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DNA detection and analysis method based on Cas9 nuclease and application thereof

An analytical method, nuclease technology, used in the field of biomedicine to achieve high specificity and sensitivity

Active Publication Date: 2018-06-22
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the Cas9 / sgRNA system has not been directly used to detect and type genomic DNA, which is the main purpose of routine nucleic acid detection

Method used

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  • DNA detection and analysis method based on Cas9 nuclease and application thereof
  • DNA detection and analysis method based on Cas9 nuclease and application thereof
  • DNA detection and analysis method based on Cas9 nuclease and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Cutting HPV 16and 18L1 gene with Cas9 / sgRNA

[0049] experimental method:

[0050] Construction of sgRNA expression plasmid: first synthesize a pair of primers, use pCas9 (Addgene) as a template, and PCR amplify the prokaryotic Cas9 gene sequence, wherein the forward primer contains J23100 promoter plus RBS sequence (total 88bp). The PCR product was cloned into pCas9 plasmid with Cas9, trancRNA and spacer RNA sequences removed. The J23119-sgRNA sequence was amplified by PCR from pgRNA-bacteria (Addgene) and cloned into a freshly prepared pCas9 vector. The new plasmid was named pCas9-sgRNA, under the control of J23100 and J23119 promoters, pCas9-sgRNA can express Cas9 protein and sgRNA respectively. This plasmid also contains the chloramphenicol gene under the control of the cat promoter. Various sgRNA sequences cut by BsaI enzyme (annealed double-stranded oligonucleotides with BsaI sites at the ends) were cloned into pCas9-sgRNA, and Cas9 protein and sgrRNA...

Embodiment 2

[0055] Example 2 Cutting the HPV L1 gene cloned into the plasmid with Cas9 / sgRNA

[0056] experimental method:

[0057] Preparation of sgRNA: sgRNA was synthesized by in vitro transcription with T7 polymerase (New England Biolabs) according to the instructions. The sgRNA DNA template was amplified by three PCRs using the oligonucleotides listed in Table 1. The first PCR was performed with F1 and R (7 cycles). Use the product of the first PCR as a template, F2 and sgR as primers for the second PCR (30 cycles), use the product of the second PCR as a template, and F3 and sgR as primers for the third PCR (30 cycles) . 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 (NewEngland Biolabs) for in vitro transcription. The in vitro transcribed RNA was mixed with Trizol solution, extracted sequentially with chloroform and isopropanol, and precipitated with...

Embodiment 3

[0061] Embodiment 3 detects HPV 16 and 18L1 gene with ctPCR

[0062] experimental method:

[0063] Preparation of sgRNA: Same as Example 1.

[0064]Detection of HPV 16 and 18L1 genes cloned in plasmids by ctPCR: To prepare T-linkers, oligooJW102 and oJW103 (Table 3) were dissolved in Tris-HCl / EDTA / NaCl(TEN) buffer and mixed in the same molarity. The mixture was heated at 95 °C for 5 minutes and cooled slowly to room temperature. A pair of sgRNA specific to HPV16 and HPV18L1 genes combined with Cas9 protein cuts the plasmids of L1 gene clones of various HPV subtypes (200ng). The plasmid (200ng) was mixed with reaction buffer containing 1×Cas9 nuclease, 1μM Cas9 nuclease, 300nM sgRNA a (16-1274 or 18-1490; Table 2), 300nM sgRNA b (16-950 or 18-1274; Table 2 ) of the preassembled Cas9 / sgRNA complex and incubated at 37°C for 5 minutes. The digestion reaction (5 μL) was mixed with 5 μL premixed Taq (Takara) and incubated at 72 °C for 5 min plus A. Add A reaction solution (10 μ...

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Abstract

The invention provides a DNA detection and analysis method based on Cas9 nuclease and application thereof. The method comprises the following three steps: (1) carrying out PCR amplification on targetDNAs; (2) treating a PCR amplification product by using a CAT method; and (3) carrying out PCR amplification with DNAs treated by using the CAT method as a template. The method can successfully detectL1 and E6 / E7 genes of HPV16 and HPV18 in human cervical carcinoma cells. According to the invention, the method successfully avoids current key bottleneck problems in nucleic acid hybridization, designing of specific PCR primers and the like in the fields of nucleic acid detection and typing.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a Cas9 nuclease-based DNA detection and analysis 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 summary, there are three main categories of DNA testing and genotyping techniques 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 the recently developed digital PCR. Because of ob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/70
CPCC12Q1/686C12Q1/708C12Q2521/327
Inventor 王进科王巧
Owner SOUTHEAST UNIV
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