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CRISPR (clustered regularly interspaced short palindromic repeat) based DNA detecting and typing method and application thereof

A typing method, DNA ligase technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., to achieve the effect of avoiding key bottleneck problems

Active Publication Date: 2018-03-23
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, among the reported Cas9-based DNA assays, the Cas9 / sgRNA system has not been directly used to detect and type genomic DNA

Method used

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  • CRISPR (clustered regularly interspaced short palindromic repeat) based DNA detecting and typing method and application thereof
  • CRISPR (clustered regularly interspaced short palindromic repeat) based DNA detecting and typing method and application thereof
  • CRISPR (clustered regularly interspaced short palindromic repeat) based DNA detecting and typing method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The schematic diagram of the principle and flow chart of CARP detection and typing of DNA molecules is as follows: figure 1 shown. CARP detection consists of three steps: (1) Cas9 endonuclease cleaves the target DNA. A pair of sgRNAs (sgRNA a and b) cleave the target DNA after binding to Cas9 nuclease; (2) the cleaved DNA is ligated by T4 DNA ligase (intermolecular and intramolecular ligation); (3) the ligated DNA is ligated by PCR Amplify. In the present invention, CARP is used to detect L1 and E6 / E7 genes of HPV16 and HPV18 to demonstrate the feasibility of CARP detection technology. In the PCR amplification step, conventional PCR (tPCR) and quantitative PCR (qPCR) were used, respectively.

[0040] The L1 and E6 / E7 genes of the target DNA HPV16 and HPV18 detected in each embodiment of the present invention, and the positions of reverse PCR primers and sgRNA designed for these genes are as follows: figure 2 shown. The indication figure 2 It is helpful to unders...

Embodiment 2

[0041] Example 2 Cutting HPV 16 and HPV 18 L1 genes with Cas9 / sgRNA

[0042] experimental method:

[0043]Preparation of sgRNA: According to the instructions of T7 polymerase (New England Biolabs), sgRNA was synthesized by in vitro transcription with T7 polymerase. The DNA template of the sgRNA was amplified by PCR three times using the oligonucleotides listed in Table 1 (SEQ ID NO.1-27). The first PCR was performed with F1 and R (7 cycles). Use the product of the first PCR as a template, and use F2 and sgR as primers for the second PCR (30 cycles); use the product of the second PCR as a template, and use F3 and sgR as primers for the third PCR (30 cycles). cycle). The purified product of the third PCR was used as a template for in vitro transcription. The purified sgRNA template was then incubated overnight at 37°C with T7 RNA polymerase (New England Biolabs) for in vitro transcription. The in vitro transcribed RNA was mixed with Trizol solution, extracted sequentially w...

Embodiment 3

[0053] Embodiment 3 detects HPV16 and 18 L1 gene with CARP

[0054] experimental method:

[0055] The preparation of sgRNA and HPV L1 gene fragment was the same as that in Example 1.

[0056] Cas9 / sgRNA cleavage of HPV16and 18 L1 gene: Recombinant Cas9 protein was purchased from New England Biolabs (NEB). Cas9 cleavage reaction (30 μL): 1× Cas9 nuclease reaction buffer, 1 μM Cas9 nuclease (NEB), 300 nM sgRNA a (16L1a1 or 18L1a1; Table 1) and 300 nM sgRNA b (16L1b1 or 18L1b1; Table 1). First, the Cas9 reaction solution was incubated at 25°C for 10 minutes. Then 200ng of HPV L1 gene fragment (substrate DNA) was added to the Cas9 reaction solution, and incubated at 37°C for 20 minutes. Finally, Cas9 was inactivated at 65 °C for 10 min. Electrophoresis was performed on 2% agarose gel.

[0057] Ligation reaction system (15 μL): 1×T4 ligase buffer, 5 U T4 DNA ligase (Thermo) and 5 μL Cas9 digested products of HPV16 and HPV18 L1 genes (as described above). Incubate at 22°C for ...

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Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeat) based DNA detecting and typing method and an application thereof. The method comprises following steps: (1),target DNA is subjected to PCR (polymerase chain reaction) amplification by a pair of universal primers; (2), the amplified target DNA is cut by Cas9 / sgRNA; (3), the cut target DNA is linked with DNAligase; (4), the linked target DNA is subjected to PCR amplification. According to the method, the target DNA can be subjected to specific detection and typing simply, rapidly and sensitively according to the specific identification cutting characteristics of the CRISPR technology on DNA, the method is one novel DNA detection method with high specificity and sensitivity, and the critical bottleneck problems about nucleic acid hybridization, specific PCR primer design and the like in current nucleic acid detection and typing fields are solved successfully. With the application of the method, L1 and E6 / E7 genes of HPV16 and HPV18 in human cervical cancer cells are detected successfully.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a CRISPR-based DNA detection and typing method and its application. Background technique [0002] DNA testing and genotyping have always been important for basic research, various testing and diagnostic applications. Therefore, DNA detection and genotyping technology has been widely concerned, thus promoting the development of this type of technology. In short, there are three main categories of DNA testing and genotyping technologies that are widely used. The first are various techniques based on the polymerase chain reaction (PCR). PCR is the most commonly used DNA detection and genotyping technique. PCR-based DNA detection and genotyping mainly rely on the design of specific primers and multiplex PCR amplification. PCR detection can be achieved by traditional PCR (tPCR), quantitative PCR (qPCR) and recently developed digital PCR. Because of obvious advantages, such as real-ti...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12Q1/70
CPCC12Q1/686C12Q1/708C12Q2521/327C12Q2521/501
Inventor 王进科张贝贝
Owner SOUTHEAST UNIV
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