CN111808934A - Circular RNA full-length identification method and kit thereof - Google Patents

Abstract

The invention relates to the field of molecular biology, and relates to a method for identifying the full length of circular RNA and a kit thereof, which comprises the following steps: s1, analyzing a splice junction sequence and predicting the comparison of the full-length sequence of the circular RNA; s2, designing CD Primers and Sjod Primers; s3.LS reverse transcription reaction: adding N6 Random Primer, RNase-Free H into 0.1-1 μ g of extracted total RNA₂Mixing O to obtain a reaction solution I; then adding 5x LS RT Buffer into the reaction liquid I, and adding LS Enzyme Mix into the reaction liquid I to obtain cDNA; s4, PCR amplification and PCR product analysis; and S5.Sanger sequencing and cross-analysis of the comparison sequence.

Description

Circular RNA full-length identification method and kit thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a method for identifying the full length of circular RNA and a kit thereof.

Background

Circular RNAs (circular RNAs) are a class of RNA molecules with closed loop structures that are widely found in many species and are more stable than linear RNAs.

At present, circular RNA information is obtained by using databases such as circBase and the like and high-throughput sequencing of own samples in research, PCR detection, cDNA/gDNA amplification verification, RNase R digestion and other experimental verifications are carried out after circular RNA to be researched is obtained by screening, and the actual existence of the circular RNA is confirmed. However, these verifications are based on the circularization site linker (Splicejunction) sequence of the circular RNA, and lack of verification of the true full-length sequence inside the circularization site, and therefore, are likely to result in distortion of the true accuracy of the detection, especially in the case where there are multiple exons inside the sequence and variable splicing is present. For example (Yibing Yang et al, Novel roll of FBXW7 Circular RNA in reproducing GliomaTumori genetics. J Natl Cancer Inst.2018 Mar 1; 110(3)) through PCR amplification and Sanger sequencing, it was determined that exon3 and exon4 of FBXW7 are reverse-sheared into 620nt Circular RNA, rather than 1227nt recorded in hsa _ circ _0001451 in the circBase database, and it was found that failure to identify the true full-length sequence of Circular RNA is very likely to cause gene recognition errors, detection and verification distortions.

The annotation of reference sequences in circular RNA information obtained using circBase et al databases and high throughput sequencing of self-contained samples is also limited to the technology itself and the analytical methods. 1) The length of reads of high-throughput sequencing is about 150nt, and the true full-length sequence of the circular RNA cannot be detected. 2) Screening, identification and annotation of circular RNA Junction reads are mapped to a reference genome and GenBank dataset, a full-length reference sequence of the circular RNA is determined only by comparative analysis of known linear genes, and a transcript with the highest comparative match is taken as the full-length sequence of the circular RNA, so that variable splicing of exons and exon combinations of the linear genes possibly different from the circular RNA cannot be distinguished. 3) Cleavage from two or more pre-mRNAs results in trans-cleaving (trans-cleaving) to form tsRNAs (trans-cleaved RNAs) which may have the same Splice Junction sequence as the circular RNAs, resulting in false positives for circular RNA screening identification.

Therefore, it is urgently needed to develop a method for identifying the full length of circular RNA, and the full length of the circular RNA which is already recorded or newly found by circBase is identified to determine the true full length sequence of the circular RNA.

Disclosure of Invention

Aiming at the defects in the prior art, the first purpose of the invention is to provide a circular RNA full-length identification method, which solves the problems of identification errors, detection and verification distortion and the like easily occurring in circular RNA identification verification and research.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for identifying the full length of circular RNA comprises the following steps:

s1, comparison and prediction of splice junction sequence analysis and circular RNA full-length sequence: firstly, comparing in UCSCgenome blast to confirm AG-GT shearing sites corresponding to the Splice junction sequence, then comparing in UCSCgenome brown to obtain a corresponding genome DNA sequence, and comparing in Ensembl to obtain a predicted transcription circular RNA full-length sequence;

s2, designing CD Primers and Sjod Primers: designing a primer by using software to search a 40bp primer selectable region in a known sequence, obtaining a sequence of a circular RNA and a Splice junction sequence from a database, selecting 21-40 bp as a forward primer, and performing reverse complementation on the sequence of 1-20 bp to obtain a reverse primer to obtain CD Primers; inputting 30bp sequences of 20bp upstream and 10bp downstream of the Splicejunction position into primer design software, searching and calculating to enable the primer to meet the conventional parameter requirements of GC content and Tm value, enabling the primer to contain sequences not less than 13bp upstream and not more than 7bp downstream so as to obtain an Sjdo upstream primer, then automatically designing an Sjdo downstream primer by using the software according to the parameters and the position of the upstream primer, and obtaining the Sjdo Primers;

s3.LS reverse transcription reaction: extracting total RNA of various tissues, cells, blood, body fluid, exosome, oral swab and alveolar lavage fluid samples, taking 0.1-1 mu g of the extracted total RNA, adding N6 Random Primer, RNase-Free H₂Mixing O, reacting at the temperature of 60-70 ℃ for 5-10 min, and rapidly carrying out ice bath for 2-3 min to obtain a reaction solution I; then adding 5x LSRT Buffer into the reaction liquid I, adding LS Enzyme Mix into the reaction liquid I, reacting for 5-10 min at the temperature of 20-30 ℃, reacting for 60-65 min at the temperature of 50-60 ℃, and reacting for 5-10 min at the temperature of 80-90 ℃ to obtain cDNA;

s4, PCR amplification and PCR product analysis: amplifying the CD Primers and Sjod Primers obtained in the step S2 and the cDNA obtained in the step S3 to obtain a PCR product, and carrying out agarose gel electrophoresis analysis on the PCR product to obtain a direct amplification band and a rolling circle product band;

and S5.Sanger sequencing and cross-analysis of the comparison sequence.

Preferably, the LS Enzyme Mix comprises reverse transcriptase LS M-MLV (RNase H-) and RNaseINHIBITOR, the reverse transcriptase LS M-MLV (RNase H-) mutant is delta N23/D108R/T306L/V433K/. DELTA.aa 524-551, and the gene sequence of the reverse transcriptase LS M-MLV (RNase H-) is shown in SEQ ID NO. 1.

Preferably, the composition of the reaction solution I in the reverse transcription in step S3 includes:

the composition of cDNA in LS reverse transcription in the step S3 comprises:

preferably, 0.1-1 μ g of total RNA extracted in step S3 is added to N6 Random Primer, RNase-Free H₂And O, mixing, reacting at 65 ℃ for 5min, and rapidly carrying out ice bath for 2min to obtain a reaction solution I.

Preferably, in the step S3, the LS Enzyme Mix is added to the reaction solution I, reacted for 5min at a temperature of 25 ℃, reacted for 60min at a temperature of 55 ℃, and reacted for 5min at a temperature of 85 ℃ to obtain cDNA.

Preferably, the PCR product is subjected to electrophoresis analysis by using agarose gel with the concentration of 1.5-2%.

Preferably, in the step S5, the direct amplification band obtained in the step S4 is recovered and directly subjected to Sanger sequencing, and the rolling circle product band is recovered and ligated into the pMD19T vector for Sanger sequencing.

Preferably, in the step S5, the direct amplification band sequence of CD Primers or Sjod Primers, which confirms and verifies the sequence of the position of the Splice junction, is cross-compared with the rolling circle product band sequence, in which the true full-length sequence of the circular RNA is located between two Splice junction sites.

The second purpose of the invention is to provide a kitComprises a reverse transcription reagent and a PCR amplification reagent, wherein the reverse transcription reagent comprises LS Enzyme Mix, 5 XLS RT Buffer, N6 Random Primer and RNase-Free H₂O; the PCR amplification reagent comprises 2 XHS Taq Mix and RNase-Free H₂O。

Preferably, the LS Enzyme Mix comprises 200U/. mu.L LS M-MLV (RNase H-), 40U/. mu.L RNaseIII inhibitor, RNase-Free H₂O and 50% glycerol; the 5 XSS RT Buffer comprises 150mM Tris-HCl, 375mM KCl, 15mM MgCl at pH8.3₂10mM dNTP and 20mM DTT; the 2 XHS Taq Mix comprises 2.5U/. mu.L HSTaq Polymerase, 10mM dNTP, 125mM Tris-HCl at pH8.3, 15mM MgCl₂250mM KCl, 500mM glycine betaine, 0.15% Tween-20, 5% DMSO, and 10% glycerol.

The invention utilizes the natural template replacement activity of the reverse transcriptase M-MLV, realizes high thermal stability and super-strong binding force through point mutation design, deletes the RNase H structural domain and avoids the digestion of a template chain in the reverse transcription process. The modified and optimized reverse transcriptase LS M-MLV (RNase H-) can be subjected to LS reverse transcription treatment at 55-60 ℃, so that the secondary structure of circular RNA can be better opened, and higher yield and yield are obtained. The nature of natural template displacement activity and super-strong binding force is matched with an optimized reaction system and conditions, and the concentration of random primers is reduced, so that the chain binding efficiency and the reverse transcription efficiency during the reverse transcription of the circular RNA template are greatly increased, a large number of products of rolling circle reverse transcription are obtained, namely a plurality of Splice junctions are connected in one cDNA chain, and the possibility of obtaining the real full-length sequence of the circular RNA by PCR amplification is provided.

The invention comprehensively uses two types of reverse Primers, namely CD Primers and Sjod Primers, uses the CD Primers to amplify possible circular RNA sequence combinations, designs the Sjod Primers by referring to the Splice junction to amplify specific circular RNA to be identified, and can efficiently and quickly determine the real full-length sequence of the circular RNA through cross comparison and analysis of the results of the two types of Primers. Wherein the PCR product of the CD Primers comprises one Splice junction, the direct amplification band sequence of the Sjod Primers comprises one Splice junction, and the rolling circle product band sequence of the Sjod Primers comprises two Splice junctions. The PCR product of CD Primers or the direct amplification band of Sjod Primers can verify the sequence of the Splicejunction position, and the true full-length sequence of the circular RNA is arranged between two Splicejunction sites in the sequence of the rolling circle product band of the Sjod Primers.

As shown in FIG. 2, the line indicates PCR products for CD Primers, the line indicates direct amplification bands for Sjod Primers, and the line indicates rolling circle product bands for Sjod Primers.

Compared with the prior art, the invention has the following beneficial effects:

the method for identifying the full length of the circular RNA can directly identify the total RNA extracted from various samples such as tissues, cells, blood, body fluid, exosomes, oral swabs, alveolar lavage fluid and the like without additional treatment such as RNase R digestion and the like, is simple and easy to implement, has standard experimental design and analysis method, is stable in matched full length identification kit system, has wide application range, and can enable researchers to obtain the real full length sequence of the circular RNA more simply and efficiently. The method for identifying the full length of the circular RNA can efficiently and specifically identify the variable shearing of the circular RNA exons and the combination of different exons of the linear gene and the circular RNA, can accurately obtain the difference of single bases, most accurately and truly obtains the full length sequence of the circular RNA, and ensures the accuracy and reliability of the identification, detection and verification of the circular RNA.

Drawings

FIG. 1 is a schematic diagram of the LS reverse transcription process of a circular RNA full-length identification method of the present invention;

FIG. 2 is a schematic representation of CD Primers or Sjod Primers and PCR products of the present invention;

FIG. 3 shows the AG-GT cleavage site corresponding to the Splice sequence of the circular RNAhsa _ circ _0007928 of the present invention;

FIG. 4 is a photograph of the PCR product of the circular RNAhsa _ circ _0007928 of the present invention by electrophoresis;

FIG. 5 is a graph of a 226bp band Sanger sequencing peak;

FIG. 6 is a plot of the KFR1 band for Sanger sequencing peaks;

FIG. 7 is a graph of the F2KR band Sanger sequencing peak;

FIG. 8 is a cross-comparison of the sequencing results of the circular RNAhsa _ circ _0007928 of the present invention;

FIG. 9 shows the AG-GT cleavage site corresponding to the Splice sequence of the circular RNArno _ novel 5139;

FIG. 10 is a diagram illustrating the comparison result of the ring RNArno _ novel5139 in Ensembl according to the present invention;

FIG. 11 is a diagram showing the electrophoretic detection of PCR products of the RNArno _ novel5139 of the present invention;

FIG. 12 is a graph of the F2R2 band Sanger sequencing peak;

FIG. 13 is a graph of the F1R1 band Sanger sequencing peak;

FIG. 14 is a cross-sectional view showing the sequencing results of the RNArno _ novel5139 according to the present invention;

FIG. 15 shows the AG-GT cleavage site corresponding to the Splice sequence of the circular RNAm 9_ circ _007585 of the present invention;

FIG. 16 is a diagram showing the comparison of circular RNA mm9_ circ _007585 in Ensembl according to the present invention;

FIG. 17 is an electrophoretic detection scheme of PCR products of the circular RNAm 9_ circ _007585 of the present invention;

FIG. 18 is a graph of a Sanger sequencing peak of the circular RNA F1R1 band of the present invention;

FIG. 19 is a graph of the F2KR band Sanger sequencing peak;

FIG. 20 is a cross-comparison of sequencing results for circular RNAm 9_ circ _007585 of the present invention;

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

As used herein, unless otherwise noted, other experimental methods may be employed as are conventional in the art, for example, with reference to the molecular cloning instructions (fourth edition) (scientific press, 2017).

The LS Enzyme Mix comprises a reverse transcriptase LS M-MLV (RNase H-) and an RNase Inhibitor, the reverse transcriptase LS M-MLV (RNase H-) mutant is delta N23/D108R/T306L/V433K/[ delta ] aa524-551, and the gene sequence of the reverse transcriptase LS M-MLV (RNase H-) is shown as SEQ ID NO. 1;

wherein N6 Random Primer is synthetic oligonucleotide 5' -N₆-3' at a concentration of 50. mu.M.

Example 1

In this example, the circular RNA is referenced to the hsa _ circ _0007928 sequence and the Splicejunction sequence in the circBase database.

Downloading the reference sequence 389nt of hsa _ circ _0007928 from the circBase database as set forth in SEQ ID No. 2;

the Splice connection sequence 100nt for hsa _ circ _0007928 was obtained from the circBase database as follows: 5'-CTGAAGGCATGGAGAAATTTTGTGAAGACATTGGTGTTGAACCAGAAAAC-3' -5'-ATTTTCAGCTGAACTCTCATCTCTCAACACTGGCAAATATTCATAAGATC-3'.

A method for identifying the full length of circular RNA comprises the following steps:

s1, comparison and prediction of splice junction sequence analysis and circular RNA full-length sequence: firstly, comparing in UCSCgenome blast to confirm AG-GT shearing sites corresponding to the Splice junction sequence (refer to figure 3), then comparing in UCSCgenome browser to obtain corresponding genome DNA sequence, and comparing in Ensembl to obtain predicted transcription circular RNA full-length sequence;

s2, designing CD Primers and Sjod Primers: CD Primers and Sjod Primers were designed from the reference 389nt sequence using Clone Mgr Suite software, some of which can be used in common, and the sequences were sent to the organism for synthesis and PAGE purification.

CD Primers：

C7928-TZ-F1:TGCGGTCTTGCAGTTCTTCAGA

C7928-TZ-R1:GACTTCCTGTTTGGTGATCGTC

Amplification size 389bp, or 389+ n 389bp

C7928-TZ-F2:CCTGCCTCTGGAGATGATTTA

C7928-TZ-R2:GTCTTCTCTTTTTCCTTGGTGGC

Amplification size 390bp, or 390+ n 389bp

Sjod Primers：

C7928-TZ-KF:GTTGAACCAGAAAACATTTTC

C7928-TZ-R1:GACTTCCTGTTTGGTGATCGTC

The amplification size is 132bp, and the amplification size of the rolling circle product is 521bp

C7928-TZ-F2:CCTGCCTCTGGAGATGATTTA

C7928-TZ-KR:GAGTTCAGCTGAAAATGTTTTC

The amplification size is 226bp, and the amplification size of the rolling circle product is 615bp

S3, RNA extraction and LS reverse transcription reaction: total RNA of human esophageal cancer cell line ECA109 was extracted using Trizol Reagent, followed by LS reverse transcription. The reverse transcription reaction system and procedure were as follows:

firstly, preparing reaction liquid according to the table 1, reacting for 5min at the temperature of 65 ℃, and rapidly carrying out ice bath for 2 min.

TABLE 1 LS reverse transcription step [ ]

② preparing reaction liquid according to the table 2, reacting for 5min at 25 ℃, heating to 55 ℃ for 60min, and then heating to 85 ℃ for 5 min.

TABLE 2 LS reverse transcription step-

S4, PCR amplification and PCR product analysis: four pairs of primers were used to perform PCR amplification using ECA109cDNA as a template, and the reaction system is shown in Table 3. The temperature of the reaction program is 95 ℃ for 5min,40 cycles (95 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 80s) and 72 ℃ for 5min, thus obtaining the PCR product.

TABLE 3 PCR amplification reaction System

According to the length of the predicted circular RNA and the size of the PCR product expected to be amplified, an appropriate agarose gel concentration is selected, when the length of the predicted circular RNA is less than 750nt, the concentration of 2% is used, when the length of the predicted cirRNA is more than 750nt, the concentration of 1.5% is used, the PCR product is subjected to agarose gel electrophoresis analysis, and an expected direct amplification band and a rolling circle product band are distinguished according to the band size analysis, and the result is shown in figure 4.

The results show that the bands of the amplification products of F1/R1 and F2/R2 are more miscellaneous, the sizes of the bands of the products of KF/R1 and F2/KR are in accordance with the expectation, wherein 132bp and 226bp are direct amplification bands, and KFR1 and F2KR are rolling circle product bands. Labeled KFR1, F2KR, and 226bp, respectively, and recovered.

And S5.Sanger sequencing and cross-analysis of the comparison sequence.

The 226bp directly amplified band was recovered and subjected to Sanger sequencing directly, which confirmed that the sequence of the Splice junction of hsa _ circ _0007928 was identical to that expected, and the sequencing peak was as shown in FIG. 5.

And recovering a rolling circle product band KFR1, connecting the band KFR1 to a pMD19T vector for Sanger sequencing to obtain a sequencing sequence, wherein a sequencing peak diagram is shown in 6, and a 521bp band is provided with primer sequences of C7928-TZ-KF and C7928-TZ-R1 and two cyclization cleavage sites, and the sequence between the two cleavage sites is a true full-length sequence of hsa _ circ _ 0007928. The underlined markers indicate the full-length sequence of the gene of interest.

Such as:

>GZ19011701145(KFR1)M13-48_J_G11

and recovering the rolling circle product band F2KR, connecting the band to a pMD19T vector for Sanger sequencing, and obtaining a sequencing sequence, wherein a sequencing peak map is shown as 7, and the 615bp band is provided with C7928-TZ-F2 and C7928-TZ-KR primer sequences and two cyclization shearing sites, and the sequence between the two shearing sites is the true full-length sequence of hsa _ circ _ 0007928. The bold label is the primer sequence and the underlined label is the full length sequence of the gene of interest.

Such as: > GZ19011701148(F2KR) M13-48_ J _ B12

Referring to FIG. 8, the sequence of the Splice junction obtained by sequencing of Sanger in the 226bp band and the full-length sequence obtained by sequencing of KFR1 and Sanger in the F2KR band are cross-compared, and analyzed to obtain the true full-length sequence 389nt of hsa _ circ _0007928 as shown in SEQ ID NO. 3.

Example 2

Obtaining a Splice junction sequence of rno _ novel5139 by high-throughput sequencing;

s1, comparison and prediction of splice junction sequence analysis and circular RNA full-length sequence:

the sequence of the Splice junction of rno _ novel5139 is known as follows.

>rno_novel5139_junction_seq

ACTGGTGGCCATCCTGTTGTGAAGATCTTGAACCAGATGATTGTGCAT

Comparison in UCSC genome blast confirmed that the corresponding AG-GT cleavage site of the sponge junction sequence of rno _ novel5139 is shown in FIG. 9.

And (3) comparing in a UCSC genome browser to obtain a corresponding genome DNA sequence shown in SEQ ID NO. 4.

Referring to FIG. 10, in Ensembl alignment, rno _ novel5139 is shown overlapping with Nfatc3 gene sequence, predicting rno _ novel5139 to comprise two exons, which are referenced to full-length sequence 1301nt as in SEQ ID NO. 5.

S2, designing CD Primers and Sjod Primers: CD Primers and Sjod Primers were designed for the reference 1301nt sequence using Clone Mgr Suite software, some of which were shared, and sequences were sent to the organism for synthesis and PAGE purification.

CD Primers：

rno5139-YZ-F2:AACAGTATGGACCTGGACATT

rno5139-YZ-R1:TGTGGTAAGCCAAGTGATGAA

Amplification size 810bp, or 810+ n 1301bp

rno5139-YZ-F2:AACAGTATGGACCTGGACATT

rno5139-YZ-R2:ATACTGTTGATGCCAGGACTC

Amplifying the size 1309bp, or 1309+ n 1301bp

Sjod Primers：

rno5139-YZ-F1:CATCCTGTTGTGAAGATCTTG

rno5139-YZ-R1:TGTGGTAAGCCAAGTGATGAA

The amplification size is 103bp, and the amplification size of the rolling circle product is 1404bp

S3, RNA extraction and LS reverse transcription reaction: total RNA from rat liver tissue was extracted using Trizol Reagent, followed by LS reverse transcription. The reverse transcription reaction system and procedure were as follows:

the reaction solution was prepared according to Table 4 under reaction conditions of 65 ℃ for 5min and rapidly ice-cooled for 2 min.

TABLE 4 LS reverse transcription step [ ]

The reaction solution was prepared according to Table 5 under the conditions of 25 deg.C for 5min,55 deg.C for 60min, and 85 deg.C for 5 min.

TABLE 5LS reverse transcription step-

S4, PCR amplification and PCR product analysis:

PCR was performed using rno cDNA as a template and three pairs of primers, respectively, and the reaction system is shown in Table 6. The reaction program was 95 ℃ for 5min,40 cycles (95 ℃ 30s,58 ℃ 30s,72 ℃ 180s), 72 ℃ for 5 min.

TABLE 6PCR amplification reaction System

And selecting proper agarose gel concentration according to the length of the predicted circular RNA and the size of the PCR product expected to be amplified, performing agarose gel electrophoresis analysis on the PCR product by using the concentration of 2% when the length of the predicted circular RNA is less than 750nt and the concentration of 1.5% when the length of the predicted cirRNA is more than 750nt, and analyzing and distinguishing an expected direct amplification band and a rolling circle product band according to the band size.

The results of the electrophoresis are shown in FIG. 11, which shows that the band size of the F2/R1 amplification product is wrong, and the amplification may be non-specific amplification. The sizes of the product bands of F2/R2 and F1/R1 are consistent with expectations, wherein 103bp is a direct amplification band, and F2R2 and F1R1 are rolling circle product bands. Labeled as F2R2 and F1R1, respectively, and recovered.

As shown in FIG. 12, the rolling circle product band F2R2 was recovered and subjected directly to Sanger sequencing, which confirmed that the sequence of the Splice junction of rno _ novel5139 was identical to that expected. The rolling circle product band F1R1 is recovered and is connected to a pMD19T vector for Sanger sequencing to obtain a sequencing sequence, a sequencing peak map is shown in figure 13, the 1404bp band is provided with rno5139-YZ-F1 and rno5139-YZ-R1 primer sequences, two cyclization shearing sites are provided, and the sequence between the two shearing sites is the real full-length sequence of rno _ novel 5139. The bold label is the primer sequence and the underlined label is the full length sequence of the gene of interest.

>GZ18061302232(5139-F1R1)M13+_-

S5.Sanger sequencing and Cross-analysis of alignment sequences

As shown in FIG. 14, the Splice junction sequence obtained by Sanger sequencing of the F2R2 band and the full-length sequence obtained by Sanger sequencing of the F1R1 band are crossly compared, and the real full-length sequence 1301nt of the rno _ novel5139 is analyzed to be as shown in SEQ ID NO. 6.

Example 3

Obtaining a reference sequence and a Splice connection sequence of mm9_ circ _007585 from a circBase database;

s1, comparison and prediction of splice junction sequence analysis and circular RNA full-length sequence: downloading the reference sequence 449bp of mm9_ circ _007585 from the circBase database as shown in SEQ ID NO. 7;

the 100bp Splice connection sequence for mm9_ circ _007585 was obtained from the circBase database as follows:

5’-TGGACAGCCTGGCTTGGCTACTCATTCTTCTGTTAACAGGGTCCTGGCAG-3’-5’-GAGGCTGCTGGAGTCTTCCCTCATTTCATTATCCCGTTATGATGGAGCAG-3’

comparison in UCSC genome blast confirmed that the corresponding AG-GT cleavage site of the bolt junction sequence of mm9_ circ _007585 is shown in FIG. 15.

The corresponding genomic DNA sequence obtained by alignment in UCSC genome browser is shown as SEQ ID No. 8.

As shown in FIG. 16, in Ensembl alignment, mm9_ circ _007585 is shown to overlap with Ambra1 gene sequence, and mm9_ circ _007585 is predicted to contain up to four exons, and its reference full-length sequence 449nt is predicted as shown in SEQ ID No.9 by the longest sequence.

S2, designing CD Primers and Sjod Primers: CD Primers and Sjod Primers were designed for the reference sequence using Clone Mgr Suite software, some of which were used in common, and the sequences were sent to the organisms for synthesis and PAGE purification.

CD Primers：

mc7585-YZ-F1:AATCGGCTTCGTTCTTCCACCTCC

mc7585-YZ-R1:ATTCTGTTGGTAGCGCATGGAGC

Amplification size 452bp, or 452+ n 449bp

Sjod Primers：

mc7585-YZ-F2:CTGCACCTTCACTTGGACGATT

mc7585-YZ-KR:AAGACTCCAGCAGCCTCCTGC

The amplification size is 149bp, and the amplification size of the rolling circle product is 598 bp.

S3, RNA extraction and LS reverse transcription reaction: total RNA of mouse NC1469 cell line was extracted using Trizol Reagent and then subjected to LS reverse transcription treatment. The reverse transcription reaction system and procedure were as follows:

the reaction solution was prepared according to Table 7 under reaction conditions of 65 ℃ for 5min and rapidly cooled in ice for 2 min.

TABLE 7 LS reverse transcription step [ ]

The reaction solution was prepared according to Table 8 under the conditions of 25 ℃ for 5min,55 ℃ for 60min and 85 ℃ for 5 min.

TABLE 8 LS reverse transcription step-

S4, PCR amplification and PCR product analysis:

PCR was carried out using NC1469 cDNA as a template and two pairs of primers, respectively, and the reaction system is shown in Table 9. The reaction program was 95 ℃ for 5min,40 cycles (95 ℃ 30s,58 ℃ 30s,72 ℃ 90s), 72 ℃ for 5 min.

TABLE 9 PCR amplification reaction System

And selecting proper agarose gel concentration according to the length of the predicted circular RNA and the size of the PCR product expected to be amplified, performing agarose gel electrophoresis analysis on the PCR product by using the concentration of 2% when the length of the predicted circular RNA is less than 750nt and the concentration of 1.5% when the length of the predicted cirRNA is more than 750nt, and analyzing and distinguishing an expected direct amplification band and a rolling circle product band according to the band size.

The results of the electrophoresis are shown in FIG. 17, and the results show that the sizes of the product bands of F1/R1 and F2/KR are consistent with the expected sizes, wherein 149bp is a direct amplification band, and F2KR is a rolling circle product band. Labeled as F1R1 and F2KR, respectively, and recovered.

S5.Sanger sequencing and Cross-analysis of alignment sequences

As shown in FIG. 18, the F1R1 band was recovered and directly subjected to Sanger sequencing, which confirmed that the Splice junction sequence of mm9_ circle _007585 was consistent with the expected sequence.

The rolling circle product band F2KR was recovered and ligated into pMD19T vector for Sanger sequencing to obtain the following sequencing sequence, the sequencing peak map is shown in FIG. 19, and shows that there are mc7585-YZ-F2 and mc7585-YZ-KR primer sequences in the 598bp band, and there are two circularization cleavage sites, and the sequence between the two cleavage sites is the true full-length sequence of mm9_ circ _ 007585. The bold label is the primer sequence and the underlined label is the full length sequence of the gene of interest.

>GZ17122700658(7585-F2KR)M13-47_J_C01

As shown in FIG. 20, the Splice junction sequence obtained by Sanger sequencing of the F1R1 band and the full-length sequence obtained by Sanger sequencing of the F2KR band are crossly compared, and the real full-length sequence 449nt of mm9_ circ _007585 is analyzed and is shown as SEQ ID NO. 10.

Example 4

A kit for identifying the full-length sequence of circular RNA, which comprises a reverse transcription reagent and a PCR amplification reagent and an instruction for use, wherein the reverse transcription reagent comprises LS Enzyme Mix, 5 XLS RT Buffer, N6 Random Primer and RNase-Free H₂O; the PCR amplification reagent comprises 2 XHS Taq Mix and RNase-Free H₂O。

The LS Enzyme Mix comprises 200U/. mu.L LS M-MLV (RNase H-), 40U/. mu.L RNaseINhibitor and RNase-Free H₂O and 50% glycerol; the 5 XSS RT Buffer comprises 150mM Tris-HCl, 375mM KCl, 15mM MgCl at pH8.3₂10mM dNTP and 20mM DTT; the 2 XHS Taq Mix comprises 2.5U/. mu.L HSTaq Polymerase, 10mM dNTP, 125mM Tris-HCl at pH8.3, 15mM MgCl₂250mM KCl, 500mM glycine betaine, 0.15% Tween-20, 5% DMSO, and 10% glycerol.

The application instructions give a using method of the kit and a standardized experimental design and analysis method for identifying the real full-length sequence of the circular RNA, and provide guidance, guidance or teaching for reverse transcription in full-length identification, sequence analysis and comparison prediction of the full-length sequence, primer design, PCR amplification and electrophoresis detection, sanger sequencing and sequence analysis.

The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.

Sequence listing

<110> Guangzhou Giselsa Biotechnology Ltd

<120> circular RNA full-length identification method and kit thereof

<130>2020.7.17

<160>10

<170>SIPOSequenceListing 1.0

<210>1

<211>623

<212>PRT

<213>SEQ ID NO.1

<400>1

Met Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly

1 5 10 15

Met Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala

20 25 30

Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala

35 40 45

Arg Leu Gly Ile Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile

50 55 60

Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys

65 70 75 80

Lys Pro Gly Thr Asn Arg Tyr Arg Pro Val Gln Asp Leu Arg Glu Val

85 90 95

Asn Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn

100 105 110

Leu Leu Ser Gly Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp

115 120 125

Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro

130 135 140

Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln

145 150 155 160

Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu

165 170 175

Phe Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His

180 185 190

Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala

195 200 205

Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr

210 215 220

LeuGly Asn Leu Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys

225 230 235 240

Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg

245 250 255

Trp Leu Thr Glu Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro

260 265 270

Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu Gly Leu Ala Gly Phe Cys

275 280 285

Arg Leu Trp Ile Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro

290 295 300

Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln Lys

305 310 315 320

Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala Leu Gly

325 330 335

Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln

340 345 350

Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg

355 360 365

Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro Val Ala Ala Gly Trp

370 375 380

Pro Pro CysLeu Arg Met Val Ala Ala Ile Ala Val Leu Thr Lys Asp

385 390 395 400

Ala Gly Lys Leu Thr Met Gly Gln Pro Leu Lys Ile Leu Ala Pro His

405 410 415

Ala Val Glu Ala Leu Val Lys Gln Pro Pro Asp Arg Trp Leu Ser Asn

420 425 430

Ala Arg Met Thr His Tyr Gln Ala Leu Leu Leu Asp Thr Asp Arg Val

435 440 445

Gln Phe Gly Pro Val Val Ala Leu Asn Pro Ala Thr Leu Leu Pro Leu

450 455 460

Pro Glu Glu Gly Leu Gln His Asn Cys Leu Asp Ile Leu Ala Glu Ala

465 470 475 480

His Gly Thr Arg Pro Asp Leu Thr Asp Gln Pro Leu Pro Asp Ala Asp

485 490 495

His Thr Trp Tyr Thr Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg Ala

500 505 510

Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys Lys

515 520 525

Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Ile

530 535 540

His Gly Glu Ile TyrArg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys

545 550 555 560

Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe

565 570 575

Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly

580 585 590

His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala Arg

595 600 605

Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile

610 615 620

<210>2

<211>389

<212>DNA

<213>SEQ ID NO.2

<400>2

attttcagct gaactctcat ctctcaacac tggcaaatat tcataagatc taccacaccc 60

ttaataagct gaacctaaca gaagacattg gccaagacga tcaccaaaca ggaagtctgc 120

ggtcttgcag ttcttcagac tgctttaata aagtgatgcc accaaggaaa aagagaagac 180

ctgcctctgg agatgattta tctgccaaga aaagtagaca tgatagcatg tatagaaaat 240

atgattcgac tagaataaag actgaagaag aagccttttc aagtaaaagg tgcttggaat 300

ggttctatga atatgcagga actgatgatg ttgtaggccc tgaaggcatg gagaaatttt 360

gtgaagacat tggtgttgaa ccagaaaac 389

<210>3

<211>389

<212>DNA

<213>SEQ ID NO.3

<400>3

attttcagct gaactctcat ctctcaacac tggcaaatat tcataagatc taccacaccc 60

ttaataagct gaacctaaca gaagacattg gccaagacga tcaccaaaca ggaagtctgc 120

ggtcttgcag ttcttcagac tgctttaata aagtgatgcc accaaggaaa aagagaagac 180

ctgcctctgg agatgattta tctgccaaga aaagtagaca tgatagcatg tatagaaaat 240

atgattcgac tagaataaag actgaagaag aagccttttc aagtaaaagg tgcttggaat 300

ggttctatga atatgcagga actgatgatg ttgtaggccc tgaaggcatg gagaaatttt 360

gtgaagacat tggtgttgaa ccagaaaac 389

<210>4

<211>4637

<212>DNA

<213>SEQ ID NO.4

<400>4

atcttgaacc agatgattgt gcatccattt acatctttaa tgtagacccg cctccatcta 60

ctttaaattc atcacttggc ttaccacatc atggactgct gcagtctcac tcttctgttt 120

tgtcaccatc atttcagctc caaggtttca aaaattatga aggaactgat gatatttctg 180

aatctaaata tagctcatta agtggtccta aaccctttga atgcccaagt attcaaatta 240

catccatctc tcctaactgt catcaagaaa cagatgctca tgaagatgac ctacatgtaa 300

atgacccaga aagggaatat ttggaaaggc cttctagaga tcatctctat ctcccacttg 360

aaccgtccta ccgggaatct tcccttagtc ctagtcctgc cagcagcgtt tcttctagga 420

gttggttctc agatgcatct tcttgtgagt ctctctcaca catttatgac gatgtggact 480

cagagttgaa tgaagctgct gcacgattta ctcttggctc acctctgact tctccaggtg 540

gctctccagg aggttgccct ggagaagagt cctggcatca acagtatgga cctggacatt 600

ccttgtcacc taggcaatct ccttgccact ctcctagatc cagtatcact gatgagaatt 660

ggctgagccc cagaccagcc tcaggaccct catccaggcc cacttctcct tgtggtaaac 720

gacggcactc cagtgctgaa gtatgttatg ctggctctct ttcaccccat cactcacctg 780

ttccgtcccc tggtcactcg cctagaggga gtgtaactga agatacctgg ctcactgctc 840

ctgtccacac tggatcaggc ctcagccctg caccgtttcc atttcagtac tgtgtagaga 900

ctgacatccc tttgaaaaca aggaagactt ctgacgatca agctgccata ctaccaggaa 960

aattagaggt ctgttcagat gatcaaggga gcttatcccc atcccgggag acatcagtag 1020

atgatggcct tggatctcag tatcctttaa agaaagattc atctggtgac caatttcttt 1080

cagttccttc accttttacc tggagcaaac caaagcctgg ccacactcct atatttcggt 1140

gagttggtag aaatggctgc tgatcagttt ttcatgctta taggtcattt gcattgtata 1200

actacattct gacctttttt cttctccttg cccttctccc tttcatttct tttctctctc 1260

ccctctccct gccctcatct tccttgtttt tcattttttt aaaaaattaa ttcttctggt 1320

caaataactt agaggtcctt tttatttttc tttaagtctt taaaagtgtt tttaaattga 1380

ctaaagttac attttaaaag tatatagatg aagaaagtat gtgatggatt gtagagaaca 1440

gtaaatgaaa ttgaaggttc cttgcggaat tccttatcta gaaaagatta ctgatgggga 1500

gtggggaggg gaaatgggga gtggggaggg gaaattgggg agtggggagg ggaaatccca 1560

tatagaatga gaggaggagg ggttaggggg atgttggcct ggaaaccggg aaagggaata 1620

acaattgaaa tgtaaataag aaatacccaa tttaataaag atggagaaaa aactgtaaat 1680

gtcattcaaa aggtattttt atataaatgt acagttaggc agtaatacct tgaaaatcca 1740

tcatttcttt ctaatttcat ttagtaaagt aaaaaatact aaaaaaaaaa aaaagattac 1800

tgtacaggtg ggttattata ttatgaatga attaactgaa tttacatatc tgaaggactg 1860

tgtgggagtg tgtaagtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1920

tgtaggccag aagacagcca tgggtattat ttctcaggag tcatacatct tgtcatcttg 1980

tttttgaaac aagttttctt gttggcctag agctcaccaa gctgctggct ttagaaaaaa 2040

aaatacgtgt gtgaatgtgc tcatgtgtga catgccctgt gtgttgtatt cagagaacgg 2100

ctttctggag gtgtagctgt tctttccttt tacctttatg tagattctgg ggattgagct 2160

caggtcctaa ggcttgtata tactgacttt tcctttgtcc ttttaatggc aagcattttc 2220

tcccacaagg tatttacttt tttttttttt ttttggttct tttttttttt tttcggagct 2280

ggggaccgaa cccagggcct tgcgcttcct aggcaagcgc tctgccactg agccaaatcc 2340

ccaaccccag gtatttactt ttaaatattt atttaattga gatgggatct tgctacattg 2400

cctgtattga cccgtgctga cctaaaactg tatgatccta ctattccagt ctcccaaagt 2460

agctgggact acagatccat gctattgtgc ttggccttct taactttttt ttcccctcca 2520

aaatctttca gcagtcttag tttctttctt tgagattagt ttctatgtag ctcagactga 2580

ccttaagttt acaacataac tggcaatggc cttgaagttc cgatcttcct acatccaccg 2640

ccaaaattct gggataacag gtgtgtgtca ctacacctag ttttatgtag tggtagggtt 2700

caaactcagg ctttctcaca tgctgctagg caagaactct acttatcagc tgagcttttt 2760

gtcctttact atttgatctg tggatatgta aataatttta cagttctatg ttttctctgt 2820

gtgtgtgcac atgtgcgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt atgtgtgtgt 2880

gtgtgtaggt ccatgtggaa gccagaggtt gacattggat atcttcctaa ttcatttgtt 2940

tctccacttg atttttgttt atttgtttgg ttggtttggc ttttcaagtc aaggtttctc 3000

tatgtagccc tggctgtcct ggaacttgct ctgtagacca ggctggcctc taagtcagag 3060

atctgcctgc ctctgccttc tgagttctag gatcaaagtt gtataccaca ctgcccagct 3120

tccccttggt tttaaaaaag gatctctctt tgaacctggt tggctggaaa gcctcctatc 3180

tccctcccca gtgctggaat tacaggcttg agccacagtg ctggttttta tgtgtgtttg 3240

ggggatttga accaaggccc tcattataga atggcaagta cccttaatac tgaacaacct 3300

tccaagcgct tgtaatattt tcaatattaa taggcctaaa ttaacgttta ggtcttcaca 3360

gtttaagttc ttactaatca agggaaatat taacctttta aaaacatact aaaatgcttc 3420

atgtctgttt caagcagtca tgcactgaaa ctaaactttt catcagtcac aggactgctt 3480

gggattagca gctcctcaga gagaagagtg gaaggaagtg gaggagacca taggcataat 3540

agattgattt ccataggtca gattagagca gagaaagatt catactgaaa gaaatataaa 3600

aatttagatt ttaaaggtac ttagtcttgg gactggatag attactcagg tttttttttt 3660

tgttttttgt tttttttctt tttcttttct cttttctttt ttttttttgg agctggggat 3720

tgaacccagg gccttgcgct tgctaggcaa gcgctctacc actgagctaa atccccaacc 3780

ccgattactc agtttttaag agtacctgct gttcctctga agacccaggc tcaattccta 3840

gcatccacat agcagctata accatctgta actctaactc tgagggatac aatgccctct 3900

tctggcctct gcagctattg ggcctgcaca tggtacacag acacacgtgc aggcaaaaca 3960

ttcatacaca taaaatgaaa atgaataaaa ttacttattc atagctacat atgtgtgtat 4020

gtgtatgtat gtatatatat atacacatac atatatacat atatgtgtat atatatacac 4080

acatagaaag atgagtatct ttgctttaag aaattgaact actagtggga ctagggcgac 4140

aaacaacttt aatccctctt gggaggcaga ggcaagtgga tttctgtgag tttgaggaca 4200

gactggccta cagattaagt tccaggacag ccaaagctac atagagaaac cctgtctcaa 4260

aaaacccaaa ccaaccaaac aaagaaaaaa aagaatagcc cattaaatac ctcccatgat 4320

tggttctgta acttactaag tgaagtaagt tgattttgga atattttttt aagccactgg 4380

aaaaaacatt gaacctggca aagagatgct ttgagttttt gaatctgctt tgcaatgtta 4440

atatgatgaa attgttcgtg tgtttttttt atagcacatc ttcattacct ccattagact 4500

ggcctttacc aactcacttt ggacaatgtg aattgaaaat agaagtgcaa cctaaaactc 4560

accatagagc ccattatgaa actgaaggta gccgaggggc agtgaaagcc tctactggtg 4620

gccatcctgt tgtgaag 4637

<210>5

<211>1301

<212>DNA

<213>SEQ ID NO.5

<400>5

atcttgaacc agatgattgt gcatccattt acatctttaa tgtagacccg cctccatcta 60

ctttaaattc atcacttggc ttaccacatc atggactgct gcagtctcac tcttctgttt 120

tgtcaccatc atttcagctc caaggtttca aaaattatga aggaactgat gatatttctg 180

aatctaaata tagctcatta agtggtccta aaccctttga atgcccaagt attcaaatta 240

catccatctc tcctaactgt catcaagaaa cagatgctca tgaagatgac ctacatgtaa 300

atgacccaga aagggaatat ttggaaaggc cttctagaga tcatctctat ctcccacttg 360

aaccgtccta ccgggaatct tcccttagtc ctagtcctgc cagcagcgtt tcttctagga 420

gttggttctc agatgcatct tcttgtgagt ctctctcaca catttatgac gatgtggact 480

cagagttgaa tgaagctgct gcacgattta ctcttggctc acctctgact tctccaggtg 540

gctctccagg aggttgccct ggagaagagt cctggcatca acagtatgga cctggacatt 600

ccttgtcacc taggcaatct ccttgccact ctcctagatc cagtatcact gatgagaatt 660

ggctgagccc cagaccagcc tcaggaccct catccaggcc cacttctcct tgtggtaaac 720

gacggcactc cagtgctgaa gtatgttatg ctggctctct ttcaccccat cactcacctg 780

ttccgtcccc tggtcactcg cctagaggga gtgtaactga agatacctgg ctcactgctc 840

ctgtccacac tggatcaggc ctcagccctg caccgtttcc atttcagtac tgtgtagaga 900

ctgacatccc tttgaaaaca aggaagactt ctgacgatca agctgccata ctaccaggaa 960

aattagaggt ctgttcagat gatcaaggga gcttatcccc atcccgggag acatcagtag 1020

atgatggcct tggatctcag tatcctttaa agaaagattc atctggtgac caatttcttt 1080

cagttccttc accttttacc tggagcaaac caaagcctgg ccacactcct atatttcgca 1140

catcttcatt acctccatta gactggcctt taccaactca ctttggacaa tgtgaattga 1200

aaatagaagt gcaacctaaa actcaccata gagcccatta tgaaactgaa ggtagccgag 1260

gggcagtgaa agcctctact ggtggccatc ctgttgtgaa g 1301

<210>6

<211>1301

<212>DNA

<213>SEQ ID NO.6

<400>6

atcttgaacc agatgattgt gcatccattt acatctttaa tgtagacccg cctccatcta 60

ctttaaattc atcacttggc ttaccacatc atggactgct gcagtctcac tcttctgttt 120

tgtcaccatc atttcagctc caaggtttca aaaattatga aggaactgat gatatttctg 180

aatctaaata tagctcatta agtggtccta aaccctttga atgcccaagt attcaaatta 240

catccatctc tcctaactgt catcaagaaa cagatgctca tgaagatgac ctacatgtaa 300

atgacccaga aagggaatat ttggaaaggc cttctagaga tcatctctat ctcccacttg 360

aaccgtccta ccgggaatct tcccttagtc ctagtcctgc cagcagcgtt tcttctagga 420

gttggttctc agatgcatct tcttgtgagt ctctctcaca catttatgac gatgtggact 480

cagagttgaa tgaagctgct gcacgattta ctcttggctc acctctgact tctccaggtg 540

gctctccagg aggttgccct ggagaagagt cctggcatca acagtatgga cctggacatt 600

ccttgtcacc taggcaatct ccttgccact ctcctagatc cagtatcact gatgagaatt 660

ggctgagccc cagaccagcc tcaggaccct catccaggcc cacttctcct tgtggtaaac 720

gacggcactc cagtgctgaa gtatgttatg ctggctctct ttcaccccat cactcacctg 780

ttccgtcccc tggtcactcg cctagaggga gtgtaactga agatacctgg ctcactgctc 840

ctgtccacac tggatcaggc ctcagccctg caccgtttcc atttcagtac tgtgtagaga 900

ctgacatccc tttgaaaaca aggaagactt ctgacgatca agctgccata ctaccaggaa 960

aattagaggt ctgttcagat gatcaaggga gcttatcccc atcccgggag acatcagtag 1020

atgatggcct tggatctcag tatcctttaa agaaagattc atctggtgac caatttcttt 1080

cagttccttc accttttacc tggagcaaac caaagcctgg ccacactcct atatttcgca 1140

catcttcatt acctccatta gactggcctt taccaactca ctttggacaa tgtgaattga 1200

aaatagaagt gcaacctaaa actcaccata gagcccatta tgaaactgaa ggtagccgag 1260

gggcagtgaa agcctctact ggtggccatc ctgttgtgaa g 1301

<210>7

<211>449

<212>DNA

<213>SEQ ID NO.7

<400>7

gaggctgctg gagtcttccc tcatttcatt atcccgttat gatggagcag gatccagaga 60

gcacccaatt tacccagacc cagcgagatt atctcctgct gcatactacg cccagaggat 120

gatccagtat ctctcaagga gagacagtat tcgccagcgc tccatgcgct accaacagaa 180

tcggcttcgt tcttccacct cctcttcttc ctcagacaac cagggtccat cagtagaggg 240

aaccgacttg gaatttgagg actttgagga caatggtgac agatctaggc accgagctcc 300

ccgaaatgcc cgaatgtctg caccttcact tggacgattt gttccaaggc gttttttgct 360

gcctgagtac ttgccttatg ctgggatttt tcatgaacgt ggacagcctg gcttggctac 420

tcattcttct gttaacaggg tcctggcag 449

<210>8

<211>20329

<212>DNA

<213>SEQ ID NO.8

<400>8

gaggctgctg gagtcttccc tcatttcatt atcccgttat gatggagcag gatccagaga 60

gcacccaatt tacccagacc cagcgaggta aggccaaaat gagaaatggc actaggaggg 120

ggggaggggc ggcccttggg gttgccaagt tgtgggaacc cccacctgtg cctctctggt 180

cctggtgctc tgttgctctt tagtcacagc attgaggcag gtgtttcagg tgattgggga 240

ttgttgcagg taagtgaatg gcatctctgg tgacagaaaa ttataattgc gctattgtcc 300

tttgggactc tgtctgtcag tggggcacta gggtacctgt ggagggcata ggtagtcctt 360

cctactactc aagagtctgt tggctgtcac atggacaacc aaaagaagag ccaaagtttt 420

gagcatttta tttgtaggga agatagttat attaaacaaa tttacaatat caaaagggta 480

aaaaaaaatc aagtcaatgt ttagacactt tcccaaagaa gagatgaagg aagcataatg 540

gaatgaaatt gtcattttac tgaagtgctt gctttattct gttttaggag acatttgaaa 600

ggttattgaa gaccctgaat gtgtttgtgt gtgttatgca tatgtgtgtt tgtggtatgg 660

gcacaggcag aggccacatg aagactttgg agtctttctc tgttcttcac attatcattt 720

ttttctcttc ctcctactcc accatcatca tcatcaccat gacatcatcg ttcatttttg 780

agatggcatt ttttactgaa ctagaaactt gccattgtat ctagacatac ctgtgatctg 840

tctttctgta tcttgtattg ctggggtcac agatacctgt gtcatgccca gtttttacat 900

gggtgctgga gagttgcaca caggcccttt tgcttttaca gcaaactccc ttatccaccc 960

agtcaaatat ttttctaagc ctgaagtttt taaaagttag gggaaagatg tttttcattt 1020

gagcctaagg gaccatccta aagtccctag tgctgatggt ggcgctgata acaaaaccaa 1080

agctggaaca ttgagttgta ttacaacatt tatcagtaaa gagcagactc aggtggttgt 1140

catagctgga tttgatgact tattagagat ctgaaaaatg ctaacttgac tattatttaa 1200

atagtatcag aaaaagctat aaggaaaagg atagtttaca gtggtgggct aggcaagtca 1260

gtagtatagg ccagataaaa ctaaatgagc tttcccgtct atctggaaac atctttaaag 1320

accaatttga ctctagagat agtaaatgat tttgttgttg ccacagttta gttgcagtag 1380

tgcccattct taccaagata gtagtggaac aaaaacttgc catggcctct tactgtcttt 1440

tccatatccc agaagtagca aatccaagga tctagacagt agagagggat ggggacatta 1500

aaaatagata ggcaattcag atggtgattg aagcaaggtc agctatatta gtctctaatt 1560

atgttggaaa acatacaagt ccttaaaaag ggaagggaaa gtttcacatt ttcgggaacc 1620

ataaaagact cgacacaact tctttccata aatagtttct tttcatgctg tgacattgca 1680

cacctttaac tccagcagtt gggaagcaga ggtaggtaga tctctgtgaa tttggggcca 1740

gcctgctcta cctagtgact ttcagatatt ggtaggaatc tcattctcag aaattttaga 1800

ctgggaaact ttaacgattt gtttcggaga cagggccaga tgtacagcct gggctacctc 1860

taggtcactg tgtagccaat ggaggctggt ttcaaacttt tgatctttct gtctctgtct 1920

cagaatgtag agattacaga tactgccctt tcatgcagtc tgttattaat tcctactctg 1980

atttctgtcc tagtgaaagg cttagatacc taagatctca ctcggaacac ttcctggttg 2040

ttggatggtc ctgtggctgc tcggtggagg gtatttgtac catcttctca cattaactca 2100

tttctttctc ctcttagttg tgttctacat tatacggata gggaaagctt ttgtgagaca 2160

ttttctaaaa catgcagaaa ccttccttcc cacctggata cataagaggg caaaactctg 2220

tcagaactct atcttccttt atttgtttat gtgtactact ggtgatttag tttcatgtcc 2280

tgtagtgtaa ttaaacgact aggggctatg tgatgctgtg cttacatctc tatacaaaag 2340

tgtagtcctt attcaatatt acatcaacag cgctatttct acagccatta tcagtaacag 2400

tccatatttt tctgttcttt ctggccatcc ataagctgag aaatgggtgt gcttgttgtc 2460

acttacgttc tcttccacat cagttctctt caattctttc ctgttacata ttttttctcc 2520

agtctaattt ctttgtgttt aatccatgtt caatttctgc tggaatattt tagtatattt 2580

ggtaacagta agcacaacat gaaggcactg acttctgtgc aatgtctggt ggttagtggt 2640

tgaatgcttg gcaagctagc ctatgaatag tgaaaatacc atgagttgag aaacttttgg 2700

aaaacagttg gtgttgtctg attagtttga tctaggcttc tgcatgcccc tgcccaccta 2760

ctgtacaaag gtcatttgga gtcattgtgc tcagtgtagc atattatttt aacatgtaaa 2820

cttttgagtt ccagtgatta ctagctagct ccttgagaat tctatttgat aagtgaccca 2880

aaggacaatt ttaatgacag ccttaggtcc cagtgcagtt gactacaaga tctcttttag 2940

cctaggaagg tagaacttct gagaacaaag tatcaaaggt actggaagtg gaactgcaag 3000

atgtttgcag ttttgggggt gcctctgtta ctaatataag ttttggggtg cctctgttac 3060

taatataagt tttgggggtg cctctgttac taatataagt tgcccaactg acctgagctg 3120

aggctcttca gcttcttgag tagctgagat taaagtcaca caggtgtgcc caagaataaa 3180

tacttttatt tcatttagat acagctattc taagtcagtt tttaagtatt ctcaataaaa 3240

agaagacaaa acacaccttc ccttttagat atctaaacat tataaaaatg taaaatctcc 3300

caggtttgta acactaggtt tatgacttct ttttaaatgc agaaaaaaaa gtgtgtgtgt 3360

gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtat gaacgacagt 3420

atatatttca ctaacccttt ctctatttta catttttctt ttggaagagt tgccttatga 3480

gtagataagt ataattgtta catgtacata catcttaaaa tgttgagaga gggtctcaag 3540

ctaacttcaa acttgtgtgg ttgagactga ccttgaactc ctgatccatc tgctctaggt 3600

ttgtagtgct gagagtatag gcttatgtca ctgtgcccag ccaattcttt ttaatggctt 3660

cctggtgttt ctctgtatgg aaataattta cttaaatctg ttggtgggtt tctgggttgt 3720

ttcttggatt ttccccactg atgacagtgt tgcagatgaa ctttctcatg taatttatat 3780

aacattcatt tatattcttg tgtatttctg tgtgatataa tttactagtg gacttgcatc 3840

attgtgagtt ctttaaagtc ttacagttgg tttagcaatc tcttaacact atcacataag 3900

acatttttct gttgtcctca gtttatagtc tctaattaca gaacagtgtt gtaaagatgg 3960

aggaaaacct taagaaaggg aaggagtatg tagaattgtg tgatattaga aattgtcagg 4020

ggcctggaga gatggctcag tggttaagag caccgactgc tcttccaaag gtcctgagtt 4080

caaatcccag caaccacatg gtggctcaca accttcagtc atgagatctg acaccctctt 4140

ctggtgcatc tgaagacaac tacagtgtac ttagatataa taataaataa atctttaaaa 4200

aaaaaaaaag aaattgtcag agagctaaga catgtcgtcc ttggtttcaa ctggagtcta 4260

tagactgatg atggtttatt ggaaccctag cagaggagat gtattctcca ttgctgtaat 4320

tctacaaatt gaaaattctt ttttctatta ggttttctat tctcccaact acaaaatgtc 4380

tctcactccc tatcctggat agttttccat gtacaaatac ctatgattaa tttgtgaggg 4440

agggaagata caggtttcaa agagacaata ggagttaggg gaaagctgga gctgtgtgtg 4500

tgttgaagaa agaagtcctg gtaaagagga tgcctctggg tacctttgtt aatcatctaa 4560

gtgttctacc gccttctggc ttgttttttc atctataaaa catccagact tgattactaa 4620

ggtctttttc atttcttaaa ctttgtgtat ggacactgag caaggaagaa ctatttaagg 4680

acacccttaa atgctaagcc aggttccata agagcctgag aaggaagaaa gacccagtga 4740

taatcctctt gggaaggggc cactcttgat cagaagatga ctggttacat ggcctatttg 4800

cctattctca gagcaggagc actggggtgg tgtggcacac attattctgc agcacacacc 4860

tcctggcgag ttcaatgctc tatagttttt gtatatccag agctgtatca gcagagagat 4920

gccatgggca agttggcttt ttctatcttt cacagtattc tccacattag tgctagtttg 4980

tgtttgtcta tctctttctc tctcagatta tctcctgctg catactacgc ccagaggatg 5040

atccagtatc tctcaaggag agacagtatt cgccagcgct ccatgcgcta ccaacagaat 5100

cggcttcgtt cttccacctc ctcttcttcc tcagacaacc agggtccatc agtagaggga 5160

accgacttgg aatttgagga ctttgagtaa gtatattttc agtttgcttc cttctttctc 5220

ccagactaag agtgcttctg agttggcctc tgtttctgta gcagtggtga gcattcttgc 5280

ctctaacccc taagttatta tttatgtgca gcaaagtggg atgaggttca gagggtttta 5340

ggtacctaga gcactcctat ttctgggagt tacattgccg attcgccaga gcctttggga 5400

agagacaacc cattgggcca caggccggag tggaaagagg atggctgctg ctctcctgac 5460

cacacaccag acaaaggaag caggaggctt ggtgcttatt ctcttaacta gagtagaggt 5520

gacaatttaa gtttcatcgt aagtatagat ttccttgaat aaactgttcc ttgagatctg 5580

actctcttgc ctcttcctgc tcctagcccc acctgtcaga ttaaggtggc ttggggagtg 5640

actccttgat gccactgtca gctggggccg atgctgcatc tcctcttcct ccactccctt 5700

ctcttttttc tttgtttcct tatgttggtg agtggggtaa agacaaggcc taagagcagg 5760

gccagcccca agttccagag tccatgcttt ccagccctac tgctgagttc tgcctctcag 5820

atattctatt cctgaccttc tagccattta ttgtgcctcc agtttgaaat tgagttttcc 5880

tagcttgtat caaattagac atttggcttt tgttttgatt ttgtattttg acatttttta 5940

acatgcaaaa gaaaaaaaga aaaagaagca tttccctccc tcttccccaa ctccctcttg 6000

gcagctgcat gaatggttcc tcacaagcta acttgcaata gagctataga gtatatttta 6060

tctttttgcc agaaagtagt ctgaaaaaag attttaatgc tcaacaaatg caggtttagc 6120

atgaagcaga cagagcatga gtgtgctggt cctgtcagta atccctgaga ggagcccagc 6180

acctctgacc cacagccttt tttctgtctg tcctcctggg taagatggct gcatgtacaa 6240

acagtgaaag gagccagata gatgtatgtt attctggagc tgtagtagtt tttctagcac 6300

aagggtcctt ttagggaagg agtttaaaca tacatgccaa tggggttggg atgtaattca 6360

cttgcttagc atgtgaggtc ctgtgtttga ttcttcagca ctgcaaatat ataaacacac 6420

agacacacac agagtgggga gtaatgcggg gggtgggggg agagaaataa acaaacaaac 6480

aaacaaacac ttaaacactg aggctacggt agtagaaaaa cctgggtgac tttagttcag 6540

ttattctgca gtgcgctagg actctgaact ctggactctg gcttgccttg cctcttcttt 6600

ctatgcatgc ttagaagttg aaactgcctc tcaaccagtg gtgtgatgtg gggatcatgc 6660

ctctcagctg taccctgcct cgtgacttgt ccttcacaat ggagttctgg gttagtttaa 6720

aatgttctgt tctcatttta ccttcccttt cttctgctgc cacctcctcg ctctcccctt 6780

ccttccccct cgattttcct ctgtcagact gccttctgtg tgtgttcccc caacccacct 6840

gtgctctcat catcttcctg ggcttcagca tgacctggtt ttacattgcc ttttttacct 6900

ttccttcatc agcttaccag gtttttcctc ggtgtcgtta ttagccatgg cctaatctcc 6960

ctaatgccgc acctatatgt ccttgtccag atgtttgcct tttggaagat ggttttggta 7020

caaactgtaa tcttgctttt gatagcagct atgactcagt taccttaata tgagtagtac 7080

aggtcttcta gagaaagagg ttgatactct cttgccacct gtttcttctt tttctatctt 7140

ctctgtcctc ctcctggtcc tatacgctga gtactggttg taattgctcc ttttcgcctc 7200

tagtgctcag gggagaggca gaaactaggg attgtagcag cctgttctaa tttatttgcc 7260

ataatcctat tccttagctt tcataactag aaaggatgtt atttaggcct gccctccctc 7320

cctcccttct ttccttcctt cttgtttttt gagacagggt ttttctgtgt agtcctggct 7380

gtcctggaac ttgctctgta gaccaggctg gcctcaaact cagagatcca tctgcctgtt 7440

tctgccatct gagtgctgag attaaaggcc tgtgccacta ccacccagtt tgggatatta 7500

gttttttaaa taatgtttta acttatgtgt atggatgttt tgcctgcatg tatatctgtg 7560

accatcgtgg ttcctggtgc ccagagagat ctgactagag tcaggtctaa tagaactgta 7620

gttacagaca ttgggagctc ccatcttcca tgtgggtggg tgcttagaac tgagcctagg 7680

tcctctgcaa gagcaacaat tgcttttaat tgctgagcca tatctccaac cctcagaaaa 7740

gagggatatg tgggggggtg gggtaggggg atgttataca gtcattttat agttatttta 7800

aaattatgtt attttaatat acagaagcag gtaaggtttt tgtgatctgg aatctgccta 7860

aatggacacc attaagagac actgttctat tgttttttgt tgttgttgtt gttgtttgtt 7920

tgtttgtttg tttgtttgtt tttctgagca cattacaatc ttacctgtaa ggcctcatat 7980

aatcctttcc actctccctt tttctaagtc atagattcat tttactgttc ttgagcagtt 8040

ttcatccaga tgcataaagt ccccaagaaa tcagatcatg tcttctttgg ttttctttca 8100

tcatcaaaaa ttatcagtta ctgaagagtt catatgcata attgttaact acttatattt 8160

atagtatata taagcatata tacagtatgt atagttatag ccaaagtatc cacctcatat 8220

atgtatatgt agcaattgct aatactttgc atacagatgt agattatttg catatctttt 8280

tatgtataaa taatagattt aaaatttttg ttgaatcctt tttaaaaata tattatttat 8340

ttattattat ttatatgagt acactgtagc tgtctgcaga cacaccagaa gagggcatca 8400

tatcctatta cagatggttg tgagccacca aatagttgct gggaattgaa ctcaggacct 8460

ctggaagagc agtcagtgat cttaaccact gagccatctc tccagccctg ttgaatccct 8520

ttaatggccc aacaagaggg ctaataaatc attctcatct gttgttcttt gactgaggat 8580

atcaccatat cagagatgta tcagattaga gaacaggaat gagtatattt attgtctaaa 8640

gatctaaaac cttttaacag tgatatacaa ttatatttgg gtcctttcac atacttgttg 8700

tggaataggt ctttccaatt tatcttctgg gaggatgccc ttggaaattg gtcaggttaa 8760

attttctatt tttttgtagt ctggattgat gactatcttc taagcacgca tcttctcctg 8820

tcattcatct ctatctgttg aagattgaaa tggcaggatc ccagtgttta tcaaaatgta 8880

caaataggat gtccagaaac ttgctgtcag caggtctttt ttgaagtcct cagctagctg 8940

tctttgcata aagttcacaa gcagggtaaa ggtgtgtctg cctattgaca ttactgtctg 9000

cctgtgttaa agcttagact gcttgcttga atgtagttgt catataagca taacaaaaat 9060

cctattttaa tttataactt tcccattaga taaaggacag gtagcttaca aaaaaagctc 9120

gagaaaagtg ctttttttcc agtagttgct aagacttgca agagaataat gtgtgtgtgt 9180

gtgtgtgtgt gtgtttatga taattctagc tagtctttat ttttaattaa ttaaattaaa 9240

taataattta ataccaggtt ttagtgacaa acatttacat gctgagctat ctcactaacc 9300

cttgattctc gctagtcttg aacctttctt tcttttatct gttattctaa ttatgattct 9360

agaatctttc tccactcatt tccctgccct tttaacttct ttttgtagct gctttgcctt 9420

ctgaggcaaa ggatgaataa cctgatccga tacttgctac tgccagaact agagatgttt 9480

cagattagat aagccacaaa taacttcctc cacacataca gccactggta attatactca 9540

gcttcttaaa gatcgttgct agcatagaag agaataaacc ctttctttat tcatggtttt 9600

gacactgggc tgtatttgga aaatctcagt gagaacctta aagttttcag tttaagatag 9660

agtctgcagg ttagttagaa ttgtttcccc ttcctctcct ctacccttcc tccttttctt 9720

cctttcttcc cttctttctt tttttggagg ggttgggtgg gaacagtagt aggaattgaa 9780

cctagggcct cttgcctgct aggcaggtta cctgatcaca gttgggccct ttgttcttat 9840

ttctagacag tctcactatt gtagcctagg ctaacttgta agtctttatt ttcatgctgc 9900

agcctcccga gtgctgagat tataggtgtg tgtaccacca tgctcaattt tttaaaaaaa 9960

ttttatttat tttatttatg agtatactgt agctatcttc agacacacca gaagagggca 10020

tcagatccca ttacagatgg ttgtgagcta ccatgtggtt gctgggattt gaactcagga 10080

ccttcagaag agcagtcagt gctcttaact actgagccat ccctccagcc cgctcaatta 10140

cttttttttg ttgttttttg ttttgttttg tttttcgaga cagagtttct ctgtgtagct 10200

ctggctgtcc tggaactcac tctgtagacc aggctggcct caaactcaga aatccacctg 10260

cctctgcctc ccaagtgctg ggattaaagg catgtgccac cactgcccag ctgctcaact 10320

acttttaaat aaatactttt aaaagattta tttactttat tcttttatac atatgtgtgt 10380

ttagcctgca tatatgtgtg taccacatgt atgcctagtc agaaggagaa cattggactg 10440

gagtttcaaa aagtggttag ctgtgagtat tgggtattaa actcaggtct ttcttctaca 10500

agagcaccaa gtattagtag cctttgagcc aactctctac cccattttgt ttctgagaca 10560

gggttttgat gtatttccaa ggcagatctc aaactttgga gtttgtgtaa tgtttccacc 10620

caagcttctc cagtagttga gactataaga ctataagcac atagttgaga ctatgtgcac 10680

cacctcacct gcccgagctt ctctttctct gctccccttg ctaggcatct tgtggcatat 10740

atgttcttaa ttttgtgttt ggtatagctg aggagagttc caaaggctat cagctctaac 10800

atctggctga atatgacagg aaaagttgtt gacctttagt ttacaatata ctcttcttcc 10860

tctgcccttt tggtataact gagatttagt ccagcctttg gcaaaggaac tatgttaaaa 10920

accacgtaag tattgcttct catttcaggt gataatcctg aatgagactc ctacatttac 10980

ttcaaatctt ggctaggaat tccatcaaag aagccacatg tcttcagtag tacttgttct 11040

tagtttcttt tctgagctag gaagttccag gatttctgta ggagctcttg acttgtcttt 11100

tattttattt tattttattt tttgacttgt ctttaataac aaatataact cagagccaag 11160

aagaagggca gcttttgcca tctgaaatcg ttagagagat atggctgtat cattttgaaa 11220

tggtctttat ttcttccttt cacatataaa ctttagaagt gtaaagatgc actataaagc 11280

actatgaaga tgttgttctt gctgggaggt gatagtgcac acctttaatc ccagctcttg 11340

gaagatggag gcaggcagat ctctgtgagt ttgaaaatag actggtctac agagcgagtt 11400

ccaggcaatc agggctacac atagagaaac cctttttcta aaaacaaaca aacaaacaaa 11460

caaacaaaca acattcaggt gttgttcttt gtaggctgct aggcaagtgc tcagcaaatt 11520

gcatctgtag ccaggcatca gtcttaaaaa aatagggtct ttcaatagtt aaacttagga 11580

gacagcaaaa caaaacaaga aaagcatagt tattttgaca gttggctttt cttgattctt 11640

gatagatgca actatattag tcactgtaaa gttcaaatct atccaaaaag atgagacttg 11700

ggagtcatag aagtgcagat cttaagagtt gtttttgcct ttgagatgag ctttatccta 11760

ttaagatggt gacattaaaa cattttagtt atagaataaa atgatcagat ttttatataa 11820

gtatatgaaa gggggctgga aagatggttc agaagtccat agcactggct ggtcttttag 11880

gggaccctgg ctcagttccc agaacccaca tggcagttcc cagctattta taactccagt 11940

tacaggcaag tgcctctggc ctctgtggtc actgcatgta catagtgtcc atacaaacat 12000

gcaggccaaa cactcatata gacataaaca ataaaataca aagtaaaaag tacatatgtg 12060

aagagagact gtgacttaaa gagtttgacc agctctgcag gaaggaagaa cagattttgg 12120

aaaaatggga gactccatgg cttgagcttg attttgaaga ctgaatagga ctactttttg 12180

ttttgtttgt ttttaaattg cgtggggttg ttggcatagt ggggaagagt tgcatcccac 12240

tgcatggatg tattgggttt caccttacct gactttggtt ctgttttgtt catagttgta 12300

tgaccaactt aggtgattga gtttctgaga attttcttgt ctctgcctat ctccctagag 12360

gggtacactg ggattctaca ccagtgacca gttatgtcct tatattgtct ccagcagaag 12420

gtgtagccca gattaaaggt gtgttccgcc acacctttaa tcccagatga aaggcgtagc 12480

ccagattaaa ggtgtgttcc ttaaactcgg agattcaatc ttctggaatc cattgccatt 12540

atggctcaag atctccatac caagatccag ataaggatct ccaagcctcc agataagggt 12600

cactggtgag ccttccaatt ctggattgta gttcattcca aatatagtca agttgacaac 12660

caggaataac cactacaatc caccccttgt caacttgaca caaataatat ctcatgttca 12720

catgaaacag taacaaggtt gtgaatacgc ctaacatgat ataactatcc ctcgtacaat 12780

cgcaaacgca tttgtcaatt tacaaggggg cattcatatt actttataat cctcgtttct 12840

gcaactggtt acgtggcctt aattggtatt atctagtttt aacatgggtt ctggggatcc 12900

aaactcaggt tgttgggctt tcgtagcagt acttacccgc tgagccattt ccccaccccc 12960

tggactttga aagatgaatc tgtgaggatg gtgtagaaag aatatgctag ataaaggcaa 13020

taacaaagta aaagctcaga aacagatttt actcaaggtt tattcagaag cttgaataga 13080

tctctgtgta gagctatagt gtgctggtga ttgagaaaga tcagaaaact gagaactgtg 13140

aagggcttgc aatgtatggc agaaaatttg gattgtgtgt tgacttggcc tcctgagtag 13200

gggcattaca agtataggtc actatgcaag cgtacttgat gttttccaag actgagaggc 13260

ctatttgtgg gtgtctttgt aaagacagcc atgtcatcta cttctccaaa tagaatgctt 13320

cctcagcctt tattaaatat gtgctatagg aagctaactg aacaaccctg attaaggtga 13380

tacaatgtga caagtcaact gatgaagaac tagttcagta gtgatgggct agtataagtg 13440

aagatgtttt aaatatttat tgtgaaagtc cttagtgttt tcatcgtaga taccctttta 13500

aaaacgaaaa ccagggtctt attttgtagc cttagggatt gccctgtttc tgcctctgga 13560

ataatgggac taaagatgct caccatcatg cttatctggg tgtcaaattt ttgttgttgt 13620

ttttcaagcc tggttttttg tatgatgctg aggctgaacc tgaatttctg ggctcatatg 13680

attggcctga ctgggcctcc tgagtagggg cattacaagt gtaggccact atgcaagcgt 13740

acttgatgtt ttccaagact gagaggccta tttgtcgatg tctttgtaaa gacagccatg 13800

tcatctactt ctccaaatag aatacttcct cagtctttga ttactattac tggagaccat 13860

tttgaagtaa gtatttgtaa gttaacaagt gatccttaga acatcattgc ttgcaattgg 13920

aatcttttat tattgttgct agctctgttg actgggttct agtcttggtt gagcctctgc 13980

tgtagaaagc ttggaacatc taactgagaa gtttggattt tgtatgtatt tggggcagcc 14040

attattcagt tttttttttt tttgtttttt gttttttgtt tttaatttag gcagcaaact 14100

ctaaaaaaac agtggctggg ggggacctac tttggaaagt gtactgatgt agaagaacaa 14160

gtttgagaac aagtcctatt cccaaggtcc cagtgtctga tagtaaagga aattgaacaa 14220

cactggtgct tcttttaacc ttgaggttct tggatctcta ttggtgttaa ctgccttaga 14280

agatttccct tgatactcag caaagagaaa ataatgagaa ttctcagaaa gtaagagggc 14340

ctcctgaaga ggaagaggga aaagctatct agtgcttgtc aagcctcaat atgtttccaa 14400

aatatctact tatgcactac tacttcttta ccaaggatta tgacctgttg cttacaaatg 14460

tgtaacattg cttgacagct aaagaaatcc tctttctgaa aatataaagt accttatgaa 14520

tgtttatttt gggttatgga gaataagaaa tgagcaaagg aaagtagaat atatctagat 14580

tgtgcctggg ctgaaaatgg aaaaatacag acagaaagaa tgaaaaatac agccaggaaa 14640

tacagggagg aagacattac ctcttagatt tatatgttgt tttatcagaa aatgcacaat 14700

atgaatataa atttgcataa tgtaggcaaa tgagattagg aggtagaata agggaggaca 14760

agaatttgta gattataatt atctcatatt acttttcctt ggagaaatct aaggctctgt 14820

tgttccagga gacgtgcatt gttaggtttc ttacaaagga tgtcaactgg gacaaaaatt 14880

taagatcttt gtaagtgtcg tctctggaaa tagaggctgt gcctcagttt ctaataggag 14940

ctagtaagaa aaggacttga ccaagatcac tgtgccattc agagacagaa atgctaagta 15000

ggtcttctga tatttgttct tagacacact ttctttttaa atatttagca aaatcaacag 15060

tactgttggg aagacaatga ccttttgagc tttttgatgg aatagattca tcctcaaggc 15120

tcatgtcaga gctctgtttg tctgaggaac aatgacaacc tgactgtctg gtgagggctc 15180

agacaacagc ctgtagggat attttttatt cctttaaatt atggtctttc tttgaagctc 15240

ttgatcctgg gcatctagca gattgctgtt gtgtctctct tttgtcctga agatgtgttg 15300

cctttcattc aacacatgtc ttagaagctt gtttgaagga tttttttttt ctttataaaa 15360

taatttgaaa caaaatttta gtcgttcccc cacccaaaaa aataccccac tgagagttaa 15420

tcaaagatgc atagagttca acaactggga aggccattaa tagtacagtc agtttctatc 15480

ctgctgtctt aaatgcaact tctgaatgtt aaaattgatg aggatttctc tgatgttttc 15540

ttaaaaagta gtccttcttg gtggaaagat tgtactagca tatttgtgta ctttgttctt 15600

catttctttc ccataaaagg ttgttggcta tcttatgtta gagcctagtt tttagcactt 15660

gctactattg ctaatgtcaa ccaactgtag atctgttcat ctggcattat cttcagtccc 15720

gttttgactt atttcactgc tttctaactc ttgcagcttg ctttccgctg accttgtgtt 15780

agcagtgaag tgaatggcag gctcctcgtc ttcttaaaat atcttatttt aagtgcatct 15840

aattcttttg gtcttttctt ggaactcttg tcattatctt tctctcaaat ctcccatgca 15900

caaaatttgt tcttctatcc ccagttgcct ctaaaccccc ttctttctag gcaataaaat 15960

cagttatttc tctcatcttt agtttatttg gagctgtgtc aaaattattt tcccgtctag 16020

tcacccactt tggcttttgg acattctgat cattgctacc ttgacacatt ctttcttttc 16080

aaggtttatc ctgttagtaa ccatttgtgc tgcttatctc tcatgctgtt ttctcctaga 16140

cggttttgct ggtttctccc ttaacatctc atttgtgtgt ttctcatatt tcatattttc 16200

ctaataatcc tggttcctgt gctgtccttt ttctctgttg ctgctggagt cctaacactg 16260

aattccgagg cttacctgca tggtgttgag ttactactca gctcctgctt acccatcctt 16320

agccagaatg ctaagattaa gacctgcata gagctttcca ctcacttgta tgctgttcca 16380

taatgccatc catttgagaa tgtgcctata tctgagctag ctatgcttac tgtctgtctc 16440

tgtctctctg tgattagtga ttgggtctta ctatgcagcc cagactggtc tcctgagtac 16500

tgggatcact atatatgtct caccatacct gatttgatct tatttttcct ttatatatca 16560

tatttcagta gtctaaacat tttaaagcta ttacctgtgg ctgtatttca gcccaccctc 16620

cccatttctt tagttggtgt ttatttgtat gtgtgtgcgc gcgcatgtgt acacattata 16680

gcactaaaat tactactttt gccccccttt taaatgttat ttattttata agcattgatt 16740

ggtgttttgc ctgaatgcat gtctgtatga ggctattaga tcccatgggg cttgagttac 16800

agacagttgt aagtttccat gtgggtcctg gaaattgaac actggtcctc tggaagagca 16860

gccagtgttc ttaaccactg atccatctct ccagccccta tcccctcttt ttttaaaaaa 16920

atagaaagaa taactgcatt taggttttag gctttaaaat agcaaaaaga ggtttgtgat 16980

aatatgtatt gtaagagtca aacatttccc cactcatcct tgtcacttgt catccttatt 17040

tctcaccagg ctttatgctc tctcaaaaag cattgcacct catcagtact gtccactacc 17100

ctgcccttca tatctgtatt cacagataat agtcccatgt tacccgaaaa ggtggatcta 17160

tggacttgta ggaacgtgag tcagctccac aggtgatttg taggaagaac agctcagtcc 17220

agagccatgg gatagaatct accctataat gctgctatct agctttgttt tcagtcttct 17280

taaggcctgg gggcctctct atgttccttt catgtcacac ttttaaactt taaaatgttt 17340

tctaaatgcc tttcatgata attgtttaca atcattaata aattcataat aacaaatgct 17400

atcattactc tagttgtact tttaattggt actttaactt atcataacca aaatcatata 17460

tttgaaaaat aacaatattt agagctgtct tatttgttta atattcagct tctttgtgcg 17520

catgttagga gctctgcaat aattatattt atgcagcttc tgatatattt gtgtatatgt 17580

ggatgtgtgt gtacatgtgt attatgtacc tctgtgtata tgtagatgta gacatctgtt 17640

gaggaggatg tgtgcacacg ctcctgcgat tggcttgaag atgatgttga gtatcttcct 17700

tggttgttcc tttacttttt gttaaggcag ggtctcctga tgaacccata gctccctgaa 17760

tctggttaac ctagctagtt agccagcttg cctttgggac cctctgtctg cctccacagc 17820

cctagcagta taagctcctc tataccagcc cagccctttt tttttttttt ttttttttgg 17880

taaataataa atgatttatt tattttgtat acaaaatgtt ctgacttgat ggctagatga 17940

gggcaccaga gctcattgta gatggttatg agccaccatg tggttgctgg gaattaaact 18000

cggaacccct ggaagaacag ccagtgctct taacctccaa gccatctctc cagcccccaa 18060

gcccagcttt tatatgactt ctgatgatct gaactccatt cctcatgctt acatggcaaa 18120

cttgtctttc tccttatcct tccttttata cccagagttt caagtcaaca gaatacaata 18180

tcttcggaaa acaagagtgt atcttttcat tgggatgggg gacccaaata cttctatcgg 18240

atcagctttc aatagccttg gtattgattg ctcttaatgt gtgaaaccct taaaagcttt 18300

tgtttctagc tggtacagct cccagtgggc cacctaaaag ccaaagtgtt taaggttgtt 18360

tccagggcta gattgaagat tatctcattg atttgaagat aattcaaaga gagaattcag 18420

atatcccaag tcaggctttc agaggttcat gtacttcttg atttgcttaa gcagctaatt 18480

aactgctctg tccagccagt gccctttaag ttattgtttc tttgatgtgg tggaaactta 18540

agccacaaga agttcctttc ctcttttctt gtaggaaaca gtaaaatgca gtgaagagat 18600

ccaaagctct ttcctgaatc atcaactaca ttctagcctg tgaccttgga tttacctgtc 18660

tgattcttta gtttccttat gtaaaatcag actaataaca cctcatggaa tagctagaga 18720

ttaaatagga tgttaacaac actgaactta gcaggcactt aataaatggt gcttaaacat 18780

ctataataaa aaattttttt caaatgacgt tgtttaggag tgcatttttc atcatggcca 18840

gttttcaaag aaggaactgt ggactttttc ttatttctgt ctttctgccc tcttgccttt 18900

ttcatgatac taaggaatga acgtgagacc ttgcatatgc caggcaggca agagctgtca 18960

cactgagtta catctctagc ttccaacccc catctctctc tgtcagtctt tctgccaagg 19020

gggtcaaatg aagaaaaagg tattcctggg caagatgtgg caatgcatgc cattagcctc 19080

agcactcagg atgcagaggc aggtggatct ctgaatttga ggccaccttg gtctacacag 19140

tgagtttcag gacagccagg gctacataga gggactctgtcttaaaaatt aaaggaaaga 19200

ataaattctt atgtagcctt tccagattct aagatttcag atactacggg gttcaaagac 19260

cacattgact cagacctgct tttgggtact gaattaaagt tagtcaatag gtaactgctt 19320

actttggatt cctttcctga gtgtaggact ttagtttgca gatgcttagt tttggtggca 19380

ggtgaataat tctgcttcag taactgtctt atatgtagtc tctctgtttt ctccagggac 19440

aatggtgaca gatctaggca ccgagctccc cgaaatgccc gaatgtctgc accttcactt 19500

ggacgatttg ttccaaggta agaactgggt tgtaaatatt gacggatcct tgatgaatgc 19560

ctaaggtttc atttttgtgt gtttggatta ctttggcttt atagagctgg aactgggacc 19620

cagggccttc cacatactag agaggcacac tgcctctgag ctacaccaac ctctggcctt 19680

tggattaccc atatatagaa ttctctccta gcctctatat tctggtcctg ccatttaaac 19740

atttcatggg agattattag ccacagtgcc tagagataga aaaatggcac caagagcata 19800

ctgttcaaag tctgtcagag tctctctttg tagttctagt tggcctgaac tcatcatgta 19860

gactgggctg gcctggaact catgtatcta cctgctgtgc ctgtgattgc tggggttaaa 19920

ggcgtgcatt actcctgact aacattactt tagaagaagg tcagtaagga gtcacacatt 19980

tataaaagga caaatcctat agattttatg gatttttagt tagtatatcc cttaggctat 20040

attttgttat aacgtatctt ttgggaccca gtctggtctt gaacttgcaa tccttcctta 20100

gtcttccatc tttataaacc atgccattta tgcccagcct tacttagcca acgattttga 20160

aactgtagtt tttcctctgt tgtttggtag tatttagttg gctttttaca tgtgattcct 20220

tcttacaggc gttttttgct gcctgagtac ttgccttatg ctgggatttt tcatgaacgt 20280

ggacagcctg gcttggctac tcattcttct gttaacaggg tcctggcag 20329

<210>9

<211>449

<212>DNA

<213>SEQ ID NO.9

<400>9

gaggctgctg gagtcttccc tcatttcatt atcccgttat gatggagcag gatccagaga 60

gcacccaatt tacccagacc cagcgagatt atctcctgct gcatactacg cccagaggat 120

gatccagtat ctctcaagga gagacagtat tcgccagcgc tccatgcgct accaacagaa 180

tcggcttcgt tcttccacct cctcttcttc ctcagacaac cagggtccat cagtagaggg 240

aaccgacttg gaatttgagg actttgagga caatggtgac agatctaggc accgagctcc 300

ccgaaatgcc cgaatgtctg caccttcact tggacgattt gttccaaggc gttttttgct 360

gcctgagtac ttgccttatg ctgggatttt tcatgaacgt ggacagcctg gcttggctac 420

tcattcttct gttaacaggg tcctggcag 449

<210>10

<211>449

<212>DNA

<213>SEQ ID NO.10

<400>10

gaggctgctg gagtcttccc tcatttcatt atcccgttat gatggagcag gatccagaga 60

gcacccaatt tacccagacc cagcgagatt atctcctgct gcatactacg cccagaggat 120

gatccagtat ctctcaagga gagacagtat tcgccagcgc tccatgcgct accaacagaa 180

tcggcttcgt tcttccacct cctcttcttc ctcagacaac cagggtccat cagtagaggg 240

aaccgacttg gaatttgagg actttgagga caatggtgac agatctaggc accgagctcc 300

ccgaaatgcc cgaatgtctg caccttcact tggacgattt gttccaaggc gttttttgct 360

gcctgagtac ttgccttatg ctgggatttt tcatgaacgt ggacagcctg gcttggctac 420

tcattcttct gttaacaggg tcctggcag 449

Claims (9)

1. A method for identifying the full length of circular RNA is characterized by comprising the following steps:

s1, comparison and prediction of splice junction sequence analysis and circular RNA full-length sequence: firstly, comparing in UCSC genome blast to confirm AG-GT shearing sites corresponding to the Splice junction sequence, then comparing in UCSC genome brown to obtain a corresponding genome DNA sequence, and comparing in Ensembl to obtain a predicted transcription circular RNA full-length sequence;

s2, designing CD Primers and Sjod Primers: designing a primer by using software to search a 40bp primer selectable region in a known sequence, obtaining a sequence of a circular RNA and a Splice junction sequence from a database, selecting 21-40 bp as a forward primer, and performing reverse complementation on the sequence of 1-20 bp to obtain a reverse primer to obtain CD Primers; inputting 30bp sequences of 20bp upstream and 10bp downstream of the Splicejunction position into primer design software, searching and calculating to enable the primer to meet the conventional parameter requirements of GC content and Tm value, enabling the primer to contain sequences not less than 13bp upstream and not more than 7bp downstream so as to obtain an Sjdo upstream primer, then automatically designing an Sjdo downstream primer by using the software according to the parameters and the position of the upstream primer, and obtaining the Sjdo Primers;

s3. inverse of LSTranscription reaction: extracting total RNA in a sample, taking 0.1-1 mu g of the extracted total RNA, adding N6 RandomPrimer, RNase-Free H₂Mixing O, reacting at the temperature of 60-70 ℃ for 5-10 min, and rapidly carrying out ice bath for 2-3 min to obtain a reaction solution I; then adding 5x LS RT Buffer into the reaction liquid I, adding LS Enzyme Mix into the reaction liquid I, reacting for 5-10 min at the temperature of 20-30 ℃, reacting for 60-65 min at the temperature of 50-60 ℃, and reacting for 5-10 min at the temperature of 80-90 ℃ to obtain cDNA;

s4, PCR amplification and PCR product analysis: amplifying the CD Primers and Sjod Primers obtained in the step S2 and the cDNA obtained in the step S3 to obtain a PCR product, and carrying out agarose gel electrophoresis analysis on the PCR product to obtain a direct amplification band and a rolling circle product band;

and S5.Sanger sequencing and cross-analysis of the comparison sequence.

2. The method for identifying the full length of circular RNA as claimed in claim 1, wherein the LS Enzyme Mix comprises reverse transcriptase LS M-MLV (RNase H-) and RNase Inhibitor, the mutant of the reverse transcriptase LS M-MLV (RNase H-) is delta N23/D108R/T306L/V433K/. DELTA.aa 524-551, and the gene sequence of the reverse transcriptase LS M-MLV (RNase H-) is shown as SEQ ID NO. 1.

3. The method for identifying the full length of circular RNA according to claim 1, wherein the composition of the reaction solution I in the reverse transcription in the step S3 comprises:

the composition of cDNA in LS reverse transcription in the step S3 comprises:

4. the method for identifying the full length of the circular RNA according to claim 1, wherein 0.1 to 1 μ g of the extracted circular RNA is extracted in step S3Adding N6 Random Primer, RNase-Free H into total RNA₂And O, mixing, reacting at 65 ℃ for 5min, and rapidly carrying out ice bath for 2min to obtain a reaction solution I.

5. The method for identifying the full length of circular RNA according to claim 1, wherein in step S3, LSenzyme Mix is added into the reaction solution I, and the mixture is reacted at 25 ℃ for 5min, at 55 ℃ for 60min, and at 85 ℃ for 5min to obtain cDNA.

6. The method for identifying the full length of the circular RNA according to claim 1, wherein in the step S5, the direct amplification band obtained in the step S4 is recovered and then directly subjected to Sanger sequencing, and the rolling circle product band is recovered and then connected to the pMD19T vector to be subjected to Sanger sequencing.

7. The method for identifying the full length of the circular RNA according to claim 1, wherein in the step S5, the direct amplification band sequence of CD Primers or Sjod Primers is cross-compared with the rolling circle product band sequence, the direct amplification band sequence confirms and verifies the sequence of the position of the Splice junction, and the real full length sequence of the circular RNA is between two Splicejunction sites in the rolling circle product band sequence.

8. A kit for identifying the full-length sequence of circular RNA, which comprises a reverse transcription reagent and a PCR amplification reagent, wherein the reverse transcription reagent comprises LS Enzyme Mix, 5 XLS RT Buffer, N6 Random Primer and RNase-Free H₂O; the PCR amplification reagent comprises 2 XHS Taq Mix and RNase-Free H₂O。

9. The kit of claim 8, wherein said LS Enzyme Mix comprises 200U/. mu.L LS M-MLV (RNase H-), 40U/. mu.L RNase Inhibitor, RNase-Free H₂O and 50% glycerol; the 5 XSS RTBuffer comprises 150mM Tris-HCl, 375mM KCl and 15mM MgCl at pH8.3₂10mM dNTP and 20mM DTT; the 2 XHS Taq MixComprises 2.5U/. mu.L HS Taq Polymerase, 10mM dNTP, 125mM Tris-HCl at pH8.3, 15mM MgCl₂250mM KCl, 500mM Glycine betaine, 0.15% Tween-20, 5% DMSO, and 10% glycerol.

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