crRNA (Clustered Regularly Interspaced Short Palindromic Repeat-Derived Ribonucleic Acid) for specifically detecting exon mutation of human KRAS genes #2 and #3 based on CRISPR technique

An exon-specific technology, applied in the field of biological material detection, can solve the problems of low sensitivity, poor repeatability, and time-consuming detection, and achieve the effect of simplifying the operation process and improving the sensitivity

Inactive Publication Date: 2018-11-27
江苏博嘉生物医学科技有限公司
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AI Technical Summary

Problems solved by technology

[0003] At present, conventional gene detection is realized by PCR technology, but the existing PCR technology has some defects such as low sensitivity,

Method used

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  • crRNA (Clustered Regularly Interspaced Short Palindromic Repeat-Derived Ribonucleic Acid) for specifically detecting exon mutation of human KRAS genes #2 and #3 based on CRISPR technique

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Embodiment Construction

[0034] In order to describe the technical content, structural features, purpose and effect of the present invention in detail, the following examples are given to illustrate in detail.

[0035] A crRNA that specifically detects mutations in exons 2 and 3 of the human KRAS gene based on CRISPR technology, including the CRISPR-Cas13a system, and also includes a crRNA that can be combined with the CRISPR-Cas13a system. The crRNA sequence format is: 5`- Cas13a protein anchor sequence-crRNA guide sequence-3'; the Cas13a protein can be Lw Cas13a, and the Cas13a protein can also be Lsh Cas13a.

[0036] When the Cas13a protein is Lw Cas13a

[0037] The anchor sequence in the crRNA sequence is GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC The crRNA sequences are respectively

[0038] KRAS-EXON2

[0039] GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUUGCCUACGCCACCAGC UCCAACUA

[0040] KRAS-EXON3

[0041] GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUACUCCUCUUGACCUG CUGUGUCGA;

[0042] The deoxynucleic acid ...

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Abstract

The invention discloses crRNA (Clustered Regularly Interspaced Short Palindromic Repeat-Derived Ribonucleic Acid) for specifically detecting exon mutation of human KRAS genes #2 and #3 based on a CRISPR technique. The crRNA is characterized by comprising a CRISPR-Cas13a system as well as crRNA, wherein the crRNA can be combined with the CRISPR-Cas13a system; and the crRNA has a sequence format of5'-Cas13a protein anchoring sequence-crRNA guide sequence-3'. The crRNA has the advantages that according to detection results, whether the human KRAS gene have mutation or not can be judged intuitively through fluorescent reading, high-flux sequencing can be avoided, and the crRNA has the advantages of being low in cost, possible in multi-time detection, high in detection speed, possible in direct result analysis, and the like, and is applicable to large-scale clinical sample detection; the nucleic acid testing technique disclosed by the invention is different from a conventional detection technique which is based on PCR (Polymerase Chain Reaction) techniques, and no complex temperature control instrument or system is needed in a whole reaction process; and three steps of reactions are carried out in a same reaction system, so that operation procedures can be further simplified, and nucleic acid fragments with specific sequences can be detected within two hours.

Description

technical field [0001] The invention relates to the detection of biological materials, in particular to a method suitable for rapid detection of specific nucleic acid fragments in biological samples based on CRISPR-Cas13a technology. Background technique [0002] Researching and diagnosing diseases at the genetic level is the key to realizing the medical concept of individualized precision medicine. Kras is a murine sarcoma virus oncogene. There are three genes in the ras gene family related to human tumors—H-ras , K-ras and N-ras are located on chromosome 11, 12 and 1, respectively. K-ras is also known as the p21 gene because it encodes a 21kD ras protein. Among the ras genes, K-Ras has the greatest impact on human cancer. It is like a molecular switch: when normal, it can control the pathways that regulate cell growth; when abnormal, it causes cells to continue to grow and prevents cells from self-destruction. It participates in intracellular signal transmission. When th...

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2525/117C12Q2521/327
Inventor 程诚
Owner 江苏博嘉生物医学科技有限公司
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