverse transcription based on the primer in step S1 to obtain the first strand of cDNA;

[0009] S3, use the first strand of cDNA in step S2 as a template for PCR amplification to obtain the target gene fragment.

[0010] On the other hand, the present invention also provides an application of the optimized method of 5'RACE technology in the 5'RACE amplification of the Arabidopsis SR34a gene.

[0011] Based on the above technical solutions, preferably, amplifying the Arabidopsis target gene SR34a includes the following steps:

[0012] S10, using the cDNA sequence of the SR34a gene transcript as an object, predict the RG4 structure of the SR34a.4 transcript using an RNA prediction tool, and design a reverse transcription primer OSY453-RT spanning the RG4 as shown in SEQ ID NO.1;

[0013] S20, prepare a reverse transcription reaction system based on the reverse transcription primer OSY453-RT in step S10 to synthesize the first strand of cDNA;

[0014] S30, prepare an amplification reaction system using the first strand of cDNA in step S20 as a template, and perform PCR amplification to obtain the transcript of the SR34a gene.

[0015] Based on the above technical solutions, preferably, the cDNA reference sequence of the transcript of the target gene SR34a is as shown in SEQ ID NO.2-8.

[0016] Based on the above technical solutions, preferably, the reverse transcription reaction system includes reaction products, 5XRT buffer, M-MLV Reverse Transcriptase, and TSO3 shown in SEQ ID NO.9; the reaction products are the products after mixing and reacting Total RNA, OSY453-RT shown in SEQ ID NO.1, dNTP Mix, and RNase-free H2O.

[0017] Based on the above technical solutions, preferably, the amplification reaction system includes: 2X Taq Plus MasterMix, OSY650 shown in SEQ ID NO.10, OSYB370 shown in SEQ ID NO.11, template and ddH2O.

[0018] The optimized method and application of a 5'RACE technology of the present invention have the following beneficial effects compared with the prior art:

[0019] (1) Based on the existing 5'RACE technology, the present invention proposes an innovative primer design strategy. By analyzing the RNA sequence characteristics, especially its complex structure RG4, a specific reverse transcription primer spanning RG4 is designed. This primer design method abandons the commonly used Oligo(dT) 20 or general specific reverse transcription primers in traditional 5'RACE experiments, thus significantly improving the efficiency of the reverse transcription process.

[0020] (2) Experimental results show that the optimized 5'RACE technology of the present invention can effectively improve the success rate of cDNA end amplification, especially showing significant advantages when dealing with RNA molecules with complex structures. For example, the traditional 5′Race technology can only amplify SR34a.1 / 2, SR34a.6 and SR34a.7. While the 5′Race technology of the present invention can amplify all 7 transcripts of SR34a. The present invention greatly improves the success rate of 5′Race and meets the requirements of molecular biology experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 For the traditional 5'Race technical solution and the 5'Race optimized solution of the present application, Figure a is the traditional 5'Race technical solution before optimization; Figure b is the 5'Race technical solution of the present application after optimization;

[0023] Figure 2To compare the 5'-end amplification maps of 7 transcripts of Arabidopsis thaliana SR34a (AT3G49430) by the traditional 5' Race technology and the optimized 5' Race technology of the present invention, Figure a is a schematic diagram of 7 transcripts of SR34a, Figure b is the transcript result of SR34a amplified by the traditional 5' Race technology, and Figure c is the transcript result of SR34a amplified by the optimized 5' Race technology of the present invention. Detailed implementation mode

[0024] Next, in combination with the implementation mode of the present invention, the technical solutions in the implementation mode of the present invention will be clearly and completely described. Obviously, the described implementation mode is only a part of the implementation modes of the present invention, rather than all the implementation modes. Based on the implementation modes in the present invention, all other implementation modes obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present invention.

[0025] In the traditional 5' RACE technology, the reverse transcription efficiency is often low due to the complex secondary structure of RNA during the amplification process, which in turn affects the success rate of the experiment. To solve this problem, based on the existing 5' RACE technology, the present invention proposes an innovative primer design strategy. By analyzing the secondary structure of the target sequence, it is analyzed whether it contains the RG4 structure and its position; then a gene-specific primer is designed upstream of RG4 to avoid the RG4 structure, thereby enriching and detecting the 5'-end sequence.

[0026] This primer design method abandons the commonly used Oligo(dT) 20 (shown in SEQ ID NO.12) or general specific reverse transcription primers in the traditional 5' RACE experiment, thereby significantly improving the efficiency of the reverse transcription process.

[0027] Taking the Arabidopsis thaliana target gene SR34a (AT3G49430) as an example to illustrate the design method of specific reverse transcription primers.

[0028] Specifically, amplifying the Arabidopsis thaliana target gene SR34a includes the following steps:

[0029] S1, Extract the RNA of Arabidopsis thaliana leaves

[0030] Collect Arabidopsis thaliana leaves, quickly freeze and grind them into powder. Use Trizol reagent or a commercially available RNA extraction kit (such as Vazyme's FastPure Universal Plant Total RNA Isolation Kit), and follow the operation manual to extract total RNA.

[0031] RNA quality detection: Detect the RNA sample by 1% agarose gel electrophoresis. If there are relatively complete 28S and 18S rRNA bands in the result, the integrity of the total RNA is good and can be used for subsequent experiments.

[0032] S2, Primer design

[0033] SR34a is an Arabidopsis gene containing 7 transcripts (SR34a.1 to SR34a.7), and the sequences of each transcript are slightly different. Obtain the complete sequences of these transcripts from a reference database (such as TAIR) or the experimental data in step S1. The cDNA reference sequences are shown in SEQ ID NO.2 to 8.

[0034] Since the SR34a.4 transcript has the longest sequence among the other 6 transcripts, the longest transcript SR34a.4 is used as the research object, and an online RNA prediction tool (https: / / bioinformatics.ramapo.edu / QGRS / analyze.php) is used to analyze the possible RG4 structures in its sequence. Figure 2 a shows that there are 5 predicted RG4 secondary structure sites in the SR34a.4 transcript.

[0035] According to the known fragment sequence or reference sequence of SR34a (SR34a.1 to SR34a.7), design a gene-specific reverse transcription primer OSY453-RT (shown in SEQ ID NO.1) that can span the predicted RG4 position.

[0036] Control primer: Design a traditional Oligo d(T) 20 (shown in SEQ ID NO.12), which has the ability to bind to the poly(A) tail of mRNA and is used for reverse transcription in the control group.

[0037] Table 1 Primer sequences

[0038]

[0039] S3, Synthesis of the first strand of cDNA:

[0040] (1) Prepare the initial reaction system

[0041] Prepare the reaction system in an RNase-free test tube. The reaction system includes Total RNA, OSY453-RT (reverse transcription primer), dNTP Mix, and RNase-free H2O.

[0042] In the control group, replace OSY453-RT with Oligo d(T) 20 to prepare the reaction system.

[0043] Component Concentration TotalRNA 1μg OSY453-RT(1μM) 1μL dNTPMix 1μL <![CDATA[RNase-free H2O]]> upto6.5μL

[0044] First, heat the mixture to 72 °C for 3 minutes to unwind the secondary structure of RNA. Then immediately cool it on ice for 2 minutes to prevent RNA from binding to non-specific regions.

[0045] (2) Prepare the reverse transcription reaction system

[0046] The reaction system includes the above reaction product, 5X RT buffer, M-MLV Reverse Transcriptase, and TSO3. The concentrations of each component are shown in the following table:

[0047] Component Concentration The above reaction product 6.5μL 5XRTbuffer 2μL M-MLV(H-)Reverse Transcriptase 1μL TSO3(10μM) 0.5μL Total 10μL

[0048] The reaction conditions are: react at 42 °C for 30 minutes to complete the first strand of cDNA by reverse transcription. React at 85 °C for 5 minutes to inactivate the enzyme and stabilize the cDNA. After the reaction, place it at 4 °C for short-term storage.

[0049] S4, PCR amplification of the target gene fragment

[0050] After reverse transcription, perform PCR amplification on the target gene fragment.

[0051] (1) Prepare the PCR reaction system

[0052] The PCR reaction system includes 2X Taq Plus Master Mix, GSP2, OSYB370, template, and ddH2O.

[0053] For the experimental group, GSP2 is OSY650; for the control group, GSP2 is OSY453. The concentrations of each component are shown in the following table:

[0054] Component Concentration 2XTaqPlus Master Mix 25μL GSP2(10μM) 2μL OSYB370(10μM) 2μL Template (first-strand cDNA) 1μL <![CDATA[ddH2O]]> upto50μL

[0055] Perform the PCR reaction, and the procedure is as follows:

[0056]

[0057] S5, Electrophoresis detection of PCR products

[0058] Electrophorese the PCR products according to the steps in the DNA gel extraction kit manual and excise and purify the target band. The results are as Figure 2 shown.

[0059] Figure 2 As shown in b, the traditional 5′Race technology can only amplify partial transcripts of SR34a, such as SR34a.1 / 2, SR34a.6, and SR34a.7. Compared with Figure 2b, Figure 2 c shows that the novel 5'RACE technology designed in this experiment can amplify all 7 transcripts of SR34a, with a wider coverage range.

[0060] S6, Cloning and sequencing of the target fragment

[0061] Ligate the purified PCR product with the T-vector (5minTA / Blunt-Zero Cloning Kit; Vazyme).

[0062] Ligation reaction system:

[0063] Component Volume 5×TA / Blunt-Zero Cloning Mix 1μL Purified PCR product 1 - 4μL (adjust the dosage according to the fragment size) <![CDATA[ddH2O]]> Make up to a total volume of 5μL

[0064] Ligation reaction conditions: React at room temperature (20 - 37 °C) for 5 minutes.

[0065] Transformation steps:

[0066] 1. Ice bath: Take 5 μL of the ligation product and add it to 50 μL of competent cells, and incubate on ice for 30 minutes.

[0067] 2. Heat shock: Incubate in a 42 °C water bath for 90 seconds, and immediately incubate on ice for 2 minutes.

[0068] 3. Recovery: Add 200 μL of LB medium and shake the bacteria at 37 °C for 1 hour (200 rpm).

[0069] 4. Coating: Take 100 μL of the bacterial solution and coat it on an LB plate containing Kan resistance.

[0070] Colony PCR rapid identification:

[0071] Reaction system:

[0072] Component Concentration 2XTaqPlus Master Mix 10μL M13 Primer Mix(10μM) 2μL <![CDATA[Bacterial suspension (resuspended in ddH2O)]]> 1μL <![CDATA[ddH2O]]> upto20μL

[0073] Perform PCR reaction, and the program is as follows:

[0074]

[0075] Electrophoresis verification: Detect the size of the target band by 1% agarose gel electrophoresis.

[0076] Positive monoclonal sequencing: Pick monoclonal colonies identified as positive by colony PCR and send them to a sequencing company for sequencing. The sequencing method is first-generation sequencing, and the sequencing primer is the universal primer M13 Forward (5'-GTTTTCCCAGT CACGAC-3').

[0077] S7, Sequence alignment

[0078] Use Snapgene to align the sequencing results with the reference sequence.

[0079] The sequencing results show that the 5′-end sequence information of the target gene matches the reference sequence, indicating the successful amplification of the 5′ RACE of the target gene.

[0080] In summary, the 5′ Race technology of the present invention shows significant advantages in dealing with RNA molecules with complex structures and can effectively improve the success rate of cDNA end amplification. In addition, the optimization method of the 5′ RACE technology of the present invention not only significantly improves the efficiency and accuracy of transcript amplification, but also provides a more efficient and economical tool for molecular biology research, with broad application prospects and important scientific significance.

[0081] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for optimizing 5'RACE technology, characterized in that: The following steps are involved: S1, primer design: predict the RG4 structure and position of RNA based on the known partial cDNA sequence of the target gene, and design a reverse transcription primer spanning RG4 upstream of the RG4 structure; S2, reverse transcription based on the primers in step S1 to obtain the first strand of cDNA; S3, performing PCR amplification using the first strand of cDNA from step S2 as a template to obtain the target gene fragment.

2. Application of the optimization method of 5'RACE technology as claimed in claim 1 in 5'RACE amplification of Arabidopsis thaliana SR34a gene.

3. The use according to claim 2, characterized in that The following steps are involved: S10, taking the cDNA sequence of the target gene SR34a transcript as the object, using RNA prediction tools to predict the RG4 structure of the SR34a.4 transcript, and designing the reverse transcription primer OSY453-RT spanning RG4 as shown in SEQ ID NO.1; S20, preparing a reverse transcription reaction system based on the reverse transcription primer OSY453-RT in step S10 to synthesize the first strand of cDNA; S30, preparing an amplification reaction system using the first strand of cDNA in step S20 as a template, and performing PCR amplification to obtain the transcript of the SR34a gene.

4. The use according to claim 3, characterized in that The cDNA reference sequence of the transcript of the target gene SR34a is shown in SEQ ID NO.2-8.

5. The use according to claim 3, characterized in that The reverse transcription reaction system includes reaction products, 5XRT buffer, M-MLV Reverse Transcriptase and TSO3 shown in SEQ ID NO.9; the reaction product is a product obtained by mixing TotalRNA, OSY453-RT shown in SEQ ID NO.1, dNTPMix and RNase-free H2O and reacting them.

6. The use according to claim 3, characterized in that The amplification reaction system comprises: 2XTaqPlusMasterMix, OSY650 shown in SEQ ID NO.10, OSYB370 shown in SEQ ID NO.11, a template and ddH2O.

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