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Isothermal amplification and detection technique based on CRISPR-chain substitution

A technology of isothermal amplification and strand substitution, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., to achieve the effects of improving specificity, rapid response, and rapid recognition response

Pending Publication Date: 2018-09-28
武汉中科先进材料科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of specific amplification of target nucleic acid starting with CRISPR technology has not been reported yet

Method used

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  • Isothermal amplification and detection technique based on CRISPR-chain substitution
  • Isothermal amplification and detection technique based on CRISPR-chain substitution
  • Isothermal amplification and detection technique based on CRISPR-chain substitution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Using the strand replacement amplification reaction based on the CRISPR-Cas9 system to specifically amplify short target DNA double strands, and detect them by traditional PAGE technology

[0070] A pair of sgRNA was designed for a short DNA double-strand (450bp) and synthesized by in vitro transcription, which specifically recognizes the upstream CCAGTGCAAGTGCAGGTGCCAGA (SEQ ID NO:1) of the short DNA double-strand (the 5'end CCA is the complement of the PAM sequence NGG Sequence) and downstream GGCCCAGACTGAGCACGTGATGG (SEQ ID NO: 2) (where the 3'end TGG is the PAM sequence). The sgRNAs that recognize upstream and downstream sequences are named sgRNA-UPS and sgRNA-DNS, respectively.

[0071] sgRNA-UPS: GUGCAAGUGCAGGUGCCAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO: 3)

[0072] sgRNA-DNS: GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO: 4)

[0073] The strand re...

Embodiment 2

[0084] Example 2. Use the strand replacement amplification reaction based on the CRISPR-Cas9 system to specifically amplify short-term fragments of human genomic DNA, and detect them by traditional PAGE technology

[0085] First, use a commercial kit to extract genomic DNA from human embryonic kidney cells (HEK293). The extracted human genomic DNA was serially diluted to 2.5ng / μL. In the sample to be tested, the concentration of human genomic DNA is approximately 692.4 aM. In the test, a solution sample of plasmid DNA (pEGFP-C2) or mouse genomic DNA was used as a negative control. The concentration of plasmid DNA (pEGFP-C2) or mouse genomic DNA in the negative control was both 30ng / μL.

[0086] Using the same method as in Example 1, for a specific region in the human genome, sgRNA pairs that specifically recognize the upstream and downstream target DNA sequences of the region are designed and synthesized. In order to verify the reproducibility of the chain substitution amplifica...

Embodiment 3

[0111] Example 3. Using the strand replacement amplification reaction based on the CRISPR-Cas9 system to specifically amplify the short target DNA double-strands, and detect them by RT-PCR technology (real-time fluorescent PCR).

[0112] Using the same method as in Example 1, for a short DNA double strand (450 bp), the sgRNA in Example 1 was used to specifically amplify the short DNA double strand sgRNA-UPS and sgRNA-DNS.

[0113] The specific experimental steps are as follows:

[0114] 1. As in the steps 1 to 3 in Example 1, under the same conditions, prepare Cas9-sgRNA protein nucleic acid complexes with targeting activity respectively to complete the upper part of the Cas9-sgRNA protein nucleic acid complex and the short DNA double strands to be detected. Recognition reaction of downstream DNA target sequence and primer binding reaction.

[0115] 2. Add the optimized chain replacement isothermal amplification reaction enzymes in equal volume (final concentration: Klenow Fragmentexo...

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Abstract

The invention discloses an isothermal amplification and detection technique based on CRISPR-chain substitution. An isothermal amplification kit based on CRISPR-chain substitution comprises a chain-substitution isothermal amplification reagent and further comprises wild-type Cas9 and / or nuclease defective Cas9 nickase, sgRNA pairs on the upstream and the downstream of a specific identification target DNA sequence, and an amplification primer pair of a chain-substitution isothermal amplification reaction, and the primer pair and a Cas9-sgRNA complex are complementary to a single-chain region exposed after being combined with target genes. The technique is easy to operate, high in anti-jamming ability, and accurate and reliable in tested result.

Description

Technical field [0001] The invention relates to a nucleic acid amplification technology and a detection technology based on the technology, in particular to an isothermal amplification and detection technology based on CRISPR-strand substitution. Background technique [0002] Nucleic acid molecules are regarded as important biomarkers based on their biological properties. The detection of nucleic acid molecules has a wide range of applications in medical diagnosis and treatment, food safety, public health and social safety. Generally, during nucleic acid detection, the concentration of target nucleic acid molecules in the sample to be tested is extremely low, while the concentration of non-target nucleic acid molecules is relatively high. Therefore, specific amplification of target nucleic acid molecules in the sample to be detected is a common method to improve the sensitivity and accuracy of nucleic acid detection reactions. [0003] At present, PCR amplification technology spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2521/327C12Q2531/119
Inventor 喻学锋周文华胡丽
Owner 武汉中科先进材料科技有限公司
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