CN110846391B - Method for detecting full-length sequence of circular RNA - Google Patents

Abstract

The invention discloses a method for detecting a full-length sequence of circular RNA, which comprises the following steps: (1) searching a database for the DNA sequence of the target circular RNA; (2) Any two nucleotides of the circular RNA corresponding to the exon and/or the intron of the DNA sequence are taken as disconnection points in the middle of the two nucleotides which are close to each other and are disconnected into two DNA sequences, and the 3 'end of one DNA sequence is connected with the 5' end of the other DNA sequence to form a new DNA sequence; (3) designing an upstream primer and a downstream primer of the novel DNA full-length sequence; (4) Verifying the upstream and downstream primers designed in the step (3) to ensure that the upstream and downstream primers cannot amplify linear RNA molecules to obtain a final primer pair; (5) Performing PCR amplification on cDNA corresponding to the circular RNA by using the final primer; (6) And (3) performing agarose electrophoresis detection and Sanger sequencing on the PCR product in the step (5) to obtain the full-length sequence of the circular RNA. The detection method can obtain the full-length sequence of the circular RNA more easily and simply.

Description

Method for detecting full-length sequence of circular RNA

Technical Field

The invention relates to the technical field of annular RNA detection, in particular to a method for detecting an annular RNA full-length sequence, and especially relates to a method for detecting the annular RNA full-length sequence without RNase R.

Background

Circular RNA (circRNA) is a single-stranded non-coding RNA that is different from linear RNA, has no cohesive ends of 3 'and 5' nucleotides, has no poly (a) tail, and is capable of forming a closed circular structure.

The current technology for detecting circular RNA mainly relies on the initial and final positions of circular RNA, i.e. the junction site (junction site) sequence to determine whether the RNA is circular RNA, but the technology can only obtain the nucleotide sequence near the junction site, thus the sequence from the initial position to the final position of circular RNA cannot be determined. Moreover, the loop RNA may undergo a cleavage event during formation, and the mature length thereof, i.e., the cleaved length (splice length), may be different from the genomic length (genomic length) corresponding thereto in the genome. Thus, it is highly necessary to know the full length sequence of a circular RNA. At present, the problem is not noticed by the scholars, and only the form of the sheared length or genome length of the circular RNA can be called in the data. Therefore, there is a strong market demand for a method for detecting the full-length sequence of circular RNA which can be more easily and simply obtained.

Disclosure of Invention

Based on the above problems, the present invention aims to overcome the above-mentioned disadvantages of the prior art and to provide a method for detecting the full-length sequence of a circular RNA without RNase R, which can be used for accurately obtaining the full-length sequence of the circular RNA and has the advantages of simplicity, easiness and convenience in operation.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a method for detecting a full-length sequence of a circular RNA, the method requiring no Rnase R, comprising the steps of:

(1) Searching a DNA sequence of the target circular RNA in a database;

(2) Taking the middle of two adjacent nucleotides of the circular RNA corresponding to the exon and/or the intron of the DNA sequence as a disconnection point to disconnect into two pieces of DNA, and connecting the 3 '-end of one DNA sequence with the 5' -end of the other DNA sequence to form a new DNA sequence;

(3) Designing an upstream primer and a downstream primer for detecting the novel DNA full-length sequence obtained in the step (2);

(4) Verifying the upstream primer and the downstream primer designed in the step (3) to ensure that the upstream primer and the downstream primer cannot amplify linear RNA molecules to obtain a final primer pair;

(5) Performing PCR amplification on cDNA corresponding to the circular RNA by using the final primer in the step (4);

(6) And (3) performing agarose electrophoresis detection and Sanger sequencing on the PCR product in the step (5) to obtain the full-length sequence of the circular RNA.

It should be noted that, in the prior art, it is often required that the sequence at the junction and near the junction is obtained by conventional PCR means after removing the linear sequence from the Rnase R, but the prior art cannot obtain the full-length sequence of the circular RNA, and cannot know whether the circular RNA will have a cleavage event, and the Rnase R has high cost and complex operation. The invention creatively provides a method for detecting the full length of the annular RNA by utilizing the structural characteristics of the annular RNA, which not only can obtain the full length sequence of the annular RNA under the condition of not adding RNase R, but also can know whether the annular RNA has a shearing event or not, and has the advantages of simple and convenient operation, easiness, low cost and the like.

In one embodiment, the step (2) break point avoids the exon, intron cleavage site; more preferably, the cleavage point is greater than 20bp from the nearest exon and/or intron cleavage site.

In one embodiment, the database in step (1) is a circBase database.

In one embodiment, the design in step (3) employs a software primer premier 5. In one embodiment, the upstream primer and the downstream primer in step (3) are 19 to 25bp in length.

In one embodiment, the GC ratio of the upstream primer and the downstream primer in step (3) is 40 to 60%.

In one embodiment, the total number of bases in the upstream and downstream primers at which hairpin and dimer occur is less than or equal to 6.

In one embodiment, the upstream primer and the downstream primer of step (3) differ by less than 5 ℃.

In one embodiment, the step (4) verifies an in silico PCR module that selects UCSCs.

In one embodiment, the PCR extension temperature in step (5) is floated within 5℃above and below the Tm value. Therefore, the preferable scheme can enhance the binding capacity of the primer and the template strand and improve the detection rate.

In one embodiment, the PCR extension time in step (5) is dependent on the length of the circular RNA, if the circular RNA length is less than 1000bp, the extension time is 1 minute; if the length of the circular RNA is between 1000 and 2000bp, the extension time is 2 minutes. And the like, if the length of the circular RNA is between 2000 and 3000bp, the extension time is 3 minutes, if the length of the circular RNA is between 3000 and 4000bp, the extension time is 4 minutes, and if the length of the circular RNA is between 4000 and 5000bp, the extension time is 5 minutes. Therefore, the optimal scheme fully considers the problem of the number of the full-length bases of the circular RNA, so that the PCR process can be better adapted to the characteristics of each circular RNA when the full length of the circular RNA is amplified, and the detection rate is improved.

In one embodiment, the number of reaction rounds of PCR in step (5) is greater than 30cycles.

In summary, the beneficial effects of the invention are as follows:

the invention provides a method for detecting the full-length sequence of the circular RNA without Rnase R, which is not convenient and easy at present, and most students only detect the sequence at the joint of the circular RNA, do not notice the difference between the cut length (speed length) and the genome length (genomic length) of the circular RNA in a database, and neglect the problem of the full-length base sequence of the circular RNA, but the detection method of the invention can detect the full-length sequence of the circular RNA rapidly and simply, and is simpler and more effective and saves time compared with the prior art.

Drawings

FIG. 1 is a schematic diagram of a cleavage mass that may be formed during the formation of circular RNA from linear RNA in example 1 of the present invention;

FIG. 2 is a schematic diagram showing a method for detecting the full-length sequence of different circular RNA shears according to example 1 of the present invention;

FIG. 3 is a flow chart showing the method for detecting the full-length sequence of circular RNA in example 1 (without RNase R) of the present invention;

FIG. 4 is a graph of the electrophoresis results of hsa_circle_ 0007766;

FIG. 5 is a graph of the electrophoresis results of hsa_circle_ 0001355;

FIG. 6 is a graph of the electrophoresis results of hsa_circle_ 0001538;

FIG. 7 is a diagram of the results of Sanger sequencing of the hsa_circ_0007766 (exon 1) primer amplification product;

FIG. 8 is a diagram of the results of Sanger sequencing of the hsa_circ_0007766 (exon 2) primer amplification product;

FIG. 9 is a diagram of the results of Sanger sequencing of the hsa_circ_0007766 (exon 3) primer amplification product;

FIG. 10 is a diagram of the results of Sanger sequencing of the hsa_circ_0007766 (exon 4) primer amplification product;

FIG. 11 is a diagram showing the results of Sanger sequencing of the hsa_circ_0007766 (exon 5) primer amplification product;

FIG. 12 is a diagram of the results of Sanger sequencing of the hsa_circ_0001355 (exon 1) primer amplification product;

FIG. 13 is a diagram showing the results of Sanger sequencing of the hsa_circ_0001355 (exon 2) primer amplification product;

FIG. 14 is a diagram showing the results of Sanger sequencing of the hsa_circ_0001538 (exon 1) primer amplification product

FIG. 15 is a graph showing the results of Sanger sequencing of the hsa_circ_0001538 (exon 2) primer amplification product.

Detailed Description

At present, no specific report is made on the method for detecting the full-length sequence of the circular RNA, and few students notice the difference between the cut length (speed length) and the genome length (genomic length) of the circular RNA in a database, and most students only depend on a plurality of bases of the sequence at the joint of the circular RNA and neglect the problem of the full-length base sequence of the circular RNA. Therefore, the invention provides a method for detecting the full-length sequence of the circular RNA without Rnase R, which not only can meet the requirement of defining the full-length sequence of the circular RNA, but also is simple, easy and convenient to operate.

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods in the present invention are all conventional methods.

Example 1

1. Design principle of circular RNA primer:

as shown in FIG. 1, as the opposite primers for several bases of the sequence at the junction of circular RNAs are conventionally designed, only the sequence at the junction can be amplified due to PCR amplification. However, in the case of circular RNA, the circular RNA may form variable cuts during formation, introns may be subtracted or retained, and such primers may only detect the sequence at the junction, but not the full-length sequence of the circular RNA. In summary, we aimed at the characteristics of circular RNAs, simply and rapidly designed primers that were able to detect circular full-length sequences with different variable cleavage patterns for circular RNAs.

2. The design scheme of the circular RNA primer comprises the following steps:

(1) Hsa_circ_0007766, hsa_circ_0001355 and hsa_circ_0001538 are exemplified, respectively. Genomic DNA sequences of circular RNAs are found in circBase (where english uppercase is exons and english lowercase is introns).

The hsa_circ_0007766 (SEQ ID NO. 1) is annotated by the circBase database as shown in Table 1 below.

TABLE 1

Full length DNA sequence (genomic length, where English uppercase is exon and English lowercase is intron)

GATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGgtggccttcaccgtcattgaaaccttctcttggttattcagagctgaccagggccactgctaaccagggggaggctttgtgtgcattagaaatggtgtcccttctgggcagacgcaggcagagcccgggaagacgccctcagaagattggaaaaagattccccttcttcctgggaagttgtagcttgcgtcagcacatataattcaatcgtgagaatgcaggctgggtttttgcccccacttggctgagtgaagtgtacagtgaacaacctatgtaactatttgctggccctggagccgactctgccccagagtctgggtgccaggtgctttgcccgcatggcccatttcagtcacgctgcagtcctgtcaggaaaaaatcagtgttattctcattctacatatgagaaaactgaggcttgcagatataagggccaaaagttacacagctagtgagtgatggggctgagtttcagactccacagtctcttaaccaccaagcagcatgcccagagtagaggtgagaaggaaggagagagctgcggtccacatgagcatctggacctagcatggacaactcactcctccctggctctcgctttgttcttgttgcgggtgtggtggtggtgggactcaaagacggtaaagatagctttctctcctccctggggaatctgggggttgtttaaaaggcctgctcctcttttagaaggcaggagggccccaagggaagcagaaggtgacagaaggggaaagggtcctctgatcattgctcaccccacagAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctgcctcttctctcagacagcctgaccccagccgcaaactcccaacttacaacccagtgcctgcccgccactgccccagccgcctacaccacccatttcctccctctctgtccctcctgccatctccctgtgcctcttcatctctggggttctctgtcttgtctccctctgcttataggttgtgcctctggtttgggggcctctcagcctgtctgggtccctcccttgctgtgcagttggcctcgtggcctctgctgctgtttgtgcctctctctgttactaacccgtcctctcgctgttagacatctctctcactgcctgtctctggttctgtcctcagGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCgtgagtctcagggaggcctggagtcagggaaggggagggctggggccgggtggaatgcaggtgtcatacaggtgacatgggaggggtgggataacaggcttgggatgtctcccctgggccaggtagtctccctagaaggtgatgctgatgagggtctggtgcccagggcgccactcagccctcatcctgccctttgcccaacagTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctctaccccccaggatgcaaggggtgggcaccctgcctggtactgccctattgcccctggcacaccagggcaaaacagcacagtgaaagccagccacctgtccccccagGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCT(SEQ ID NO.1)

hsa_circ_0001355 (SEQ ID NO. 2) the circBase database notes are shown in Table 2 below.

TABLE 2

Full length DNA sequence (genomic length, where English uppercase is exon and English lowercase is intron)

AAATGGGACGTCGGTCATCAGATACTGAAGAAGAAAGCAGAAGCAAGAGAAAAAAGAAACACCGTAGACGGTCCTCCTCGAGCAGTTCTTCAGATAGTAGAACATACAGCCGAAAGAAAGGAGGAAGGAAATCAAGATCAAAGTCAAGATCTTGGTCCAGAGATCTTCAGCCTCGTTCACATTCTTATGATAGAAGgtgatttttgtaatttttatttatatagtaatgaggataacattctttaaaattcatgtttttgtatttttaattttgctagataaaacatttttcttttactagctatatatttatgaaagtcccaacccagagtttttttgttccaccaggtccagtccgtgggtctagtgtcctcaacctttgtctgttcttagtaggcaaatagatagaaagtggtgtagtttccttttttaatttaatttaattttttttctagtgcatttatttatttatttttctaatgaattattttttattttattatactttaagttctagtacacaacatgcaggtttgttacatatgtatacatgtgccatgttggtgtgctgcatctgttaactcgtcatttacattaggtgtatctcctaatgctatccctcccctagccccccaccccatgacaggccccagtgtgtgatgttccccttcctgtgtccaagagttctcattgttcaattcccacctatgaatgagaacatgcggtgtttggttttttgtccttgcgatagtttgctgagaatgatggtttccagcttcatctatgtgcctacaaaggatgtgaactcatccttttttatggctgcgtagtattccatggtgtatatgtgccacattttcttaatccagtctgtcattgatggacatttgagttggttccaagcctttgctattgtgaacagtgctgcaacggagttttgctcttgttgtccaggctggagtacaatggcgtgatctcagctcactgcagcctctgcctcccaggttcaagcagttcttctcagccttccaagtagccgggattccaggcatgcaccactatgcccagctagttttgtatttttagtagagacagggtttcaccatgttggtcaggctggtcttgaactcctgacctcaggtgatctgccctcctcagcctcccaaaatgttgggattacaggcgtgagccaccgtgcctggccttaattttaaatttttttagagacaagatcttgctctgtcactcaggcgggagtgcagcggtgcagtcatatcttttggactcaagggatcctcccacctcagcttcctgaatagctagtactagaggcatgtgccaccatgcctagctaagtttttttatttttattttttgtggagacttggccttgctgtgttgcccaagctggtatcgaattcttggcctcaagcgatcctcctgcctcggcctcccaacatgttgggattacaggcatgagctactgagcctggccttggtttagttttcataccatataatctgatgatatttggaaagattctaagacccttaataccttaattagtactgatccaatcactaagttatgaaccagtcatatgactgttcaagagaaataacattttaatttatctctgtttgcttccaggaaacagtgagacagtaaaaatcagatgattctttagaatctagaaggtttttccttaatgctcataataaaaatttttatagcagtgatctttgattatattttatttcagACGCAGGCATCGATCAAGCAGTAGCTCTTCTTATGGCTCCAGAAGGAAACGAAGTCGAAGTCGTTCAAGGGGTCGAGGGAAATCCTATAGAGTTCAGAGGTCTAGGTCAAAAAGCAGAACAAGAAG(SEQ ID NO.2)

hsa_circ_0001538 (SEQ ID NO. 3) the circBase database notes are shown in Table 3 below.

TABLE 3 Table 3

Full length DNA sequence (genomic length, where English uppercase is exon and English lowercase is intron)

GTTAAAAACGACAACCAACATCAGCCATGAAAGATCCAAGTCGCAGCAGTACTAGCCCAAGCATCATCAATGAAGATGTGATTATTAACGGTCATTCTCATGAAGATGACAATCCATTTGCAGAGTACATGTGGATGGAAAATGAAGAAGAATTCAACAGACAAgttagttttttgtacaaacatgtttttatagtccattctggccctcaatatgattgtacttgtccctgaaatgtgtttatcttgtcctttatgtataaagtatgtagactgatgtgccaccactcctgtttatttggctgctgattactaccaggcttaactgcttacggaaataacctggggagcttgtttcagggtcccaccctagagatgtctgaatcaggaggatgagggtggagcctgggaaactttttcagagctctcagatggttccattgagcagtcagatgtggaaaccactgccttatgccagtaagattgctggttaccgtgaaagtcaagagaattcaggagtcaggagcagacaggcacagaaaaaagagacaggcatctctttttacttcttccctgccaccctcaatggaaaaaagaatgttctactaataaaagtaaaaacatagaaaaaccctaaacagatagatcttcgccggtcattatggtaagcattttttttccttagcatggaagcagaaaagaaagtatgactttgtgcctttaaatggtttagttcaagatattttaagccattcctagtgatatctaccaagtgtgctataaattatcctttattataaatctagATAGAAGAGGAGTTATGGGAAGAAGAATTTATTGAACGCTGTTTCCAAGAAATGCTGGAAGAGGAAGAAGAGCATGAATGGTTTATTCCAGCTCGAGATCTCCCACAAACTATGGACCAAATCCAAGACCAGTTTAATGACCTTGTTATCAGTGATGGCTCTTCTCTGGAAGATCTTGTG(SEQ ID NO.3)

(2) The same sequence is respectively disconnected in an exon region and an intron region, the disconnection position is at least 20nt away from the cutting sites of the exons and the introns, the 3 'end of one DNA sequence is linked with the 5' end of the other DNA sequence to form a new DNA sequence, and the following new DNA sequences are formed after disconnection (wherein English is in uppercase as the exons, english is in lowercase as the introns, the underlined part is the donor sequence, and the non-underlined part is the receiver sequence):

hsa_circ_0007766

hsa_circ_0007766(exon1):

CCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGgtggccttcaccgtcattgaaaccttctcttggttattcagagctgaccagggccactgctaaccagggggaggctttgtgtgcattagaaatggtgtcccttctgggcagacgcaggcagagcccgggaagacgccctcagaagattggaaaaagattccccttcttcctgggaagttgtagcttgcgtcagcacatataattcaatcgtgagaatgcaggctgggtttttgcccccacttggctgagtgaagtgtacagtgaacaacctatgtaactatttgctggccctggagccgactctgccccagagtctgggtgccaggtgctttgcccgcatggcccatttcagtcacgctgcagtcctgtcaggaaaaaatcagtgttattctcattctacatatgagaaaactgaggcttgcagatataagggccaaaagttacacagctagtgagtgatggggctgagtttcagactccacagtctcttaaccaccaagcagcatgcccagagtagaggtgagaaggaaggagagagctgcggtccacatgagcatctggacctagcatggacaactcactcctccctggctctcgctttgttcttgttgcgggtgtggtggtggtgggactcaaagacggtaaagatagctttctctcctccctggggaatctgggggttgtttaaaaggcctgctcctcttttagaaggcaggagggccccaagggaagcagaaggtgacagaaggggaaagggtcctctgatcattgctcaccccacagAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctgcctcttctctcagacagcctgaccccagccgcaaactcccaacttacaacccagtgcctgcccgccactgccccagccgcctacaccacccatttcctccctctctgtccctcctgccatctccctgtgcctcttcatctctggggttctctgtcttgtctccctctgcttataggttgtgcctctggtttgggggcctctcagcctgtctgggtccctcccttgctgtgcagttggcctcgtggcctctgctgctgtttgtgcctctctctgttactaacccgtcctctcgctgttagacatctctctcactgcctgtctctggttctgtcctcagGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCgtgagtctcagggaggcctggagtcagggaaggggagggctggggccgggtggaatgcaggtgtcatacaggtgacatgggaggggtgggataacaggcttgggatgtctcccctgggccaggtagtctccctagaaggtgatgctgatgagggtctggtgcccagggcgccactcagccctcatcctgccctttgcccaacagTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctctaccccccaggatgcaaggggtgggcaccctgcctggtactgccctattgcccctggcacaccagggcaaaacagcacagtgaaagccagccacctgtccccccagGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCG GATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGC

hsa_circ_0007766(exon2):

ACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctgcctcttctctcagacagcctgaccccagccgcaaactcccaacttacaacccagtgcctgcccgccactgccccagccgcctacaccacccatttcctccctctctgtccctcctgccatctccctgtgcctcttcatctctggggttctctgtcttgtctccctctgcttataggttgtgcctctggtttgggggcctctcagcctgtctgggtccctcccttgctgtgcagttggcctcgtggcctctgctgctgtttgtgcctctctctgttactaacccgtcctctcgctgttagacatctctctcactgcctgtctctggttctgtcctcagGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCgtgagtctcagggaggcctggagtcagggaaggggagggctggggccgggtggaatgcaggtgtcatacaggtgacatgggaggggtgggataacaggcttgggatgtctcccctgggccaggtagtctccctagaaggtgatgctgatgagggtctggtgcccagggcgccactcagccctcatcctgccctttgcccaacagTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctctaccccccaggatgcaaggggtgggcaccctgcctggtactgccctattgcccctggcacaccagggcaaaacagcacagtgaaagccagccacctgtccccccagGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTG ATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGAT TGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACC ACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGgtggccttcaccgtc attgaaaccttctcttggttattcagagctgaccagggccactgctaaccagggggaggctttgtgtgcattagaa atggtgtcccttctgggcagacgcaggcagagcccgggaagacgccctcagaagattggaaaaagattccccttct tcctgggaagttgtagcttgcgtcagcacatataattcaatcgtgagaatgcaggctgggtttttgcccccacttg gctgagtgaagtgtacagtgaacaacctatgtaactatttgctggccctggagccgactctgccccagagtctggg tgccaggtgctttgcccgcatggcccatttcagtcacgctgcagtcctgtcaggaaaaaatcagtgttattctcat tctacatatgagaaaactgaggcttgcagatataagggccaaaagttacacagctagtgagtgatggggctgagtt tcagactccacagtctcttaaccaccaagcagcatgcccagagtagaggtgagaaggaaggagagagctgcggtcc acatgagcatctggacctagcatggacaactcactcctccctggctctcgctttgttcttgttgcgggtgtggtgg tggtgggactcaaagacggtaaagatagctttctctcctccctggggaatctgggggttgtttaaaaggcctgctc ctcttttagaaggcaggagggccccaagggaagcagaaggtgacagaaggggaaagggtcctctgatcattgctca ccccacagAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGA

hsa_circ_0007766(exon3):

TAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCgtgagtctcagggaggcctggagtcagggaaggggagggctggggccgggtggaatgcaggtgtcatacaggtgacatgggaggggtgggataacaggcttgggatgtctcccctgggccaggtagtctccctagaaggtgatgctgatgagggtctggtgcccagggcgccactcagccctcatcctgccctttgcccaacagTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctctaccccccaggatgcaaggggtgggcaccctgcctggtactgccctattgcccctggcacaccagggcaaaacagcacagtgaaagccagccacctgtccccccagGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTGATATCCAGGAGGTGCAGGGCTACGTGCT CATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGAC AACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAG GCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGgtggccttcaccgtcattgaaaccttctcttggttattcagag ctgaccagggccactgctaaccagggggaggctttgtgtgcattagaaatggtgtcccttctgggcagacgcaggc agagcccgggaagacgccctcagaagattggaaaaagattccccttcttcctgggaagttgtagcttgcgtcagca catataattcaatcgtgagaatgcaggctgggtttttgcccccacttggctgagtgaagtgtacagtgaacaacct atgtaactatttgctggccctggagccgactctgccccagagtctgggtgccaggtgctttgcccgcatggcccat ttcagtcacgctgcagtcctgtcaggaaaaaatcagtgttattctcattctacatatgagaaaactgaggcttgca gatataagggccaaaagttacacagctagtgagtgatggggctgagtttcagactccacagtctcttaaccaccaa gcagcatgcccagagtagaggtgagaaggaaggagagagctgcggtccacatgagcatctggacctagcatggaca actcactcctccctggctctcgctttgttcttgttgcgggtgtggtggtggtgggactcaaagacggtaaagatag ctttctctcctccctggggaatctgggggttgtttaaaaggcctgctcctcttttagaaggcaggagggccccaag ggaagcagaaggtgacagaaggggaaagggtcctctgatcattgctcaccccacagAGATCTTGAAAGGAGGGGTC TTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGC TGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctgcctcttctctcaga cagcctgaccccagccgcaaactcccaacttacaacccagtgcctgcccgccactgccccagccgcctacaccacc catttcctccctctctgtccctcctgccatctccctgtgcctcttcatctctggggttctctgtcttgtctccctc tgcttataggttgtgcctctggtttgggggcctctcagcctgtctgggtccctcccttgctgtgcagttggcctcg tggcctctgctgctgtttgtgcctctctctgttactaacccgtcctctcgctgttagacatctctctcactgcctg tctctggttctgtcctcagGCCACCCCTGTTCTCCGATGTG

hsa_circ_0007766(exon4):

GTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctctaccccccaggatgcaaggggtgggcaccctgcctggtactgccctattgcccctggcacaccagggcaaaacagcacagtgaaagccagccacctgtccccccagGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGA GGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCT AGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTT CGAAGCCTCACAGgtggccttcaccgtcattgaaaccttctcttggttattcagagctgaccagggccactgctaa ccagggggaggctttgtgtgcattagaaatggtgtcccttctgggcagacgcaggcagagcccgggaagacgccct cagaagattggaaaaagattccccttcttcctgggaagttgtagcttgcgtcagcacatataattcaatcgtgaga atgcaggctgggtttttgcccccacttggctgagtgaagtgtacagtgaacaacctatgtaactatttgctggccc tggagccgactctgccccagagtctgggtgccaggtgctttgcccgcatggcccatttcagtcacgctgcagtcct gtcaggaaaaaatcagtgttattctcattctacatatgagaaaactgaggcttgcagatataagggccaaaagtta cacagctagtgagtgatggggctgagtttcagactccacagtctcttaaccaccaagcagcatgcccagagtagag gtgagaaggaaggagagagctgcggtccacatgagcatctggacctagcatggacaactcactcctccctggctct cgctttgttcttgttgcgggtgtggtggtggtgggactcaaagacggtaaagatagctttctctcctccctgggga atctgggggttgtttaaaaggcctgctcctcttttagaaggcaggagggccccaagggaagcagaaggtgacagaa ggggaaagggtcctctgatcattgctcaccccacagAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCA GCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGAC ACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctgcctcttctctcagacagcctgaccccagccgcaa actcccaacttacaacccagtgcctgcccgccactgccccagccgcctacaccacccatttcctccctctctgtcc ctcctgccatctccctgtgcctcttcatctctggggttctctgtcttgtctccctctgcttataggttgtgcctct ggtttgggggcctctcagcctgtctgggtccctcccttgctgtgcagttggcctcgtggcctctgctgctgtttgt gcctctctctgttactaacccgtcctctcgctgttagacatctctctcactgcctgtctctggttctgtcctcagG CCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCgtgagtct cagggaggcctggagtcagggaaggggagggctggggccgggtggaatgcaggtgtcatacaggtgacatgggagg ggtgggataacaggcttgggatgtctcccctgggccaggtagtctccctagaaggtgatgctgatgagggtctggt gcccagggcgccactcagccctcatcctgccctttgcccaacagTGACGCGCACTGTCTGTGCCGGTGGCTGTGCC CGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCA

hsa_circ_0007766(exon5):

AGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTGATATCCAGGAGGTGCAGGGCTA CGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTT GAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCC CAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGgtggccttcaccgtcattgaaaccttctcttggttat tcagagctgaccagggccactgctaaccagggggaggctttgtgtgcattagaaatggtgtcccttctgggcagac gcaggcagagcccgggaagacgccctcagaagattggaaaaagattccccttcttcctgggaagttgtagcttgcg tcagcacatataattcaatcgtgagaatgcaggctgggtttttgcccccacttggctgagtgaagtgtacagtgaa caacctatgtaactatttgctggccctggagccgactctgccccagagtctgggtgccaggtgctttgcccgcatg gcccatttcagtcacgctgcagtcctgtcaggaaaaaatcagtgttattctcattctacatatgagaaaactgagg cttgcagatataagggccaaaagttacacagctagtgagtgatggggctgagtttcagactccacagtctcttaac caccaagcagcatgcccagagtagaggtgagaaggaaggagagagctgcggtccacatgagcatctggacctagca tggacaactcactcctccctggctctcgctttgttcttgttgcgggtgtggtggtggtgggactcaaagacggtaa agatagctttctctcctccctggggaatctgggggttgtttaaaaggcctgctcctcttttagaaggcaggagggc cccaagggaagcagaaggtgacagaaggggaaagggtcctctgatcattgctcaccccacagAGATCTTGAAAGGA GGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACA ACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctgcctcttct ctcagacagcctgaccccagccgcaaactcccaacttacaacccagtgcctgcccgccactgccccagccgcctac accacccatttcctccctctctgtccctcctgccatctccctgtgcctcttcatctctggggttctctgtcttgtc tccctctgcttataggttgtgcctctggtttgggggcctctcagcctgtctgggtccctcccttgctgtgcagttg gcctcgtggcctctgctgctgtttgtgcctctctctgttactaacccgtcctctcgctgttagacatctctctcac tgcctgtctctggttctgtcctcagGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTT CTGAGGATTGTCAGAGCCgtgagtctcagggaggcctggagtcagggaaggggagggctggggccgggtggaatgc aggtgtcatacaggtgacatgggaggggtgggataacaggcttgggatgtctcccctgggccaggtagtctcccta gaaggtgatgctgatgagggtctggtgcccagggcgccactcagccctcatcctgccctttgcccaacagTGACGC GCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGC CGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctctaccccccag gatgcaaggggtgggcaccctgcctggtactgccctattgcccctggcacaccagggcaaaacagcacagtgaaag ccagccacctgtccccccagGCCTGCCTCCACTTCAACCAC

hsa_circ_0007766(intron1):

tcgctttgttcttgttgcgggtgtggtggtggtgggactcaaagacggtaaagatagctttctctcctccctggggaatctgggggttgtttaaaaggcctgctcctcttttagaaggcaggagggccccaagggaagcagaaggtgacagaaggggaaagggtcctctgatcattgctcaccccacagAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctgcctcttctctcagacagcctgaccccagccgcaaactcccaacttacaacccagtgcctgcccgccactgccccagccgcctacaccacccatttcctccctctctgtccctcctgccatctccctgtgcctcttcatctctggggttctctgtcttgtctccctctgcttataggttgtgcctctggtttgggggcctctcagcctgtctgggtccctcccttgctgtgcagttggcctcgtggcctctgctgctgtttgtgcctctctctgttactaacccgtcctctcgctgttagacatctctctcactgcctgtctctggttctgtcctcagGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCgtgagtctcagggaggcctggagtcagggaaggggagggctggggccgggtggaatgcaggtgtcatacaggtgacatgggaggggtgggataacaggcttgggatgtctcccctgggccaggtagtctccctagaaggtgatgctgatgagggtctggtgcccagggcgccactcagccctcatcctgccctttgcccaacagTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctctaccccccaggatgcaaggggtgggcaccctgcctggtactgccctattgcccctggcacaccagggcaaaacagcacagtgaaagccagccacctgtccccccagGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTGATATCC AGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCG AGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCT GTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGgtggccttcaccgtcattgaa accttctcttggttattcagagctgaccagggccactgctaaccagggggaggctttgtgtgcattagaaatggtg tcccttctgggcagacgcaggcagagcccgggaagacgccctcagaagattggaaaaagattccccttcttcctgg gaagttgtagcttgcgtcagcacatataattcaatcgtgagaatgcaggctgggtttttgcccccacttggctgag tgaagtgtacagtgaacaacctatgtaactatttgctggccctggagccgactctgccccagagtctgggtgccag gtgctttgcccgcatggcccatttcagtcacgctgcagtcctgtcaggaaaaaatcagtgttattctcattctaca tatgagaaaactgaggcttgcagatataagggccaaaagttacacagctagtgagtgatggggctgagtttcagac tccacagtctcttaaccaccaagcagcatgcccagagtagaggtgagaaggaaggagagagctgcggtccacatga gcatctggacctagcatggacaactcactcctccctggctc

hsa_circ_0007766(intron2):

tctctgtcttgtctccctctgcttataggttgtgcctctggtttgggggcctctcagcctgtctgggtccctcccttgctgtgcagttggcctcgtggcctctgctgctgtttgtgcctctctctgttactaacccgtcctctcgctgttagacatctctctcactgcctgtctctggttctgtcctcagGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCgtgagtctcagggaggcctggagtcagggaaggggagggctggggccgggtggaatgcaggtgtcatacaggtgacatgggaggggtgggataacaggcttgggatgtctcccctgggccaggtagtctccctagaaggtgatgctgatgagggtctggtgcccagggcgccactcagccctcatcctgccctttgcccaacagTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctctaccccccaggatgcaaggggtgggcaccctgcctggtactgccctattgcccctggcacaccagggcaaaacagcacagtgaaagccagccacctgtccccccagGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAA GTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCG TGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCA GCTTCGAAGCCTCACAGgtggccttcaccgtcattgaaaccttctcttggttattcagagctgaccagggccactg ctaaccagggggaggctttgtgtgcattagaaatggtgtcccttctgggcagacgcaggcagagcccgggaagacg ccctcagaagattggaaaaagattccccttcttcctgggaagttgtagcttgcgtcagcacatataattcaatcgt gagaatgcaggctgggtttttgcccccacttggctgagtgaagtgtacagtgaacaacctatgtaactatttgctg gccctggagccgactctgccccagagtctgggtgccaggtgctttgcccgcatggcccatttcagtcacgctgcag tcctgtcaggaaaaaatcagtgttattctcattctacatatgagaaaactgaggcttgcagatataagggccaaaa gttacacagctagtgagtgatggggctgagtttcagactccacagtctcttaaccaccaagcagcatgcccagagt agaggtgagaaggaaggagagagctgcggtccacatgagcatctggacctagcatggacaactcactcctccctgg ctctcgctttgttcttgttgcgggtgtggtggtggtgggactcaaagacggtaaagatagctttctctcctccctg gggaatctgggggttgtttaaaaggcctgctcctcttttagaaggcaggagggccccaagggaagcagaaggtgac agaaggggaaagggtcctctgatcattgctcaccccacagAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACC CCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGAT AGACACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctgcctcttctctcagacagcctgaccccagcc gcaaactcccaacttacaacccagtgcctgcccgccactgccccagccgcctacaccacccatttcctccctctct gtccctcctgccatctccctgtgcctcttcatctctggggt

hsa_circ_0007766(intron3):

gtctcccctgggccaggtagtctccctagaaggtgatgctgatgagggtctggtgcccagggcgccactcagccctcatcctgccctttgcccaacagTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctctaccccccaggatgcaaggggtgggcaccctgcctggtactgccctattgcccctggcacaccagggcaaaacagcacagtgaaagccagccacctgtccccccagGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTGATATCCAGGAGGTGCAGGGCTACGTGCTC ATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACA ACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGG CCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGgtggccttcaccgtcattgaaaccttctcttggttattcagagc tgaccagggccactgctaaccagggggaggctttgtgtgcattagaaatggtgtcccttctgggcagacgcaggca gagcccgggaagacgccctcagaagattggaaaaagattccccttcttcctgggaagttgtagcttgcgtcagcac atataattcaatcgtgagaatgcaggctgggtttttgcccccacttggctgagtgaagtgtacagtgaacaaccta tgtaactatttgctggccctggagccgactctgccccagagtctgggtgccaggtgctttgcccgcatggcccatt tcagtcacgctgcagtcctgtcaggaaaaaatcagtgttattctcattctacatatgagaaaactgaggcttgcag atataagggccaaaagttacacagctagtgagtgatggggctgagtttcagactccacagtctcttaaccaccaag cagcatgcccagagtagaggtgagaaggaaggagagagctgcggtccacatgagcatctggacctagcatggacaa ctcactcctccctggctctcgctttgttcttgttgcgggtgtggtggtggtgggactcaaagacggtaaagatagc tttctctcctccctggggaatctgggggttgtttaaaaggcctgctcctcttttagaaggcaggagggccccaagg gaagcagaaggtgacagaaggggaaagggtcctctgatcattgctcaccccacagAGATCTTGAAAGGAGGGGTCT TGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCT GGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctgcctcttctctcagac agcctgaccccagccgcaaactcccaacttacaacccagtgcctgcccgccactgccccagccgcctacaccaccc atttcctccctctctgtccctcctgccatctccctgtgcctcttcatctctggggttctctgtcttgtctccctct gcttataggttgtgcctctggtttgggggcctctcagcctgtctgggtccctcccttgctgtgcagttggcctcgt ggcctctgctgctgtttgtgcctctctctgttactaacccgtcctctcgctgttagacatctctctcactgcctgt ctctggttctgtcctcagGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGA TTGTCAGAGCCgtgagtctcagggaggcctggagtcagggaaggggagggctggggccgggtggaatgcaggtgtc atacaggtgacatgggaggggtgggataacaggcttgggat

hsa_circ_0007766(intron4):

cctattgcccctggcacaccagggcaaaacagcacagtgaaagccagccacctgtccccccagGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTGATATCCAGGAGGTG CAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCC AGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGG GGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGgtggccttcaccgtcattgaaaccttctc ttggttattcagagctgaccagggccactgctaaccagggggaggctttgtgtgcattagaaatggtgtcccttct gggcagacgcaggcagagcccgggaagacgccctcagaagattggaaaaagattccccttcttcctgggaagttgt agcttgcgtcagcacatataattcaatcgtgagaatgcaggctgggtttttgcccccacttggctgagtgaagtgt acagtgaacaacctatgtaactatttgctggccctggagccgactctgccccagagtctgggtgccaggtgctttg cccgcatggcccatttcagtcacgctgcagtcctgtcaggaaaaaatcagtgttattctcattctacatatgagaa aactgaggcttgcagatataagggccaaaagttacacagctagtgagtgatggggctgagtttcagactccacagt ctcttaaccaccaagcagcatgcccagagtagaggtgagaaggaaggagagagctgcggtccacatgagcatctgg acctagcatggacaactcactcctccctggctctcgctttgttcttgttgcgggtgtggtggtggtgggactcaaa gacggtaaagatagctttctctcctccctggggaatctgggggttgtttaaaaggcctgctcctcttttagaaggc aggagggccccaagggaagcagaaggtgacagaaggggaaagggtcctctgatcattgctcaccccacagAGATCT TGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCA CAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTgtaagccatgcccctccctgctg cctcttctctcagacagcctgaccccagccgcaaactcccaacttacaacccagtgcctgcccgccactgccccag ccgcctacaccacccatttcctccctctctgtccctcctgccatctccctgtgcctcttcatctctggggttctct gtcttgtctccctctgcttataggttgtgcctctggtttgggggcctctcagcctgtctgggtccctcccttgctg tgcagttggcctcgtggcctctgctgctgtttgtgcctctctctgttactaacccgtcctctcgctgttagacatc tctctcactgcctgtctctggttctgtcctcagGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGG AGAGAGTTCTGAGGATTGTCAGAGCCgtgagtctcagggaggcctggagtcagggaaggggagggctggggccggg tggaatgcaggtgtcatacaggtgacatgggaggggtgggataacaggcttgggatgtctcccctgggccaggtag tctccctagaaggtgatgctgatgagggtctggtgcccagggcgccactcagccctcatcctgccctttgcccaac agTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAG TGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGgtatgtgcctctgctttgtgcccaatgtgctcta ccccccaggatgcaaggggtgggcaccctgcctggtactgc

hsa_circ_0001355

hsa_circ_0001355(exon1):

CTCGAGCAGTTCTTCAGATAGTAGAACATACAGCCGAAAGAAAGGAGGAAGGAAATCAAGATCAAAGTCAAGATCTTGGTCCAGAGATCTTCAGCCTCGTTCACATTCTTATGATAGAAGgtgatttttgtaatttttatttatatagtaatgaggataacattctttaaaattcatgtttttgtatttttaattttgctagataaaacatttttcttttactagctatatatttatgaaagtcccaacccagagtttttttgttccaccaggtccagtccgtgggtctagtgtcctcaacctttgtctgttcttagtaggcaaatagatagaaagtggtgtagtttccttttttaatttaatttaattttttttctagtgcatttatttatttatttttctaatgaattattttttattttattatactttaagttctagtacacaacatgcaggtttgttacatatgtatacatgtgccatgttggtgtgctgcatctgttaactcgtcatttacattaggtgtatctcctaatgctatccctcccctagccccccaccccatgacaggccccagtgtgtgatgttccccttcctgtgtccaagagttctcattgttcaattcccacctatgaatgagaacatgcggtgtttggttttttgtccttgcgatagtttgctgagaatgatggtttccagcttcatctatgtgcctacaaaggatgtgaactcatccttttttatggctgcgtagtattccatggtgtatatgtgccacattttcttaatccagtctgtcattgatggacatttgagttggttccaagcctttgctattgtgaacagtgctgcaacggagttttgctcttgttgtccaggctggagtacaatggcgtgatctcagctcactgcagcctctgcctcccaggttcaagcagttcttctcagccttccaagtagccgggattccaggcatgcaccactatgcccagctagttttgtatttttagtagagacagggtttcaccatgttggtcaggctggtcttgaactcctgacctcaggtgatctgccctcctcagcctcccaaaatgttgggattacaggcgtgagccaccgtgcctggccttaattttaaatttttttagagacaagatcttgctctgtcactcaggcgggagtgcagcggtgcagtcatatcttttggactcaagggatcctcccacctcagcttcctgaatagctagtactagaggcatgtgccaccatgcctagctaagtttttttatttttattttttgtggagacttggccttgctgtgttgcccaagctggtatcgaattcttggcctcaagcgatcctcctgcctcggcctcccaacatgttgggattacaggcatgagctactgagcctggccttggtttagttttcataccatataatctgatgatatttggaaagattctaagacccttaataccttaattagtactgatccaatcactaagttatgaaccagtcatatgactgttcaagagaaataacattttaatttatctctgtttgcttccaggaaacagtgagacagtaaaaatcagatgattctttagaatctagaaggtttttccttaatgctcataataaaaatttttatagcagtgatctttgattatattttatttcagACGCAGGCATCGATCAAGCAGTAGCTCTTCTTATGGCTCCAGAAGGAAACGAAGTCGAAGTCGTTCAAGGGGTCGAGGGAAATCCTATAGAGTTCAGAGGTCTAGGTCAAAAAGCAGAACAAGAAGAAAT GGGACGTCGGTCATCAGATACTGAAGAAGAAAGCAGAAGCAAGAGAAAAAAGAAACACCGTAGACGGTCCTC

hsa_circ_0001355(exon2):

GGTCGAGGGAAATCCTATAGAGTTCAGAGGTCTAGGTCAAAAAGCAGAACAAGAAGAAATGGGACGTC GGTCATCAGATACTGAAGAAGAAAGCAGAAGCAAGAGAAAAAAGAAACACCGTAGACGGTCCTCCTCGAGCAGTTC TTCAGATAGTAGAACATACAGCCGAAAGAAAGGAGGAAGGAAATCAAGATCAAAGTCAAGATCTTGGTCCAGAGAT CTTCAGCCTCGTTCACATTCTTATGATAGAAGgtgatttttgtaatttttatttatatagtaatgaggataacatt ctttaaaattcatgtttttgtatttttaattttgctagataaaacatttttcttttactagctatatatttatgaa agtcccaacccagagtttttttgttccaccaggtccagtccgtgggtctagtgtcctcaacctttgtctgttctta gtaggcaaatagatagaaagtggtgtagtttccttttttaatttaatttaattttttttctagtgcatttatttat ttatttttctaatgaattattttttattttattatactttaagttctagtacacaacatgcaggtttgttacatat gtatacatgtgccatgttggtgtgctgcatctgttaactcgtcatttacattaggtgtatctcctaatgctatccc tcccctagccccccaccccatgacaggccccagtgtgtgatgttccccttcctgtgtccaagagttctcattgttc aattcccacctatgaatgagaacatgcggtgtttggttttttgtccttgcgatagtttgctgagaatgatggtttc cagcttcatctatgtgcctacaaaggatgtgaactcatccttttttatggctgcgtagtattccatggtgtatatg tgccacattttcttaatccagtctgtcattgatggacatttgagttggttccaagcctttgctattgtgaacagtg ctgcaacggagttttgctcttgttgtccaggctggagtacaatggcgtgatctcagctcactgcagcctctgcctc ccaggttcaagcagttcttctcagccttccaagtagccgggattccaggcatgcaccactatgcccagctagtttt gtatttttagtagagacagggtttcaccatgttggtcaggctggtcttgaactcctgacctcaggtgatctgccct cctcagcctcccaaaatgttgggattacaggcgtgagccaccgtgcctggccttaattttaaatttttttagagac aagatcttgctctgtcactcaggcgggagtgcagcggtgcagtcatatcttttggactcaagggatcctcccacct cagcttcctgaatagctagtactagaggcatgtgccaccatgcctagctaagtttttttatttttattttttgtgg agacttggccttgctgtgttgcccaagctggtatcgaattcttggcctcaagcgatcctcctgcctcggcctccca acatgttgggattacaggcatgagctactgagcctggccttggtttagttttcataccatataatctgatgatatt tggaaagattctaagacccttaataccttaattagtactgatccaatcactaagttatgaaccagtcatatgactg ttcaagagaaataacattttaatttatctctgtttgcttccaggaaacagtgagacagtaaaaatcagatgattct ttagaatctagaaggtttttccttaatgctcataataaaaatttttatagcagtgatctttgattatattttattt cagACGCAGGCATCGATCAAGCAGTAGCTCTTCTTATGGCTCCAGAAGGAAACGAAGTCGAAGTCGTTCAAGG

hsa_circ_0001355(intron1):

cccacctatgaatgagaacatgcggtgtttggttttttgtccttgcgatagtttgctgagaatgatggtttccagcttcatctatgtgcctacaaaggatgtgaactcatccttttttatggctgcgtagtattccatggtgtatatgtgccacattttcttaatccagtctgtcattgatggacatttgagttggttccaagcctttgctattgtgaacagtgctgcaacggagttttgctcttgttgtccaggctggagtacaatggcgtgatctcagctcactgcagcctctgcctcccaggttcaagcagttcttctcagccttccaagtagccgggattccaggcatgcaccactatgcccagctagttttgtatttttagtagagacagggtttcaccatgttggtcaggctggtcttgaactcctgacctcaggtgatctgccctcctcagcctcccaaaatgttgggattacaggcgtgagccaccgtgcctggccttaattttaaatttttttagagacaagatcttgctctgtcactcaggcgggagtgcagcggtgcagtcatatcttttggactcaagggatcctcccacctcagcttcctgaatagctagtactagaggcatgtgccaccatgcctagctaagtttttttatttttattttttgtggagacttggccttgctgtgttgcccaagctggtatcgaattcttggcctcaagcgatcctcctgcctcggcctcccaacatgttgggattacaggcatgagctactgagcctggccttggtttagttttcataccatataatctgatgatatttggaaagattctaagacccttaataccttaattagtactgatccaatcactaagttatgaaccagtcatatgactgttcaagagaaataacattttaatttatctctgtttgcttccaggaaacagtgagacagtaaaaatcagatgattctttagaatctagaaggtttttccttaatgctcataataaaaatttttatagcagtgatctttgattatattttatttcagACGCAGGCATCGATCAAGCAGTAGCTCTTCTTATGGCTCCAGAAGGAAACGAAGTCGAAGTCGTTCAAGGGGTCGAGGGAAATCCTATAGAGTTCAGAGGTCTAGGTCAAAAAGCAGAACAAGAAGAAATGGGACGTCGGTCATC AGATACTGAAGAAGAAAGCAGAAGCAAGAGAAAAAAGAAACACCGTAGACGGTCCTCCTCGAGCAGTTCTTCAGAT AGTAGAACATACAGCCGAAAGAAAGGAGGAAGGAAATCAAGATCAAAGTCAAGATCTTGGTCCAGAGATCTTCAGC CTCGTTCACATTCTTATGATAGAAGgtgatttttgtaatttttatttatatagtaatgaggataacattctttaaa attcatgtttttgtatttttaattttgctagataaaacatttttcttttactagctatatatttatgaaagtccca acccagagtttttttgttccaccaggtccagtccgtgggtctagtgtcctcaacctttgtctgttcttagtaggca aatagatagaaagtggtgtagtttccttttttaatttaatttaattttttttctagtgcatttatttatttatttt tctaatgaattattttttattttattatactttaagttctagtacacaacatgcaggtttgttacatatgtataca tgtgccatgttggtgtgctgcatctgttaactcgtcatttacattaggtgtatctcctaatgctatccctccccta gccccccaccccatgacaggccccagtgtgtgatgttccccttcctgtgtccaagagttctcattgttcaatt

hsa_circ_0001538

hsa_circ_0001538(exon1):

TGGATGGAAAATGAAGAAGAATTCAACAGACAAgttagttttttgtacaaacatgtttttatagtccattctggccctcaatatgattgtacttgtccctgaaatgtgtttatcttgtcctttatgtataaagtatgtagactgatgtgccaccactcctgtttatttggctgctgattactaccaggcttaactgcttacggaaataacctggggagcttgtttcagggtcccaccctagagatgtctgaatcaggaggatgagggtggagcctgggaaactttttcagagctctcagatggttccattgagcagtcagatgtggaaaccactgccttatgccagtaagattgctggttaccgtgaaagtcaagagaattcaggagtcaggagcagacaggcacagaaaaaagagacaggcatctctttttacttcttccctgccaccctcaatggaaaaaagaatgttctactaataaaagtaaaaacatagaaaaaccctaaacagatagatcttcgccggtcattatggtaagcattttttttccttagcatggaagcagaaaagaaagtatgactttgtgcctttaaatggtttagttcaagatattttaagccattcctagtgatatctaccaagtgtgctataaattatcctttattataaatctagATAGAAGAGGAGTTATGGGAAGAAGAATTTATTGAACGCTGTTTCCAAGAAATGCTGGAAGAGGAAGAAGAGCATGAATGGTTTATTCCAGCTCGAGATCTCCCACAAACTATGGACCAAATCCAAGACCAGTTTAATGACCTTGTTATCAGTGATGGCTCTTCTCTGGAAGATCTTGTGGTTAAAAACGACAACCAACATCAGCCATGAAAGATCCAAGTCGCAGCAGTACT AGCCCAAGCATCATCAATGAAGATGTGATTATTAACGGTCATTCTCATGAAGATGACAATCCATTTGCAGAGTACAT G

hsa_circ_0001538(exon2)

AAGAGCATGAATGGTTTATTCCAGCTCGAGATCTCCCACAAACTATGGACCAAATCCAAGACCAGTTTAATGACCTTGTTATCAGTGATGGCTCTTCTCTGGAAGATCTTGTGGTTAAAAACGACAACCAACATCAGCCATGAA AGATCCAAGTCGCAGCAGTACTAGCCCAAGCATCATCAATGAAGATGTGATTATTAACGGTCATTCTCATGAAGAT GACAATCCATTTGCAGAGTACATGTGGATGGAAAATGAAGAAGAATTCAACAGACAAgttagttttttgtacaaac atgtttttatagtccattctggccctcaatatgattgtacttgtccctgaaatgtgtttatcttgtcctttatgta taaagtatgtagactgatgtgccaccactcctgtttatttggctgctgattactaccaggcttaactgcttacgga aataacctggggagcttgtttcagggtcccaccctagagatgtctgaatcaggaggatgagggtggagcctgggaa actttttcagagctctcagatggttccattgagcagtcagatgtggaaaccactgccttatgccagtaagattgct ggttaccgtgaaagtcaagagaattcaggagtcaggagcagacaggcacagaaaaaagagacaggcatctcttttt acttcttccctgccaccctcaatggaaaaaagaatgttctactaataaaagtaaaaacatagaaaaaccctaaaca gatagatcttcgccggtcattatggtaagcattttttttccttagcatggaagcagaaaagaaagtatgactttgt gcctttaaatggtttagttcaagatattttaagccattcctagtgatatctaccaagtgtgctataaattatcctt tattataaatctagATAGAAGAGGAGTTATGGGAAGAAGAATTTATTGAACGCTGTTTCCAAGAAATGCTGGAAGA GGAAG

hsa_circ_0001538(intron1)

gacaggcacagaaaaaagagacaggcatctctttttacttcttccctgccaccctcaatggaaaaaagaatgttctactaataaaagtaaaaacatagaaaaaccctaaacagatagatcttcgccggtcattatggtaagcattttttttccttagcatggaagcagaaaagaaagtatgactttgtgcctttaaatggtttagttcaagatattttaagccattcctagtgatatctaccaagtgtgctataaattatcctttattataaatctagATAGAAGAGGAGTTATGGGAAGAAGAATTTATTGAACGCTGTTTCCAAGAAATGCTGGAAGAGGAAGAAGAGCATGAATGGTTTATTCCAGCTCGAGATCTCCCACAAACTATGGACCAAATCCAAGACCAGTTTAATGACCTTGTTATCAGTGATGGCTCTTCTCTGGAAGATCTTGTGGTTAAAAACGACAACCAACATCAGCCATGAAAGATCCAAGTCGCAGCAGTACTAGCCCAAGCATCA TCAATGAAGATGTGATTATTAACGGTCATTCTCATGAAGATGACAATCCATTTGCAGAGTACATGTGGATGGAAAA TGAAGAAGAATTCAACAGACAAgttagttttttgtacaaacatgtttttatagtccattctggccctcaatatgat tgtacttgtccctgaaatgtgtttatcttgtcctttatgtataaagtatgtagactgatgtgccaccactcctgtt tatttggctgctgattactaccaggcttaactgcttacggaaataacctggggagcttgtttcagggtcccaccct agagatgtctgaatcaggaggatgagggtggagcctgggaaactttttcagagctctcagatggttccattgagca gtcagatgtggaaaccactgccttatgccagtaagattgctggttaccgtgaaagtcaagagaattcaggagtcag gagca

(3) Primer design using primer premier 5 software: the new sequence is put into primer premier 5 software and is set as follows: detecting full-length primer sequences, wherein the primer lengths are 19-25bp respectively, the GC proportion of the primers is 40% -60%, the primers are free from occurrence of more hairpin and dimer, and the difference between the Tm values of the upstream primer and the downstream primer is less than 5 ℃.

hsa_circ_0007766 (upstream and downstream primer sequences)

hsa_circ_0007766(exon1)

Upstream primer (5 '-3') GACCCGCTGAACAATACCACCC

Downstream primer (5 '-3') TCCATTGTCTAGCACGGCCAGG

hsa_circ_0007766(exon2)

Upstream primer (5 '-3') ACCCCCAGCTCTGCTACCAGG

Downstream primer (5 '-3') TCCGCTGGATCAAGACCCCTCC

hsa_circ_0007766(exon3)

Upstream primer (5 '-3') TAAGGGCTCCCGCTGCTGGGGA

Downstream primer (5 '-3') CACATCGGAGAACAGGGGTGGC

hsa_circ_0007766(exon4)

Upstream primer (5 '-3') TGTGCTGCCGGCTGCACGGG

Downstream primer (5 '-3') GCTCATGGCAGCAGTCAGTGG

hsa_circ_0007766(exon5)

Upstream primer (5 '-3') GTGGCATCTGTGAGCTGCACT

Downstream primer (5 '-3') GTGGTTGAAGTGGAGGCAGGC

hsa_circ_0007766(intron1)

Upstream primer (5 '-3') TCGCTTTGTTCTTGTTGCGGG

Downstream primer (5 '-3') GAGCCAGGGAGGAGTGAGTTGTC

hsa_circ_0007766(intron2)

Upstream primer (5 '-3') TTATAGGTTGTGCCTCTGGTTTG

Downstream primer (5 '-3') GCAGAGGGAGACAAGACAGAGA

hsa_circ_0007766(intron3)

Upstream primer (5 '-3') GTCTCCCCTGGGCCAGGTAGTCTC

Downstream primer (5 '-3') ATCCCAAGCCTGTTATCCCACCCC

hsa_circ_0007766(intron4)

Upstream primer (5 '-3') CCTATTGCCCCTGGCACACCA

Downstream primer (5 '-3') GCAGTACCAGGCAGGGTGCC

hsa_circ_0001355 (upstream and downstream primer sequences):

hsa_circ_0001355(exon1)

upstream primer (5 '-3') CTCGAGCAGTTCTTCAGATAGTA

Downstream primer (5 '-3') GAGGACCGTCTACGGTGTTT

hsa_circ_0001355(exon2)

Upstream primer (5 '-3') CTCTTCTTATGGCTCCAGAAG

Downstream primer (5 '-3') CTACTGCTTGATCGATGCCT

hsa_circ_0001355(intron1)

Upstream primer (5 '-3') AGCTTCATCTATGTGCCTACAA

Downstream primer (5 '-3') GGAAACCATCATTCTCAGCA

hsa_circ_0001538 (upstream and downstream primer sequences):

hsa_circ_0001538(exon1)

upstream primer (5 '-3') TGGATGGAAAATGAAGAAGAAT

Downstream primer (5 '-3') CATGTACTCTGCAAATGGATTG

hsa_circ_0001538(exon2)

Upstream primer (5 '-3') AAGAGCATGAATGGTTTATTCC

Downstream primer (5 '-3') CTTCCTCTTCCAGCATTTCTT

hsa_circ_0001538(intron1)

Upstream primer (5 '-3') ACTCCTGTTTATTTGGCTGCT

Downstream primer (5 '-3') GGTGGCACATCAGTCTACATAC

(4) The designed upstream and downstream primers were subjected to in silico PCR, confirming that the primer pair was unable to amplify linear RNA molecules and other RNA molecules. The primer pair is the final primer.

(5) Reverse transcribing RNA and performing PCR. Preparing 5-10ug total RNA of each RNA sample, performing reverse transcription (reverse transcription method is determined by reverse transcription kit according to different formulas), and performing PCR reaction (the PCR reaction system can be set according to specific requirements) by using the designed primer after reverse transcription, wherein the PCR reaction steps are as follows:

(6) Agarose electrophoresis confirmed that the designed primers were able to amplify the full-length sequence of circular RNA without the addition of RNase R.

RNase R is an enzyme that digests linear RNA. Under normal conditions, it was necessary to add RNase R to the extracted RNA and to detect the sequence at the junction of the circular RNA by PCR in combination with Sanger sequencing to confirm that the circular RNA was indeed a closed circular structure. Thus, the primers designed according to the method of the present invention were directly subjected to PCR without adding RNase R, combined with Sanger sequencing, and were able to detect the junction sequence of circular RNA and to amplify a band size that was expected to be consistent with the genomic length (genomic length) or post-cut length (splice length) of circular RNA, indicating that the primers were indeed able to amplify circular RNA and were able to obtain the full-length sequence of circular RNA.

The experimental procedure was as follows: a0.8% TAE agarose electrophoresis gel was prepared, and the PCR reaction product was subjected to electrophoresis, wherein the DNA marker used for electrophoresis was DNA 2000plus.

The result of electrophoresis of hsa_circle_ 0007766 is shown in FIG. 4.

Experimental results: lane 1: DNA 2000plus marker, lane 2: hsa_circ_0007766 (exon 1) primer PCR product

Lane 3: hsa_circ_0007766 (exon 2) primer PCR product, lane 4: hsa_circ_0007766 (exon 3) primer PCR product, lane 5: hsa_circ_0007766 (exon 4) primer PCR product, lane 6: hsa_circ_0007766 (exon 5) primer PCR product, lane 7: hsa_circ_0007766 (intron 1) primer PCR product, lane 8: hsa_circ_0007766 (intron 2) primer PCR product, lane 9: hsa_circ_0007766 (intron 3) primer PCR product, lane 10: hsa_circ_0007766 (intron 4) primer PCR product. As a result of the electrophoresis of the above graph, lanes 2-6 can see a series of relatively homogeneous bands all located at 500-750nt, the fragment size of which is similar to the sheared length (676 nt) of hsa_circ_0007766, and basically judging that hsa_circ_0007766 is full length, while lanes 7-10 have no bands with better consistency and even no product amplification, representing that hsa_circ_0007766 will completely trim the intron during formation.

The result of electrophoresis of hsa_circle_ 0001355 is shown in FIG. 5.

Experimental results: lane 1: DNA 2000plus marker, lane 2: hsa_circ_0001355 (exon 1) primer PCR product

Lane 3: hsa_circ_0001355 (exon 2) primer PCR product, lane 4: hsa_circ_0001355 (intron 1) primer PCR product, lane 5: as a result of the above-described electropherograms, lanes 2-3 can each see a series of relatively homogeneous bands located at 250-500nt each, with fragment sizes substantially similar to the sheared length (322 nt) of hsa_circ_0001355, although some non-specific amplification occurred in lane 2, both sizes and positions were different from the sheared length or genomic length of hsa_circ_0001355, whereas lane 4 had no product amplification, and the full length sequence of hsa_circ_0001355 was substantially determined to be 322nt.

The result of electrophoresis of hsa_circle_ 0001538 is shown in FIG. 6.

Experimental results: lane 1: DNA 2000plus marker, lane 2: hsa_circ_0001538 (exon 1) primer PCR product

Lane 3: hsa_circ_0001538 (exon 2) primer PCR product, lane 4: hsa_circ_0001538 (intron 1) primer PCR product, lane 5: as a result of the above-described electrophoresis, it can be seen that lanes 2-3 each have a series of relatively homogeneous bands located at 250-500nt, which have a fragment size substantially similar to the sheared length (344 nt) of hsa_circ_0001538, and that although some very small amounts of non-specific amplification occur in lanes 2-3, they all have a size different from the sheared length or genomic length of hsa_circ_0001538, whereas lane 4 has no product amplification, which essentially determines that the full length sequence of hsa_circ_0001538 is 344nt.

(7) Sanger sequencing verifies that the designed primers were able to amplify the full length sequence of the circular RNA without the addition of RNase R. Agarose electrophoresis can only preliminarily judge the size of the amplified product, and in order to further confirm the accuracy of the primer design method for amplifying the circular RNA, the inventor performs Sanger sequencing to detect the characteristic structure of the circular RNA in the amplified product, namely the junction site sequence. Sanger sequencing results are shown in FIGS. 7-15.

hsa_circ_0007766：

The results of Sanger sequencing of the hsa_circ_0007766 (exon 1) primer amplification product are shown in FIG. 7:

wherein: GTCCC (GTCCC)TGATATC(TGAt the joint).

The results of Sanger sequencing of the hsa_circ_0007766 (exon 2) primer amplification product are shown in FIG. 8:

wherein: GTCCC (GTCCC)TGATATC(TGAt the joint).

The results of Sanger sequencing of the hsa_circ_0007766 (exon 3) primer amplification product are shown in FIG. 9:

wherein: GTCCC (GTCCC)TGATATC(TGAt the joint).

The results of Sanger sequencing of the hsa_circ_0007766 (exon 4) primer amplification product are shown in FIG. 10:

wherein: GTCCC (GTCCC)TGATATC(TGAt the joint).

The results of Sanger sequencing of hsa_circ_0007766 (exon 5) primer amplification product are shown in FIG. 11:

wherein: GTCCC (GTCCC)TGATATC(TGAt the joint).

Experimental results: as shown in the sequencing results of FIGS. 7-11, the full-length primers designed according to the methods of the present invention amplified hsa_circ_0007766 without adding RNase R to the full-length sequence of hsa_circ_0007766, confirming the sequence at the characteristic junction of hsa_circ_0007766, and confirming that the full-length sequence of hsa_circ_0007766 is 676nt rather than 2161nt in genome length.

hsa_circ_0001355：

The results of Sanger sequencing of the hsa_circ_0001355 (exon 1) primer amplification product are shown in FIG. 12:

wherein: AAGAAGAAATGG(GAIs the joint position

The results of Sanger sequencing of the hsa_circ_0001355 (exon 2) primer amplification product are shown in FIG. 13:

wherein: AAGAAGAAATGG(GAIs the joint position

Experimental results: as shown in the sequencing results of FIGS. 12 and 13, the full-length primer for amplifying hsa_circ_0001355 designed according to the method of the present invention can amplify the full-length sequence of hsa_circ_0001355 without adding RNase R, confirm the sequence at the characteristic adaptor of hsa_circ_0001355, and confirm that the full-length sequence of hsa_circ_0001355 is 322nt instead of the genomic length 1889nt thereof by Sanger sequencing.

hsa_circ_0001538

The results of Sanger sequencing of the hsa_circ_0001538 (exon 1) primer amplification product are shown in FIG. 14:

wherein: CTTGTGGTTAAA(GGIs the joint position

The results of Sanger sequencing of hsa_circ_0001538 (exon 2) primer amplification product are shown in FIG. 15:

wherein: CTTGTGGTTAAA(GGIs the joint position

Experimental results: as shown in the sequencing results of FIGS. 14 and 15, the full-length primer for amplifying hsa_circ_0001538 designed according to the method of the present invention can amplify the full-length sequence of hsa_circ_0001538 without adding RNase R, confirm the sequence at the characteristic adaptor of hsa_circ_0001538, and confirm that the full-length sequence of hsa_circ_0001538 is 344nt instead of the genomic length 985nt thereof by Sanger sequencing.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> university of Zhongshan Sun Yixian commemorative Hospital

<120> method for detecting full-length sequence of circular RNA

<130> 2019

<160> 48

<170> PatentIn version 3.5

<210> 1

<211> 2161

<212> DNA

<213> hsa

<400> 1

gatatccagg aggtgcaggg ctacgtgctc atcgctcaca accaagtgag gcaggtccca 60

ctgcagaggc tgcggattgt gcgaggcacc cagctctttg aggacaacta tgccctggcc 120

gtgctagaca atggagaccc gctgaacaat accacccctg tcacaggggc ctccccagga 180

ggcctgcggg agctgcagct tcgaagcctc acaggtggcc ttcaccgtca ttgaaacctt 240

ctcttggtta ttcagagctg accagggcca ctgctaacca gggggaggct ttgtgtgcat 300

tagaaatggt gtcccttctg ggcagacgca ggcagagccc gggaagacgc cctcagaaga 360

ttggaaaaag attccccttc ttcctgggaa gttgtagctt gcgtcagcac atataattca 420

atcgtgagaa tgcaggctgg gtttttgccc ccacttggct gagtgaagtg tacagtgaac 480

aacctatgta actatttgct ggccctggag ccgactctgc cccagagtct gggtgccagg 540

tgctttgccc gcatggccca tttcagtcac gctgcagtcc tgtcaggaaa aaatcagtgt 600

tattctcatt ctacatatga gaaaactgag gcttgcagat ataagggcca aaagttacac 660

agctagtgag tgatggggct gagtttcaga ctccacagtc tcttaaccac caagcagcat 720

gcccagagta gaggtgagaa ggaaggagag agctgcggtc cacatgagca tctggaccta 780

gcatggacaa ctcactcctc cctggctctc gctttgttct tgttgcgggt gtggtggtgg 840

tgggactcaa agacggtaaa gatagctttc tctcctccct ggggaatctg ggggttgttt 900

aaaaggcctg ctcctctttt agaaggcagg agggccccaa gggaagcaga aggtgacaga 960

aggggaaagg gtcctctgat cattgctcac cccacagaga tcttgaaagg aggggtcttg 1020

atccagcgga acccccagct ctgctaccag gacacgattt tgtggaagga catcttccac 1080

aagaacaacc agctggctct cacactgata gacaccaacc gctctcgggc ctgtaagcca 1140

tgcccctccc tgctgcctct tctctcagac agcctgaccc cagccgcaaa ctcccaactt 1200

acaacccagt gcctgcccgc cactgcccca gccgcctaca ccacccattt cctccctctc 1260

tgtccctcct gccatctccc tgtgcctctt catctctggg gttctctgtc ttgtctccct 1320

ctgcttatag gttgtgcctc tggtttgggg gcctctcagc ctgtctgggt ccctcccttg 1380

ctgtgcagtt ggcctcgtgg cctctgctgc tgtttgtgcc tctctctgtt actaacccgt 1440

cctctcgctg ttagacatct ctctcactgc ctgtctctgg ttctgtcctc aggccacccc 1500

tgttctccga tgtgtaaggg ctcccgctgc tggggagaga gttctgagga ttgtcagagc 1560

cgtgagtctc agggaggcct ggagtcaggg aaggggaggg ctggggccgg gtggaatgca 1620

ggtgtcatac aggtgacatg ggaggggtgg gataacaggc ttgggatgtc tcccctgggc 1680

caggtagtct ccctagaagg tgatgctgat gagggtctgg tgcccagggc gccactcagc 1740

cctcatcctg ccctttgccc aacagtgacg cgcactgtct gtgccggtgg ctgtgcccgc 1800

tgcaaggggc cactgcccac tgactgctgc catgagcagt gtgctgccgg ctgcacgggc 1860

cccaagcact ctgactgcct ggtatgtgcc tctgctttgt gcccaatgtg ctctaccccc 1920

caggatgcaa ggggtgggca ccctgcctgg tactgcccta ttgcccctgg cacaccaggg 1980

caaaacagca cagtgaaagc cagccacctg tccccccagg cctgcctcca cttcaaccac 2040

agtggcatct gtgagctgca ctgcccagcc ctggtcacct acaacacaga cacgtttgag 2100

tccatgccca atcccgaggg ccggtataca ttcggcgcca gctgtgtgac tgcctgtccc 2160

t 2161

<210> 2

<211> 1889

<212> DNA

<213> hsa

<400> 2

aaatgggacg tcggtcatca gatactgaag aagaaagcag aagcaagaga aaaaagaaac 60

accgtagacg gtcctcctcg agcagttctt cagatagtag aacatacagc cgaaagaaag 120

gaggaaggaa atcaagatca aagtcaagat cttggtccag agatcttcag cctcgttcac 180

attcttatga tagaaggtga tttttgtaat ttttatttat atagtaatga ggataacatt 240

ctttaaaatt catgtttttg tatttttaat tttgctagat aaaacatttt tcttttacta 300

gctatatatt tatgaaagtc ccaacccaga gtttttttgt tccaccaggt ccagtccgtg 360

ggtctagtgt cctcaacctt tgtctgttct tagtaggcaa atagatagaa agtggtgtag 420

tttccttttt taatttaatt taattttttt tctagtgcat ttatttattt atttttctaa 480

tgaattattt tttattttat tatactttaa gttctagtac acaacatgca ggtttgttac 540

atatgtatac atgtgccatg ttggtgtgct gcatctgtta actcgtcatt tacattaggt 600

gtatctccta atgctatccc tcccctagcc ccccacccca tgacaggccc cagtgtgtga 660

tgttcccctt cctgtgtcca agagttctca ttgttcaatt cccacctatg aatgagaaca 720

tgcggtgttt ggttttttgt ccttgcgata gtttgctgag aatgatggtt tccagcttca 780

tctatgtgcc tacaaaggat gtgaactcat ccttttttat ggctgcgtag tattccatgg 840

tgtatatgtg ccacattttc ttaatccagt ctgtcattga tggacatttg agttggttcc 900

aagcctttgc tattgtgaac agtgctgcaa cggagttttg ctcttgttgt ccaggctgga 960

gtacaatggc gtgatctcag ctcactgcag cctctgcctc ccaggttcaa gcagttcttc 1020

tcagccttcc aagtagccgg gattccaggc atgcaccact atgcccagct agttttgtat 1080

ttttagtaga gacagggttt caccatgttg gtcaggctgg tcttgaactc ctgacctcag 1140

gtgatctgcc ctcctcagcc tcccaaaatg ttgggattac aggcgtgagc caccgtgcct 1200

ggccttaatt ttaaattttt ttagagacaa gatcttgctc tgtcactcag gcgggagtgc 1260

agcggtgcag tcatatcttt tggactcaag ggatcctccc acctcagctt cctgaatagc 1320

tagtactaga ggcatgtgcc accatgccta gctaagtttt tttattttta ttttttgtgg 1380

agacttggcc ttgctgtgtt gcccaagctg gtatcgaatt cttggcctca agcgatcctc 1440

ctgcctcggc ctcccaacat gttgggatta caggcatgag ctactgagcc tggccttggt 1500

ttagttttca taccatataa tctgatgata tttggaaaga ttctaagacc cttaatacct 1560

taattagtac tgatccaatc actaagttat gaaccagtca tatgactgtt caagagaaat 1620

aacattttaa tttatctctg tttgcttcca ggaaacagtg agacagtaaa aatcagatga 1680

ttctttagaa tctagaaggt ttttccttaa tgctcataat aaaaattttt atagcagtga 1740

tctttgatta tattttattt cagacgcagg catcgatcaa gcagtagctc ttcttatggc 1800

tccagaagga aacgaagtcg aagtcgttca aggggtcgag ggaaatccta tagagttcag 1860

aggtctaggt caaaaagcag aacaagaag 1889

<210> 3

<211> 985

<212> DNA

<213> hsa

<400> 3

gttaaaaacg acaaccaaca tcagccatga aagatccaag tcgcagcagt actagcccaa 60

gcatcatcaa tgaagatgtg attattaacg gtcattctca tgaagatgac aatccatttg 120

cagagtacat gtggatggaa aatgaagaag aattcaacag acaagttagt tttttgtaca 180

aacatgtttt tatagtccat tctggccctc aatatgattg tacttgtccc tgaaatgtgt 240

ttatcttgtc ctttatgtat aaagtatgta gactgatgtg ccaccactcc tgtttatttg 300

gctgctgatt actaccaggc ttaactgctt acggaaataa cctggggagc ttgtttcagg 360

gtcccaccct agagatgtct gaatcaggag gatgagggtg gagcctggga aactttttca 420

gagctctcag atggttccat tgagcagtca gatgtggaaa ccactgcctt atgccagtaa 480

gattgctggt taccgtgaaa gtcaagagaa ttcaggagtc aggagcagac aggcacagaa 540

aaaagagaca ggcatctctt tttacttctt ccctgccacc ctcaatggaa aaaagaatgt 600

tctactaata aaagtaaaaa catagaaaaa ccctaaacag atagatcttc gccggtcatt 660

atggtaagca ttttttttcc ttagcatgga agcagaaaag aaagtatgac tttgtgcctt 720

taaatggttt agttcaagat attttaagcc attcctagtg atatctacca agtgtgctat 780

aaattatcct ttattataaa tctagataga agaggagtta tgggaagaag aatttattga 840

acgctgtttc caagaaatgc tggaagagga agaagagcat gaatggttta ttccagctcg 900

agatctccca caaactatgg accaaatcca agaccagttt aatgaccttg ttatcagtga 960

tggctcttct ctggaagatc ttgtg 985

<210> 4

<211> 2161

<212> DNA

<213> artificial sequence

<400> 4

cctggccgtg ctagacaatg gagacccgct gaacaatacc acccctgtca caggggcctc 60

cccaggaggc ctgcgggagc tgcagcttcg aagcctcaca ggtggccttc accgtcattg 120

aaaccttctc ttggttattc agagctgacc agggccactg ctaaccaggg ggaggctttg 180

tgtgcattag aaatggtgtc ccttctgggc agacgcaggc agagcccggg aagacgccct 240

cagaagattg gaaaaagatt ccccttcttc ctgggaagtt gtagcttgcg tcagcacata 300

taattcaatc gtgagaatgc aggctgggtt tttgccccca cttggctgag tgaagtgtac 360

agtgaacaac ctatgtaact atttgctggc cctggagccg actctgcccc agagtctggg 420

tgccaggtgc tttgcccgca tggcccattt cagtcacgct gcagtcctgt caggaaaaaa 480

tcagtgttat tctcattcta catatgagaa aactgaggct tgcagatata agggccaaaa 540

gttacacagc tagtgagtga tggggctgag tttcagactc cacagtctct taaccaccaa 600

gcagcatgcc cagagtagag gtgagaagga aggagagagc tgcggtccac atgagcatct 660

ggacctagca tggacaactc actcctccct ggctctcgct ttgttcttgt tgcgggtgtg 720

gtggtggtgg gactcaaaga cggtaaagat agctttctct cctccctggg gaatctgggg 780

gttgtttaaa aggcctgctc ctcttttaga aggcaggagg gccccaaggg aagcagaagg 840

tgacagaagg ggaaagggtc ctctgatcat tgctcacccc acagagatct tgaaaggagg 900

ggtcttgatc cagcggaacc cccagctctg ctaccaggac acgattttgt ggaaggacat 960

cttccacaag aacaaccagc tggctctcac actgatagac accaaccgct ctcgggcctg 1020

taagccatgc ccctccctgc tgcctcttct ctcagacagc ctgaccccag ccgcaaactc 1080

ccaacttaca acccagtgcc tgcccgccac tgccccagcc gcctacacca cccatttcct 1140

ccctctctgt ccctcctgcc atctccctgt gcctcttcat ctctggggtt ctctgtcttg 1200

tctccctctg cttataggtt gtgcctctgg tttgggggcc tctcagcctg tctgggtccc 1260

tcccttgctg tgcagttggc ctcgtggcct ctgctgctgt ttgtgcctct ctctgttact 1320

aacccgtcct ctcgctgtta gacatctctc tcactgcctg tctctggttc tgtcctcagg 1380

ccacccctgt tctccgatgt gtaagggctc ccgctgctgg ggagagagtt ctgaggattg 1440

tcagagccgt gagtctcagg gaggcctgga gtcagggaag gggagggctg gggccgggtg 1500

gaatgcaggt gtcatacagg tgacatggga ggggtgggat aacaggcttg ggatgtctcc 1560

cctgggccag gtagtctccc tagaaggtga tgctgatgag ggtctggtgc ccagggcgcc 1620

actcagccct catcctgccc tttgcccaac agtgacgcgc actgtctgtg ccggtggctg 1680

tgcccgctgc aaggggccac tgcccactga ctgctgccat gagcagtgtg ctgccggctg 1740

cacgggcccc aagcactctg actgcctggt atgtgcctct gctttgtgcc caatgtgctc 1800

taccccccag gatgcaaggg gtgggcaccc tgcctggtac tgccctattg cccctggcac 1860

accagggcaa aacagcacag tgaaagccag ccacctgtcc ccccaggcct gcctccactt 1920

caaccacagt ggcatctgtg agctgcactg cccagccctg gtcacctaca acacagacac 1980

gtttgagtcc atgcccaatc ccgagggccg gtatacattc ggcgccagct gtgtgactgc 2040

ctgtccctga tatccaggag gtgcagggct acgtgctcat cgctcacaac caagtgaggc 2100

aggtcccact gcagaggctg cggattgtgc gaggcaccca gctctttgag gacaactatg 2160

c 2161

<210> 5

<211> 2161

<212> DNA

<213> artificial sequence

<400> 5

acccccagct ctgctaccag gacacgattt tgtggaagga catcttccac aagaacaacc 60

agctggctct cacactgata gacaccaacc gctctcgggc ctgtaagcca tgcccctccc 120

tgctgcctct tctctcagac agcctgaccc cagccgcaaa ctcccaactt acaacccagt 180

gcctgcccgc cactgcccca gccgcctaca ccacccattt cctccctctc tgtccctcct 240

gccatctccc tgtgcctctt catctctggg gttctctgtc ttgtctccct ctgcttatag 300

gttgtgcctc tggtttgggg gcctctcagc ctgtctgggt ccctcccttg ctgtgcagtt 360

ggcctcgtgg cctctgctgc tgtttgtgcc tctctctgtt actaacccgt cctctcgctg 420

ttagacatct ctctcactgc ctgtctctgg ttctgtcctc aggccacccc tgttctccga 480

tgtgtaaggg ctcccgctgc tggggagaga gttctgagga ttgtcagagc cgtgagtctc 540

agggaggcct ggagtcaggg aaggggaggg ctggggccgg gtggaatgca ggtgtcatac 600

aggtgacatg ggaggggtgg gataacaggc ttgggatgtc tcccctgggc caggtagtct 660

ccctagaagg tgatgctgat gagggtctgg tgcccagggc gccactcagc cctcatcctg 720

ccctttgccc aacagtgacg cgcactgtct gtgccggtgg ctgtgcccgc tgcaaggggc 780

cactgcccac tgactgctgc catgagcagt gtgctgccgg ctgcacgggc cccaagcact 840

ctgactgcct ggtatgtgcc tctgctttgt gcccaatgtg ctctaccccc caggatgcaa 900

ggggtgggca ccctgcctgg tactgcccta ttgcccctgg cacaccaggg caaaacagca 960

cagtgaaagc cagccacctg tccccccagg cctgcctcca cttcaaccac agtggcatct 1020

gtgagctgca ctgcccagcc ctggtcacct acaacacaga cacgtttgag tccatgccca 1080

atcccgaggg ccggtataca ttcggcgcca gctgtgtgac tgcctgtccc tgatatccag 1140

gaggtgcagg gctacgtgct catcgctcac aaccaagtga ggcaggtccc actgcagagg 1200

ctgcggattg tgcgaggcac ccagctcttt gaggacaact atgccctggc cgtgctagac 1260

aatggagacc cgctgaacaa taccacccct gtcacagggg cctccccagg aggcctgcgg 1320

gagctgcagc ttcgaagcct cacaggtggc cttcaccgtc attgaaacct tctcttggtt 1380

attcagagct gaccagggcc actgctaacc agggggaggc tttgtgtgca ttagaaatgg 1440

tgtcccttct gggcagacgc aggcagagcc cgggaagacg ccctcagaag attggaaaaa 1500

gattcccctt cttcctggga agttgtagct tgcgtcagca catataattc aatcgtgaga 1560

atgcaggctg ggtttttgcc cccacttggc tgagtgaagt gtacagtgaa caacctatgt 1620

aactatttgc tggccctgga gccgactctg ccccagagtc tgggtgccag gtgctttgcc 1680

cgcatggccc atttcagtca cgctgcagtc ctgtcaggaa aaaatcagtg ttattctcat 1740

tctacatatg agaaaactga ggcttgcaga tataagggcc aaaagttaca cagctagtga 1800

gtgatggggc tgagtttcag actccacagt ctcttaacca ccaagcagca tgcccagagt 1860

agaggtgaga aggaaggaga gagctgcggt ccacatgagc atctggacct agcatggaca 1920

actcactcct ccctggctct cgctttgttc ttgttgcggg tgtggtggtg gtgggactca 1980

aagacggtaa agatagcttt ctctcctccc tggggaatct gggggttgtt taaaaggcct 2040

gctcctcttt tagaaggcag gagggcccca agggaagcag aaggtgacag aaggggaaag 2100

ggtcctctga tcattgctca ccccacagag atcttgaaag gaggggtctt gatccagcgg 2160

a 2161

<210> 6

<211> 2161

<212> DNA

<213> artificial sequence

<400> 6

taagggctcc cgctgctggg gagagagttc tgaggattgt cagagccgtg agtctcaggg 60

aggcctggag tcagggaagg ggagggctgg ggccgggtgg aatgcaggtg tcatacaggt 120

gacatgggag gggtgggata acaggcttgg gatgtctccc ctgggccagg tagtctccct 180

agaaggtgat gctgatgagg gtctggtgcc cagggcgcca ctcagccctc atcctgccct 240

ttgcccaaca gtgacgcgca ctgtctgtgc cggtggctgt gcccgctgca aggggccact 300

gcccactgac tgctgccatg agcagtgtgc tgccggctgc acgggcccca agcactctga 360

ctgcctggta tgtgcctctg ctttgtgccc aatgtgctct accccccagg atgcaagggg 420

tgggcaccct gcctggtact gccctattgc ccctggcaca ccagggcaaa acagcacagt 480

gaaagccagc cacctgtccc cccaggcctg cctccacttc aaccacagtg gcatctgtga 540

gctgcactgc ccagccctgg tcacctacaa cacagacacg tttgagtcca tgcccaatcc 600

cgagggccgg tatacattcg gcgccagctg tgtgactgcc tgtccctgat atccaggagg 660

tgcagggcta cgtgctcatc gctcacaacc aagtgaggca ggtcccactg cagaggctgc 720

ggattgtgcg aggcacccag ctctttgagg acaactatgc cctggccgtg ctagacaatg 780

gagacccgct gaacaatacc acccctgtca caggggcctc cccaggaggc ctgcgggagc 840

tgcagcttcg aagcctcaca ggtggccttc accgtcattg aaaccttctc ttggttattc 900

agagctgacc agggccactg ctaaccaggg ggaggctttg tgtgcattag aaatggtgtc 960

ccttctgggc agacgcaggc agagcccggg aagacgccct cagaagattg gaaaaagatt 1020

ccccttcttc ctgggaagtt gtagcttgcg tcagcacata taattcaatc gtgagaatgc 1080

aggctgggtt tttgccccca cttggctgag tgaagtgtac agtgaacaac ctatgtaact 1140

atttgctggc cctggagccg actctgcccc agagtctggg tgccaggtgc tttgcccgca 1200

tggcccattt cagtcacgct gcagtcctgt caggaaaaaa tcagtgttat tctcattcta 1260

catatgagaa aactgaggct tgcagatata agggccaaaa gttacacagc tagtgagtga 1320

tggggctgag tttcagactc cacagtctct taaccaccaa gcagcatgcc cagagtagag 1380

gtgagaagga aggagagagc tgcggtccac atgagcatct ggacctagca tggacaactc 1440

actcctccct ggctctcgct ttgttcttgt tgcgggtgtg gtggtggtgg gactcaaaga 1500

cggtaaagat agctttctct cctccctggg gaatctgggg gttgtttaaa aggcctgctc 1560

ctcttttaga aggcaggagg gccccaaggg aagcagaagg tgacagaagg ggaaagggtc 1620

ctctgatcat tgctcacccc acagagatct tgaaaggagg ggtcttgatc cagcggaacc 1680

cccagctctg ctaccaggac acgattttgt ggaaggacat cttccacaag aacaaccagc 1740

tggctctcac actgatagac accaaccgct ctcgggcctg taagccatgc ccctccctgc 1800

tgcctcttct ctcagacagc ctgaccccag ccgcaaactc ccaacttaca acccagtgcc 1860

tgcccgccac tgccccagcc gcctacacca cccatttcct ccctctctgt ccctcctgcc 1920

atctccctgt gcctcttcat ctctggggtt ctctgtcttg tctccctctg cttataggtt 1980

gtgcctctgg tttgggggcc tctcagcctg tctgggtccc tcccttgctg tgcagttggc 2040

ctcgtggcct ctgctgctgt ttgtgcctct ctctgttact aacccgtcct ctcgctgtta 2100

gacatctctc tcactgcctg tctctggttc tgtcctcagg ccacccctgt tctccgatgt 2160

g 2161

<210> 7

<211> 2161

<212> DNA

<213> artificial sequence

<400> 7

gtgtgctgcc ggctgcacgg gccccaagca ctctgactgc ctggtatgtg cctctgcttt 60

gtgcccaatg tgctctaccc cccaggatgc aaggggtggg caccctgcct ggtactgccc 120

tattgcccct ggcacaccag ggcaaaacag cacagtgaaa gccagccacc tgtcccccca 180

ggcctgcctc cacttcaacc acagtggcat ctgtgagctg cactgcccag ccctggtcac 240

ctacaacaca gacacgtttg agtccatgcc caatcccgag ggccggtata cattcggcgc 300

cagctgtgtg actgcctgtc cctgatatcc aggaggtgca gggctacgtg ctcatcgctc 360

acaaccaagt gaggcaggtc ccactgcaga ggctgcggat tgtgcgaggc acccagctct 420

ttgaggacaa ctatgccctg gccgtgctag acaatggaga cccgctgaac aataccaccc 480

ctgtcacagg ggcctcccca ggaggcctgc gggagctgca gcttcgaagc ctcacaggtg 540

gccttcaccg tcattgaaac cttctcttgg ttattcagag ctgaccaggg ccactgctaa 600

ccagggggag gctttgtgtg cattagaaat ggtgtccctt ctgggcagac gcaggcagag 660

cccgggaaga cgccctcaga agattggaaa aagattcccc ttcttcctgg gaagttgtag 720

cttgcgtcag cacatataat tcaatcgtga gaatgcaggc tgggtttttg cccccacttg 780

gctgagtgaa gtgtacagtg aacaacctat gtaactattt gctggccctg gagccgactc 840

tgccccagag tctgggtgcc aggtgctttg cccgcatggc ccatttcagt cacgctgcag 900

tcctgtcagg aaaaaatcag tgttattctc attctacata tgagaaaact gaggcttgca 960

gatataaggg ccaaaagtta cacagctagt gagtgatggg gctgagtttc agactccaca 1020

gtctcttaac caccaagcag catgcccaga gtagaggtga gaaggaagga gagagctgcg 1080

gtccacatga gcatctggac ctagcatgga caactcactc ctccctggct ctcgctttgt 1140

tcttgttgcg ggtgtggtgg tggtgggact caaagacggt aaagatagct ttctctcctc 1200

cctggggaat ctgggggttg tttaaaaggc ctgctcctct tttagaaggc aggagggccc 1260

caagggaagc agaaggtgac agaaggggaa agggtcctct gatcattgct caccccacag 1320

agatcttgaa aggaggggtc ttgatccagc ggaaccccca gctctgctac caggacacga 1380

ttttgtggaa ggacatcttc cacaagaaca accagctggc tctcacactg atagacacca 1440

accgctctcg ggcctgtaag ccatgcccct ccctgctgcc tcttctctca gacagcctga 1500

ccccagccgc aaactcccaa cttacaaccc agtgcctgcc cgccactgcc ccagccgcct 1560

acaccaccca tttcctccct ctctgtccct cctgccatct ccctgtgcct cttcatctct 1620

ggggttctct gtcttgtctc cctctgctta taggttgtgc ctctggtttg ggggcctctc 1680

agcctgtctg ggtccctccc ttgctgtgca gttggcctcg tggcctctgc tgctgtttgt 1740

gcctctctct gttactaacc cgtcctctcg ctgttagaca tctctctcac tgcctgtctc 1800

tggttctgtc ctcaggccac ccctgttctc cgatgtgtaa gggctcccgc tgctggggag 1860

agagttctga ggattgtcag agccgtgagt ctcagggagg cctggagtca gggaagggga 1920

gggctggggc cgggtggaat gcaggtgtca tacaggtgac atgggagggg tgggataaca 1980

ggcttgggat gtctcccctg ggccaggtag tctccctaga aggtgatgct gatgagggtc 2040

tggtgcccag ggcgccactc agccctcatc ctgccctttg cccaacagtg acgcgcactg 2100

tctgtgccgg tggctgtgcc cgctgcaagg ggccactgcc cactgactgc tgccatgagc 2160

a 2161

<210> 8

<211> 2161

<212> DNA

<213> artificial sequence

<400> 8

agtggcatct gtgagctgca ctgcccagcc ctggtcacct acaacacaga cacgtttgag 60

tccatgccca atcccgaggg ccggtataca ttcggcgcca gctgtgtgac tgcctgtccc 120

tgatatccag gaggtgcagg gctacgtgct catcgctcac aaccaagtga ggcaggtccc 180

actgcagagg ctgcggattg tgcgaggcac ccagctcttt gaggacaact atgccctggc 240

cgtgctagac aatggagacc cgctgaacaa taccacccct gtcacagggg cctccccagg 300

aggcctgcgg gagctgcagc ttcgaagcct cacaggtggc cttcaccgtc attgaaacct 360

tctcttggtt attcagagct gaccagggcc actgctaacc agggggaggc tttgtgtgca 420

ttagaaatgg tgtcccttct gggcagacgc aggcagagcc cgggaagacg ccctcagaag 480

attggaaaaa gattcccctt cttcctggga agttgtagct tgcgtcagca catataattc 540

aatcgtgaga atgcaggctg ggtttttgcc cccacttggc tgagtgaagt gtacagtgaa 600

caacctatgt aactatttgc tggccctgga gccgactctg ccccagagtc tgggtgccag 660

gtgctttgcc cgcatggccc atttcagtca cgctgcagtc ctgtcaggaa aaaatcagtg 720

ttattctcat tctacatatg agaaaactga ggcttgcaga tataagggcc aaaagttaca 780

cagctagtga gtgatggggc tgagtttcag actccacagt ctcttaacca ccaagcagca 840

tgcccagagt agaggtgaga aggaaggaga gagctgcggt ccacatgagc atctggacct 900

agcatggaca actcactcct ccctggctct cgctttgttc ttgttgcggg tgtggtggtg 960

gtgggactca aagacggtaa agatagcttt ctctcctccc tggggaatct gggggttgtt 1020

taaaaggcct gctcctcttt tagaaggcag gagggcccca agggaagcag aaggtgacag 1080

aaggggaaag ggtcctctga tcattgctca ccccacagag atcttgaaag gaggggtctt 1140

gatccagcgg aacccccagc tctgctacca ggacacgatt ttgtggaagg acatcttcca 1200

caagaacaac cagctggctc tcacactgat agacaccaac cgctctcggg cctgtaagcc 1260

atgcccctcc ctgctgcctc ttctctcaga cagcctgacc ccagccgcaa actcccaact 1320

tacaacccag tgcctgcccg ccactgcccc agccgcctac accacccatt tcctccctct 1380

ctgtccctcc tgccatctcc ctgtgcctct tcatctctgg ggttctctgt cttgtctccc 1440

tctgcttata ggttgtgcct ctggtttggg ggcctctcag cctgtctggg tccctccctt 1500

gctgtgcagt tggcctcgtg gcctctgctg ctgtttgtgc ctctctctgt tactaacccg 1560

tcctctcgct gttagacatc tctctcactg cctgtctctg gttctgtcct caggccaccc 1620

ctgttctccg atgtgtaagg gctcccgctg ctggggagag agttctgagg attgtcagag 1680

ccgtgagtct cagggaggcc tggagtcagg gaaggggagg gctggggccg ggtggaatgc 1740

aggtgtcata caggtgacat gggaggggtg ggataacagg cttgggatgt ctcccctggg 1800

ccaggtagtc tccctagaag gtgatgctga tgagggtctg gtgcccaggg cgccactcag 1860

ccctcatcct gccctttgcc caacagtgac gcgcactgtc tgtgccggtg gctgtgcccg 1920

ctgcaagggg ccactgccca ctgactgctg ccatgagcag tgtgctgccg gctgcacggg 1980

ccccaagcac tctgactgcc tggtatgtgc ctctgctttg tgcccaatgt gctctacccc 2040

ccaggatgca aggggtgggc accctgcctg gtactgccct attgcccctg gcacaccagg 2100

gcaaaacagc acagtgaaag ccagccacct gtccccccag gcctgcctcc acttcaacca 2160

c 2161

<210> 9

<211> 2161

<212> DNA

<213> artificial sequence

<400> 9

tcgctttgtt cttgttgcgg gtgtggtggt ggtgggactc aaagacggta aagatagctt 60

tctctcctcc ctggggaatc tgggggttgt ttaaaaggcc tgctcctctt ttagaaggca 120

ggagggcccc aagggaagca gaaggtgaca gaaggggaaa gggtcctctg atcattgctc 180

accccacaga gatcttgaaa ggaggggtct tgatccagcg gaacccccag ctctgctacc 240

aggacacgat tttgtggaag gacatcttcc acaagaacaa ccagctggct ctcacactga 300

tagacaccaa ccgctctcgg gcctgtaagc catgcccctc cctgctgcct cttctctcag 360

acagcctgac cccagccgca aactcccaac ttacaaccca gtgcctgccc gccactgccc 420

cagccgccta caccacccat ttcctccctc tctgtccctc ctgccatctc cctgtgcctc 480

ttcatctctg gggttctctg tcttgtctcc ctctgcttat aggttgtgcc tctggtttgg 540

gggcctctca gcctgtctgg gtccctccct tgctgtgcag ttggcctcgt ggcctctgct 600

gctgtttgtg cctctctctg ttactaaccc gtcctctcgc tgttagacat ctctctcact 660

gcctgtctct ggttctgtcc tcaggccacc cctgttctcc gatgtgtaag ggctcccgct 720

gctggggaga gagttctgag gattgtcaga gccgtgagtc tcagggaggc ctggagtcag 780

ggaaggggag ggctggggcc gggtggaatg caggtgtcat acaggtgaca tgggaggggt 840

gggataacag gcttgggatg tctcccctgg gccaggtagt ctccctagaa ggtgatgctg 900

atgagggtct ggtgcccagg gcgccactca gccctcatcc tgccctttgc ccaacagtga 960

cgcgcactgt ctgtgccggt ggctgtgccc gctgcaaggg gccactgccc actgactgct 1020

gccatgagca gtgtgctgcc ggctgcacgg gccccaagca ctctgactgc ctggtatgtg 1080

cctctgcttt gtgcccaatg tgctctaccc cccaggatgc aaggggtggg caccctgcct 1140

ggtactgccc tattgcccct ggcacaccag ggcaaaacag cacagtgaaa gccagccacc 1200

tgtcccccca ggcctgcctc cacttcaacc acagtggcat ctgtgagctg cactgcccag 1260

ccctggtcac ctacaacaca gacacgtttg agtccatgcc caatcccgag ggccggtata 1320

cattcggcgc cagctgtgtg actgcctgtc cctgatatcc aggaggtgca gggctacgtg 1380

ctcatcgctc acaaccaagt gaggcaggtc ccactgcaga ggctgcggat tgtgcgaggc 1440

acccagctct ttgaggacaa ctatgccctg gccgtgctag acaatggaga cccgctgaac 1500

aataccaccc ctgtcacagg ggcctcccca ggaggcctgc gggagctgca gcttcgaagc 1560

ctcacaggtg gccttcaccg tcattgaaac cttctcttgg ttattcagag ctgaccaggg 1620

ccactgctaa ccagggggag gctttgtgtg cattagaaat ggtgtccctt ctgggcagac 1680

gcaggcagag cccgggaaga cgccctcaga agattggaaa aagattcccc ttcttcctgg 1740

gaagttgtag cttgcgtcag cacatataat tcaatcgtga gaatgcaggc tgggtttttg 1800

cccccacttg gctgagtgaa gtgtacagtg aacaacctat gtaactattt gctggccctg 1860

gagccgactc tgccccagag tctgggtgcc aggtgctttg cccgcatggc ccatttcagt 1920

cacgctgcag tcctgtcagg aaaaaatcag tgttattctc attctacata tgagaaaact 1980

gaggcttgca gatataaggg ccaaaagtta cacagctagt gagtgatggg gctgagtttc 2040

agactccaca gtctcttaac caccaagcag catgcccaga gtagaggtga gaaggaagga 2100

gagagctgcg gtccacatga gcatctggac ctagcatgga caactcactc ctccctggct 2160

c 2161

<210> 10

<211> 2161

<212> DNA

<213> artificial sequence

<400> 10

tctctgtctt gtctccctct gcttataggt tgtgcctctg gtttgggggc ctctcagcct 60

gtctgggtcc ctcccttgct gtgcagttgg cctcgtggcc tctgctgctg tttgtgcctc 120

tctctgttac taacccgtcc tctcgctgtt agacatctct ctcactgcct gtctctggtt 180

ctgtcctcag gccacccctg ttctccgatg tgtaagggct cccgctgctg gggagagagt 240

tctgaggatt gtcagagccg tgagtctcag ggaggcctgg agtcagggaa ggggagggct 300

ggggccgggt ggaatgcagg tgtcatacag gtgacatggg aggggtggga taacaggctt 360

gggatgtctc ccctgggcca ggtagtctcc ctagaaggtg atgctgatga gggtctggtg 420

cccagggcgc cactcagccc tcatcctgcc ctttgcccaa cagtgacgcg cactgtctgt 480

gccggtggct gtgcccgctg caaggggcca ctgcccactg actgctgcca tgagcagtgt 540

gctgccggct gcacgggccc caagcactct gactgcctgg tatgtgcctc tgctttgtgc 600

ccaatgtgct ctacccccca ggatgcaagg ggtgggcacc ctgcctggta ctgccctatt 660

gcccctggca caccagggca aaacagcaca gtgaaagcca gccacctgtc cccccaggcc 720

tgcctccact tcaaccacag tggcatctgt gagctgcact gcccagccct ggtcacctac 780

aacacagaca cgtttgagtc catgcccaat cccgagggcc ggtatacatt cggcgccagc 840

tgtgtgactg cctgtccctg atatccagga ggtgcagggc tacgtgctca tcgctcacaa 900

ccaagtgagg caggtcccac tgcagaggct gcggattgtg cgaggcaccc agctctttga 960

ggacaactat gccctggccg tgctagacaa tggagacccg ctgaacaata ccacccctgt 1020

cacaggggcc tccccaggag gcctgcggga gctgcagctt cgaagcctca caggtggcct 1080

tcaccgtcat tgaaaccttc tcttggttat tcagagctga ccagggccac tgctaaccag 1140

ggggaggctt tgtgtgcatt agaaatggtg tcccttctgg gcagacgcag gcagagcccg 1200

ggaagacgcc ctcagaagat tggaaaaaga ttccccttct tcctgggaag ttgtagcttg 1260

cgtcagcaca tataattcaa tcgtgagaat gcaggctggg tttttgcccc cacttggctg 1320

agtgaagtgt acagtgaaca acctatgtaa ctatttgctg gccctggagc cgactctgcc 1380

ccagagtctg ggtgccaggt gctttgcccg catggcccat ttcagtcacg ctgcagtcct 1440

gtcaggaaaa aatcagtgtt attctcattc tacatatgag aaaactgagg cttgcagata 1500

taagggccaa aagttacaca gctagtgagt gatggggctg agtttcagac tccacagtct 1560

cttaaccacc aagcagcatg cccagagtag aggtgagaag gaaggagaga gctgcggtcc 1620

acatgagcat ctggacctag catggacaac tcactcctcc ctggctctcg ctttgttctt 1680

gttgcgggtg tggtggtggt gggactcaaa gacggtaaag atagctttct ctcctccctg 1740

gggaatctgg gggttgttta aaaggcctgc tcctctttta gaaggcagga gggccccaag 1800

ggaagcagaa ggtgacagaa ggggaaaggg tcctctgatc attgctcacc ccacagagat 1860

cttgaaagga ggggtcttga tccagcggaa cccccagctc tgctaccagg acacgatttt 1920

gtggaaggac atcttccaca agaacaacca gctggctctc acactgatag acaccaaccg 1980

ctctcgggcc tgtaagccat gcccctccct gctgcctctt ctctcagaca gcctgacccc 2040

agccgcaaac tcccaactta caacccagtg cctgcccgcc actgccccag ccgcctacac 2100

cacccatttc ctccctctct gtccctcctg ccatctccct gtgcctcttc atctctgggg 2160

t 2161

<210> 11

<211> 2161

<212> DNA

<213> artificial sequence

<400> 11

gtctcccctg ggccaggtag tctccctaga aggtgatgct gatgagggtc tggtgcccag 60

ggcgccactc agccctcatc ctgccctttg cccaacagtg acgcgcactg tctgtgccgg 120

tggctgtgcc cgctgcaagg ggccactgcc cactgactgc tgccatgagc agtgtgctgc 180

cggctgcacg ggccccaagc actctgactg cctggtatgt gcctctgctt tgtgcccaat 240

gtgctctacc ccccaggatg caaggggtgg gcaccctgcc tggtactgcc ctattgcccc 300

tggcacacca gggcaaaaca gcacagtgaa agccagccac ctgtcccccc aggcctgcct 360

ccacttcaac cacagtggca tctgtgagct gcactgccca gccctggtca cctacaacac 420

agacacgttt gagtccatgc ccaatcccga gggccggtat acattcggcg ccagctgtgt 480

gactgcctgt ccctgatatc caggaggtgc agggctacgt gctcatcgct cacaaccaag 540

tgaggcaggt cccactgcag aggctgcgga ttgtgcgagg cacccagctc tttgaggaca 600

actatgccct ggccgtgcta gacaatggag acccgctgaa caataccacc cctgtcacag 660

gggcctcccc aggaggcctg cgggagctgc agcttcgaag cctcacaggt ggccttcacc 720

gtcattgaaa ccttctcttg gttattcaga gctgaccagg gccactgcta accaggggga 780

ggctttgtgt gcattagaaa tggtgtccct tctgggcaga cgcaggcaga gcccgggaag 840

acgccctcag aagattggaa aaagattccc cttcttcctg ggaagttgta gcttgcgtca 900

gcacatataa ttcaatcgtg agaatgcagg ctgggttttt gcccccactt ggctgagtga 960

agtgtacagt gaacaaccta tgtaactatt tgctggccct ggagccgact ctgccccaga 1020

gtctgggtgc caggtgcttt gcccgcatgg cccatttcag tcacgctgca gtcctgtcag 1080

gaaaaaatca gtgttattct cattctacat atgagaaaac tgaggcttgc agatataagg 1140

gccaaaagtt acacagctag tgagtgatgg ggctgagttt cagactccac agtctcttaa 1200

ccaccaagca gcatgcccag agtagaggtg agaaggaagg agagagctgc ggtccacatg 1260

agcatctgga cctagcatgg acaactcact cctccctggc tctcgctttg ttcttgttgc 1320

gggtgtggtg gtggtgggac tcaaagacgg taaagatagc tttctctcct ccctggggaa 1380

tctgggggtt gtttaaaagg cctgctcctc ttttagaagg caggagggcc ccaagggaag 1440

cagaaggtga cagaagggga aagggtcctc tgatcattgc tcaccccaca gagatcttga 1500

aaggaggggt cttgatccag cggaaccccc agctctgcta ccaggacacg attttgtgga 1560

aggacatctt ccacaagaac aaccagctgg ctctcacact gatagacacc aaccgctctc 1620

gggcctgtaa gccatgcccc tccctgctgc ctcttctctc agacagcctg accccagccg 1680

caaactccca acttacaacc cagtgcctgc ccgccactgc cccagccgcc tacaccaccc 1740

atttcctccc tctctgtccc tcctgccatc tccctgtgcc tcttcatctc tggggttctc 1800

tgtcttgtct ccctctgctt ataggttgtg cctctggttt gggggcctct cagcctgtct 1860

gggtccctcc cttgctgtgc agttggcctc gtggcctctg ctgctgtttg tgcctctctc 1920

tgttactaac ccgtcctctc gctgttagac atctctctca ctgcctgtct ctggttctgt 1980

cctcaggcca cccctgttct ccgatgtgta agggctcccg ctgctgggga gagagttctg 2040

aggattgtca gagccgtgag tctcagggag gcctggagtc agggaagggg agggctgggg 2100

ccgggtggaa tgcaggtgtc atacaggtga catgggaggg gtgggataac aggcttggga 2160

t 2161

<210> 12

<211> 2161

<212> DNA

<213> artificial sequence

<400> 12

cctattgccc ctggcacacc agggcaaaac agcacagtga aagccagcca cctgtccccc 60

caggcctgcc tccacttcaa ccacagtggc atctgtgagc tgcactgccc agccctggtc 120

acctacaaca cagacacgtt tgagtccatg cccaatcccg agggccggta tacattcggc 180

gccagctgtg tgactgcctg tccctgatat ccaggaggtg cagggctacg tgctcatcgc 240

tcacaaccaa gtgaggcagg tcccactgca gaggctgcgg attgtgcgag gcacccagct 300

ctttgaggac aactatgccc tggccgtgct agacaatgga gacccgctga acaataccac 360

ccctgtcaca ggggcctccc caggaggcct gcgggagctg cagcttcgaa gcctcacagg 420

tggccttcac cgtcattgaa accttctctt ggttattcag agctgaccag ggccactgct 480

aaccaggggg aggctttgtg tgcattagaa atggtgtccc ttctgggcag acgcaggcag 540

agcccgggaa gacgccctca gaagattgga aaaagattcc ccttcttcct gggaagttgt 600

agcttgcgtc agcacatata attcaatcgt gagaatgcag gctgggtttt tgcccccact 660

tggctgagtg aagtgtacag tgaacaacct atgtaactat ttgctggccc tggagccgac 720

tctgccccag agtctgggtg ccaggtgctt tgcccgcatg gcccatttca gtcacgctgc 780

agtcctgtca ggaaaaaatc agtgttattc tcattctaca tatgagaaaa ctgaggcttg 840

cagatataag ggccaaaagt tacacagcta gtgagtgatg gggctgagtt tcagactcca 900

cagtctctta accaccaagc agcatgccca gagtagaggt gagaaggaag gagagagctg 960

cggtccacat gagcatctgg acctagcatg gacaactcac tcctccctgg ctctcgcttt 1020

gttcttgttg cgggtgtggt ggtggtggga ctcaaagacg gtaaagatag ctttctctcc 1080

tccctgggga atctgggggt tgtttaaaag gcctgctcct cttttagaag gcaggagggc 1140

cccaagggaa gcagaaggtg acagaagggg aaagggtcct ctgatcattg ctcaccccac 1200

agagatcttg aaaggagggg tcttgatcca gcggaacccc cagctctgct accaggacac 1260

gattttgtgg aaggacatct tccacaagaa caaccagctg gctctcacac tgatagacac 1320

caaccgctct cgggcctgta agccatgccc ctccctgctg cctcttctct cagacagcct 1380

gaccccagcc gcaaactccc aacttacaac ccagtgcctg cccgccactg ccccagccgc 1440

ctacaccacc catttcctcc ctctctgtcc ctcctgccat ctccctgtgc ctcttcatct 1500

ctggggttct ctgtcttgtc tccctctgct tataggttgt gcctctggtt tgggggcctc 1560

tcagcctgtc tgggtccctc ccttgctgtg cagttggcct cgtggcctct gctgctgttt 1620

gtgcctctct ctgttactaa cccgtcctct cgctgttaga catctctctc actgcctgtc 1680

tctggttctg tcctcaggcc acccctgttc tccgatgtgt aagggctccc gctgctgggg 1740

agagagttct gaggattgtc agagccgtga gtctcaggga ggcctggagt cagggaaggg 1800

gagggctggg gccgggtgga atgcaggtgt catacaggtg acatgggagg ggtgggataa 1860

caggcttggg atgtctcccc tgggccaggt agtctcccta gaaggtgatg ctgatgaggg 1920

tctggtgccc agggcgccac tcagccctca tcctgccctt tgcccaacag tgacgcgcac 1980

tgtctgtgcc ggtggctgtg cccgctgcaa ggggccactg cccactgact gctgccatga 2040

gcagtgtgct gccggctgca cgggccccaa gcactctgac tgcctggtat gtgcctctgc 2100

tttgtgccca atgtgctcta ccccccagga tgcaaggggt gggcaccctg cctggtactg 2160

c 2161

<210> 13

<211> 1889

<212> DNA

<213> artificial sequence

<400> 13

ctcgagcagt tcttcagata gtagaacata cagccgaaag aaaggaggaa ggaaatcaag 60

atcaaagtca agatcttggt ccagagatct tcagcctcgt tcacattctt atgatagaag 120

gtgatttttg taatttttat ttatatagta atgaggataa cattctttaa aattcatgtt 180

tttgtatttt taattttgct agataaaaca tttttctttt actagctata tatttatgaa 240

agtcccaacc cagagttttt ttgttccacc aggtccagtc cgtgggtcta gtgtcctcaa 300

cctttgtctg ttcttagtag gcaaatagat agaaagtggt gtagtttcct tttttaattt 360

aatttaattt tttttctagt gcatttattt atttattttt ctaatgaatt attttttatt 420

ttattatact ttaagttcta gtacacaaca tgcaggtttg ttacatatgt atacatgtgc 480

catgttggtg tgctgcatct gttaactcgt catttacatt aggtgtatct cctaatgcta 540

tccctcccct agccccccac cccatgacag gccccagtgt gtgatgttcc ccttcctgtg 600

tccaagagtt ctcattgttc aattcccacc tatgaatgag aacatgcggt gtttggtttt 660

ttgtccttgc gatagtttgc tgagaatgat ggtttccagc ttcatctatg tgcctacaaa 720

ggatgtgaac tcatcctttt ttatggctgc gtagtattcc atggtgtata tgtgccacat 780

tttcttaatc cagtctgtca ttgatggaca tttgagttgg ttccaagcct ttgctattgt 840

gaacagtgct gcaacggagt tttgctcttg ttgtccaggc tggagtacaa tggcgtgatc 900

tcagctcact gcagcctctg cctcccaggt tcaagcagtt cttctcagcc ttccaagtag 960

ccgggattcc aggcatgcac cactatgccc agctagtttt gtatttttag tagagacagg 1020

gtttcaccat gttggtcagg ctggtcttga actcctgacc tcaggtgatc tgccctcctc 1080

agcctcccaa aatgttggga ttacaggcgt gagccaccgt gcctggcctt aattttaaat 1140

ttttttagag acaagatctt gctctgtcac tcaggcggga gtgcagcggt gcagtcatat 1200

cttttggact caagggatcc tcccacctca gcttcctgaa tagctagtac tagaggcatg 1260

tgccaccatg cctagctaag tttttttatt tttatttttt gtggagactt ggccttgctg 1320

tgttgcccaa gctggtatcg aattcttggc ctcaagcgat cctcctgcct cggcctccca 1380

acatgttggg attacaggca tgagctactg agcctggcct tggtttagtt ttcataccat 1440

ataatctgat gatatttgga aagattctaa gacccttaat accttaatta gtactgatcc 1500

aatcactaag ttatgaacca gtcatatgac tgttcaagag aaataacatt ttaatttatc 1560

tctgtttgct tccaggaaac agtgagacag taaaaatcag atgattcttt agaatctaga 1620

aggtttttcc ttaatgctca taataaaaat ttttatagca gtgatctttg attatatttt 1680

atttcagacg caggcatcga tcaagcagta gctcttctta tggctccaga aggaaacgaa 1740

gtcgaagtcg ttcaaggggt cgagggaaat cctatagagt tcagaggtct aggtcaaaaa 1800

gcagaacaag aagaaatggg acgtcggtca tcagatactg aagaagaaag cagaagcaag 1860

agaaaaaaga aacaccgtag acggtcctc 1889

<210> 14

<211> 1889

<212> DNA

<213> artificial sequence

<400> 14

ggtcgaggga aatcctatag agttcagagg tctaggtcaa aaagcagaac aagaagaaat 60

gggacgtcgg tcatcagata ctgaagaaga aagcagaagc aagagaaaaa agaaacaccg 120

tagacggtcc tcctcgagca gttcttcaga tagtagaaca tacagccgaa agaaaggagg 180

aaggaaatca agatcaaagt caagatcttg gtccagagat cttcagcctc gttcacattc 240

ttatgataga aggtgatttt tgtaattttt atttatatag taatgaggat aacattcttt 300

aaaattcatg tttttgtatt tttaattttg ctagataaaa catttttctt ttactagcta 360

tatatttatg aaagtcccaa cccagagttt ttttgttcca ccaggtccag tccgtgggtc 420

tagtgtcctc aacctttgtc tgttcttagt aggcaaatag atagaaagtg gtgtagtttc 480

cttttttaat ttaatttaat tttttttcta gtgcatttat ttatttattt ttctaatgaa 540

ttatttttta ttttattata ctttaagttc tagtacacaa catgcaggtt tgttacatat 600

gtatacatgt gccatgttgg tgtgctgcat ctgttaactc gtcatttaca ttaggtgtat 660

ctcctaatgc tatccctccc ctagcccccc accccatgac aggccccagt gtgtgatgtt 720

ccccttcctg tgtccaagag ttctcattgt tcaattccca cctatgaatg agaacatgcg 780

gtgtttggtt ttttgtcctt gcgatagttt gctgagaatg atggtttcca gcttcatcta 840

tgtgcctaca aaggatgtga actcatcctt ttttatggct gcgtagtatt ccatggtgta 900

tatgtgccac attttcttaa tccagtctgt cattgatgga catttgagtt ggttccaagc 960

ctttgctatt gtgaacagtg ctgcaacgga gttttgctct tgttgtccag gctggagtac 1020

aatggcgtga tctcagctca ctgcagcctc tgcctcccag gttcaagcag ttcttctcag 1080

ccttccaagt agccgggatt ccaggcatgc accactatgc ccagctagtt ttgtattttt 1140

agtagagaca gggtttcacc atgttggtca ggctggtctt gaactcctga cctcaggtga 1200

tctgccctcc tcagcctccc aaaatgttgg gattacaggc gtgagccacc gtgcctggcc 1260

ttaattttaa atttttttag agacaagatc ttgctctgtc actcaggcgg gagtgcagcg 1320

gtgcagtcat atcttttgga ctcaagggat cctcccacct cagcttcctg aatagctagt 1380

actagaggca tgtgccacca tgcctagcta agttttttta tttttatttt ttgtggagac 1440

ttggccttgc tgtgttgccc aagctggtat cgaattcttg gcctcaagcg atcctcctgc 1500

ctcggcctcc caacatgttg ggattacagg catgagctac tgagcctggc cttggtttag 1560

ttttcatacc atataatctg atgatatttg gaaagattct aagaccctta ataccttaat 1620

tagtactgat ccaatcacta agttatgaac cagtcatatg actgttcaag agaaataaca 1680

ttttaattta tctctgtttg cttccaggaa acagtgagac agtaaaaatc agatgattct 1740

ttagaatcta gaaggttttt ccttaatgct cataataaaa atttttatag cagtgatctt 1800

tgattatatt ttatttcaga cgcaggcatc gatcaagcag tagctcttct tatggctcca 1860

gaaggaaacg aagtcgaagt cgttcaagg 1889

<210> 15

<211> 1889

<212> DNA

<213> artificial sequence

<400> 15

cccacctatg aatgagaaca tgcggtgttt ggttttttgt ccttgcgata gtttgctgag 60

aatgatggtt tccagcttca tctatgtgcc tacaaaggat gtgaactcat ccttttttat 120

ggctgcgtag tattccatgg tgtatatgtg ccacattttc ttaatccagt ctgtcattga 180

tggacatttg agttggttcc aagcctttgc tattgtgaac agtgctgcaa cggagttttg 240

ctcttgttgt ccaggctgga gtacaatggc gtgatctcag ctcactgcag cctctgcctc 300

ccaggttcaa gcagttcttc tcagccttcc aagtagccgg gattccaggc atgcaccact 360

atgcccagct agttttgtat ttttagtaga gacagggttt caccatgttg gtcaggctgg 420

tcttgaactc ctgacctcag gtgatctgcc ctcctcagcc tcccaaaatg ttgggattac 480

aggcgtgagc caccgtgcct ggccttaatt ttaaattttt ttagagacaa gatcttgctc 540

tgtcactcag gcgggagtgc agcggtgcag tcatatcttt tggactcaag ggatcctccc 600

acctcagctt cctgaatagc tagtactaga ggcatgtgcc accatgccta gctaagtttt 660

tttattttta ttttttgtgg agacttggcc ttgctgtgtt gcccaagctg gtatcgaatt 720

cttggcctca agcgatcctc ctgcctcggc ctcccaacat gttgggatta caggcatgag 780

ctactgagcc tggccttggt ttagttttca taccatataa tctgatgata tttggaaaga 840

ttctaagacc cttaatacct taattagtac tgatccaatc actaagttat gaaccagtca 900

tatgactgtt caagagaaat aacattttaa tttatctctg tttgcttcca ggaaacagtg 960

agacagtaaa aatcagatga ttctttagaa tctagaaggt ttttccttaa tgctcataat 1020

aaaaattttt atagcagtga tctttgatta tattttattt cagacgcagg catcgatcaa 1080

gcagtagctc ttcttatggc tccagaagga aacgaagtcg aagtcgttca aggggtcgag 1140

ggaaatccta tagagttcag aggtctaggt caaaaagcag aacaagaaga aatgggacgt 1200

cggtcatcag atactgaaga agaaagcaga agcaagagaa aaaagaaaca ccgtagacgg 1260

tcctcctcga gcagttcttc agatagtaga acatacagcc gaaagaaagg aggaaggaaa 1320

tcaagatcaa agtcaagatc ttggtccaga gatcttcagc ctcgttcaca ttcttatgat 1380

agaaggtgat ttttgtaatt tttatttata tagtaatgag gataacattc tttaaaattc 1440

atgtttttgt atttttaatt ttgctagata aaacattttt cttttactag ctatatattt 1500

atgaaagtcc caacccagag tttttttgtt ccaccaggtc cagtccgtgg gtctagtgtc 1560

ctcaaccttt gtctgttctt agtaggcaaa tagatagaaa gtggtgtagt ttcctttttt 1620

aatttaattt aatttttttt ctagtgcatt tatttattta tttttctaat gaattatttt 1680

ttattttatt atactttaag ttctagtaca caacatgcag gtttgttaca tatgtataca 1740

tgtgccatgt tggtgtgctg catctgttaa ctcgtcattt acattaggtg tatctcctaa 1800

tgctatccct cccctagccc cccaccccat gacaggcccc agtgtgtgat gttccccttc 1860

ctgtgtccaa gagttctcat tgttcaatt 1889

<210> 16

<211> 985

<212> DNA

<213> artificial sequence

<400> 16

tggatggaaa atgaagaaga attcaacaga caagttagtt ttttgtacaa acatgttttt 60

atagtccatt ctggccctca atatgattgt acttgtccct gaaatgtgtt tatcttgtcc 120

tttatgtata aagtatgtag actgatgtgc caccactcct gtttatttgg ctgctgatta 180

ctaccaggct taactgctta cggaaataac ctggggagct tgtttcaggg tcccacccta 240

gagatgtctg aatcaggagg atgagggtgg agcctgggaa actttttcag agctctcaga 300

tggttccatt gagcagtcag atgtggaaac cactgcctta tgccagtaag attgctggtt 360

accgtgaaag tcaagagaat tcaggagtca ggagcagaca ggcacagaaa aaagagacag 420

gcatctcttt ttacttcttc cctgccaccc tcaatggaaa aaagaatgtt ctactaataa 480

aagtaaaaac atagaaaaac cctaaacaga tagatcttcg ccggtcatta tggtaagcat 540

tttttttcct tagcatggaa gcagaaaaga aagtatgact ttgtgccttt aaatggttta 600

gttcaagata ttttaagcca ttcctagtga tatctaccaa gtgtgctata aattatcctt 660

tattataaat ctagatagaa gaggagttat gggaagaaga atttattgaa cgctgtttcc 720

aagaaatgct ggaagaggaa gaagagcatg aatggtttat tccagctcga gatctcccac 780

aaactatgga ccaaatccaa gaccagttta atgaccttgt tatcagtgat ggctcttctc 840

tggaagatct tgtggttaaa aacgacaacc aacatcagcc atgaaagatc caagtcgcag 900

cagtactagc ccaagcatca tcaatgaaga tgtgattatt aacggtcatt ctcatgaaga 960

tgacaatcca tttgcagagt acatg 985

<210> 17

<211> 985

<212> DNA

<213> artificial sequence

<400> 17

aagagcatga atggtttatt ccagctcgag atctcccaca aactatggac caaatccaag 60

accagtttaa tgaccttgtt atcagtgatg gctcttctct ggaagatctt gtggttaaaa 120

acgacaacca acatcagcca tgaaagatcc aagtcgcagc agtactagcc caagcatcat 180

caatgaagat gtgattatta acggtcattc tcatgaagat gacaatccat ttgcagagta 240

catgtggatg gaaaatgaag aagaattcaa cagacaagtt agttttttgt acaaacatgt 300

ttttatagtc cattctggcc ctcaatatga ttgtacttgt ccctgaaatg tgtttatctt 360

gtcctttatg tataaagtat gtagactgat gtgccaccac tcctgtttat ttggctgctg 420

attactacca ggcttaactg cttacggaaa taacctgggg agcttgtttc agggtcccac 480

cctagagatg tctgaatcag gaggatgagg gtggagcctg ggaaactttt tcagagctct 540

cagatggttc cattgagcag tcagatgtgg aaaccactgc cttatgccag taagattgct 600

ggttaccgtg aaagtcaaga gaattcagga gtcaggagca gacaggcaca gaaaaaagag 660

acaggcatct ctttttactt cttccctgcc accctcaatg gaaaaaagaa tgttctacta 720

ataaaagtaa aaacatagaa aaaccctaaa cagatagatc ttcgccggtc attatggtaa 780

gcattttttt tccttagcat ggaagcagaa aagaaagtat gactttgtgc ctttaaatgg 840

tttagttcaa gatattttaa gccattccta gtgatatcta ccaagtgtgc tataaattat 900

cctttattat aaatctagat agaagaggag ttatgggaag aagaatttat tgaacgctgt 960

ttccaagaaa tgctggaaga ggaag 985

<210> 18

<211> 985

<212> DNA

<213> artificial sequence

<400> 18

gacaggcaca gaaaaaagag acaggcatct ctttttactt cttccctgcc accctcaatg 60

gaaaaaagaa tgttctacta ataaaagtaa aaacatagaa aaaccctaaa cagatagatc 120

ttcgccggtc attatggtaa gcattttttt tccttagcat ggaagcagaa aagaaagtat 180

gactttgtgc ctttaaatgg tttagttcaa gatattttaa gccattccta gtgatatcta 240

ccaagtgtgc tataaattat cctttattat aaatctagat agaagaggag ttatgggaag 300

aagaatttat tgaacgctgt ttccaagaaa tgctggaaga ggaagaagag catgaatggt 360

ttattccagc tcgagatctc ccacaaacta tggaccaaat ccaagaccag tttaatgacc 420

ttgttatcag tgatggctct tctctggaag atcttgtggt taaaaacgac aaccaacatc 480

agccatgaaa gatccaagtc gcagcagtac tagcccaagc atcatcaatg aagatgtgat 540

tattaacggt cattctcatg aagatgacaa tccatttgca gagtacatgt ggatggaaaa 600

tgaagaagaa ttcaacagac aagttagttt tttgtacaaa catgttttta tagtccattc 660

tggccctcaa tatgattgta cttgtccctg aaatgtgttt atcttgtcct ttatgtataa 720

agtatgtaga ctgatgtgcc accactcctg tttatttggc tgctgattac taccaggctt 780

aactgcttac ggaaataacc tggggagctt gtttcagggt cccaccctag agatgtctga 840

atcaggagga tgagggtgga gcctgggaaa ctttttcaga gctctcagat ggttccattg 900

agcagtcaga tgtggaaacc actgccttat gccagtaaga ttgctggtta ccgtgaaagt 960

caagagaatt caggagtcag gagca 985

<210> 19

<211> 22

<212> DNA

<213> artificial sequence

<400> 19

gacccgctga acaataccac cc 22

<210> 20

<211> 22

<212> DNA

<213> artificial sequence

<400> 20

tccattgtct agcacggcca gg 22

<210> 21

<211> 21

<212> DNA

<213> artificial sequence

<400> 21

acccccagct ctgctaccag g 21

<210> 22

<211> 22

<212> DNA

<213> artificial sequence

<400> 22

tccgctggat caagacccct cc 22

<210> 23

<211> 22

<212> DNA

<213> artificial sequence

<400> 23

taagggctcc cgctgctggg ga 22

<210> 24

<211> 22

<212> DNA

<213> artificial sequence

<400> 24

cacatcggag aacaggggtg gc 22

<210> 25

<211> 20

<212> DNA

<213> artificial sequence

<400> 25

tgtgctgccg gctgcacggg 20

<210> 26

<211> 21

<212> DNA

<213> artificial sequence

<400> 26

gctcatggca gcagtcagtg g 21

<210> 27

<211> 21

<212> DNA

<213> artificial sequence

<400> 27

gtggcatctg tgagctgcac t 21

<210> 28

<211> 21

<212> DNA

<213> artificial sequence

<400> 28

gtggttgaag tggaggcagg c 21

<210> 29

<211> 21

<212> DNA

<213> artificial sequence

<400> 29

tcgctttgtt cttgttgcgg g 21

<210> 30

<211> 23

<212> DNA

<213> artificial sequence

<400> 30

gagccaggga ggagtgagtt gtc 23

<210> 31

<211> 23

<212> DNA

<213> artificial sequence

<400> 31

ttataggttg tgcctctggt ttg 23

<210> 32

<211> 22

<212> DNA

<213> artificial sequence

<400> 32

gcagagggag acaagacaga ga 22

<210> 33

<211> 24

<212> DNA

<213> artificial sequence

<400> 33

gtctcccctg ggccaggtag tctc 24

<210> 34

<211> 24

<212> DNA

<213> artificial sequence

<400> 34

atcccaagcc tgttatccca cccc 24

<210> 35

<211> 21

<212> DNA

<213> artificial sequence

<400> 35

cctattgccc ctggcacacc a 21

<210> 36

<211> 20

<212> DNA

<213> artificial sequence

<400> 36

gcagtaccag gcagggtgcc 20

<210> 37

<211> 23

<212> DNA

<213> artificial sequence

<400> 37

ctcgagcagt tcttcagata gta 23

<210> 38

<211> 20

<212> DNA

<213> artificial sequence

<400> 38

gaggaccgtc tacggtgttt 20

<210> 39

<211> 21

<212> DNA

<213> artificial sequence

<400> 39

ctcttcttat ggctccagaa g 21

<210> 40

<211> 20

<212> DNA

<213> artificial sequence

<400> 40

ctactgcttg atcgatgcct 20

<210> 41

<211> 22

<212> DNA

<213> artificial sequence

<400> 41

agcttcatct atgtgcctac aa 22

<210> 42

<211> 20

<212> DNA

<213> artificial sequence

<400> 42

ggaaaccatc attctcagca 20

<210> 43

<211> 22

<212> DNA

<213> artificial sequence

<400> 43

tggatggaaa atgaagaaga at 22

<210> 44

<211> 22

<212> DNA

<213> artificial sequence

<400> 44

catgtactct gcaaatggat tg 22

<210> 45

<211> 22

<212> DNA

<213> artificial sequence

<400> 45

aagagcatga atggtttatt cc 22

<210> 46

<211> 21

<212> DNA

<213> artificial sequence

<400> 46

cttcctcttc cagcatttct t 21

<210> 47

<211> 21

<212> DNA

<213> artificial sequence

<400> 47

actcctgttt atttggctgc t 21

<210> 48

<211> 22

<212> DNA

<213> artificial sequence

<400> 48

ggtggcacat cagtctacat ac 22

Claims (6)

1. A method for detecting the full-length sequence of a circular RNA, comprising the steps of:

(1) Searching a DNA sequence of the target circular RNA in a database;

(2) Taking the middle of two adjacent nucleotides of the circular RNA corresponding to the exon and/or the intron of the DNA sequence as a disconnection point to disconnect into two pieces of DNA, and connecting the 3 '-end of one DNA sequence with the 5' -end of the other DNA sequence to form a new DNA sequence;

(3) Designing an upstream primer and a downstream primer for detecting the novel DNA full-length sequence obtained in the step (2);

(4) Verifying the upstream primer and the downstream primer designed in the step (3) to ensure that the upstream primer and the downstream primer cannot amplify linear RNA molecules to obtain a final primer pair;

(5) Performing PCR amplification on cDNA corresponding to the circular RNA by using the final primer in the step (4);

(6) Performing agarose electrophoresis detection and Sanger sequencing on the PCR product in the step (5) to obtain the full-length sequence of the circular RNA; the break point of the step (2) avoids the exon and intron shearing positions; the cleavage point is more than 20bp from the nearest exon and/or intron cleavage site; the length of the upstream primer and the downstream primer in the step (3) is 19-25 bp; the GC ratio of the upstream primer to the downstream primer in the step (3) is 40-60%; the total number of bases in which the hairpin and the dimer occur in the upstream primer and the downstream primer is less than or equal to 6; the Tm values of the upstream primer and the downstream primer in step (3) differ by less than 5 ℃.

2. The assay of claim 1, wherein the database in step (1) is the circBase database.

3. The assay method of claim 1, wherein the in silico PCR module for selecting UCSC is validated in step (4).

4. The detection method according to claim 1, wherein the PCR extension temperature in the step (5) is floated within 5℃above and below the Tm value.

5. The detection method of claim 1, wherein the PCR extension time in the step (5) depends on the length of the circular RNA, and if the circular RNA length is less than 1000bp, the extension time is 1 minute; if the length of the circular RNA is between 1000 and 2000bp, the extension time is 2 minutes.

6. The detection method of claim 1, wherein the number of reaction cycles of the PCR in the step (5) is more than 30cycles.