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Method for detecting full-length sequence of circular RNA

A detection method and DNA sequence technology are applied in the field of circular RNA detection to achieve the effect of quick and easy detection

Pending Publication Date: 2020-02-28
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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Problems solved by technology

At present, few scholars have paid attention to this problem, and they can only rely on the form of the cut length of the circular RNA or the length of the genome in the data.

Method used

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  • Method for detecting full-length sequence of circular RNA
  • Method for detecting full-length sequence of circular RNA
  • Method for detecting full-length sequence of circular RNA

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Embodiment 1

[0044] 1. Circular RNA primer design principle:

[0045] like figure 1As shown, if the primers directed at several bases of the sequence at the joint of the circular RNA are conventionally designed, only the sequence at the joint can be amplified due to PCR amplification. But for circular RNA, the circular RNA can form alternative cuts during the formation process, subtract or retain the intron, such primers can only detect the sequence at the junction, but cannot detect the circular RNA full-length sequence. In summary, according to the characteristics of circular RNAs, we simply and quickly designed primers for different alternative splicing modes of circular RNAs and capable of detecting the full-length circular sequences.

[0046] 2. Circular RNA primer design scheme of the present invention:

[0047] (1) Take hsa_circ_0007766, hsa_circ_0001355 and hsa_circ_0001538 as examples respectively. Find the genomic DNA sequence of the circular RNA in circBase (where English up...

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Abstract

The invention discloses a detection method of a full-length sequence of circular RNA, which comprises the steps of: (1) searching for a DNA sequence of a target circular RNA in a database; (2) randomly taking the middle of two nucleotides, corresponding to exons and / or intron regions of the DNA sequence, of the circular RNA as a disconnection point to disconnect the circular RNA into two DNAs, andconnecting the 3' end of one DNA sequence with the 5' end of the other sequence to form a new DNA sequence; (3) designing upstream and downstream primers 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) carryingout PCR (polymerase chain reaction) amplification on cDNA corresponding to the circular RNA via the final prime pair; and (6) carrying out 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 is suitable for acquiring the full-length sequence of the circular RNA more easily and simply.

Description

technical field [0001] The invention relates to the technical field of circular RNA detection, in particular to a method for detecting the full-length sequence of circular RNA, in particular to a method for detecting the full-length sequence of circular RNA without RNase R. Background technique [0002] Circular RNA (circular RNA, circRNA) is a single-stranded non-coding RNA that is different from linear RNA, has no 3' and 5' nucleotide sticky ends, no poly (A) tail, and can form a closed circular structure. RNA. [0003] The current technical means for detecting circular RNA mainly relies on the start and end positions of the circular RNA, that is, the sequence of the junction site (junction site) to judge whether the RNA is a circular RNA, but this method can only obtain the nucleosides near the junction. Therefore, it is impossible to determine the sequence between the start position and the end position of the circular RNA. Moreover, splicing events can occur during th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/101C12Q2531/113C12Q2525/191
Inventor 龚畅刘子豪梁格豪
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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