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Sequence-specific nuclear acid binding protein based nucleic acid detecting and ribotyping method and application thereof

A protein-binding and specific technology, applied in the field of biomedicine, can solve problems such as underutilization of value

Active Publication Date: 2018-06-01
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TALE has been fully developed for gene editing and gene regulation, but rarely reported for nucleic acid detection, and its value for nucleic acid detection has not been fully exploited

Method used

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  • Sequence-specific nuclear acid binding protein based nucleic acid detecting and ribotyping method and application thereof
  • Sequence-specific nuclear acid binding protein based nucleic acid detecting and ribotyping method and application thereof
  • Sequence-specific nuclear acid binding protein based nucleic acid detecting and ribotyping method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Detection of HPV18 L1 gene fragments by PABE method

[0068] CRISPR nuclease complex Cas9 / sgRNA is a sequence-specific DNA or RNA binding protein currently used for targeted binding to a specific nucleic acid sequence, so it can be used in the PABE detection of the present invention. The detection principle is as follows: figure 2 shown. In this example 1 and examples 2-7, the 3' end of the sgRNA is added with a sequence that can anneal and hybridize with the capture sequence immobilized on the surface of the microsphere to realize the PABE detection based on Cas9 / sgRNA ( Figure 4 ).

[0069] experimental method:

[0070] Selection of Cas9 / sgRNA binding sites on the HPV18 L1 gene: design two HPV18 L1 gene-specific sgRNA targets, the sequences of the two targets and their PAM sequences are: 5′-GCA TCA TAT TGC CCA GGT ACA GG- 3' (HPV18L1-1; Table 2) and 5'-AAA CCA AAT TTA TTT GGG TCA GG-3' (HPV18L1-2; Table 2). The distance between the two targets is 839bp...

Embodiment 2

[0079] Example 2 Quantitative detection of HPV18 L1 gene fragments by PABE method

[0080] experimental method:

[0081] Selection of the Cas9 / sgRNA binding site on the HPV18 L1 gene: the same as in Example 1.

[0082] Preparation of sgRNA by in vitro transcription method: same as Example 1.

[0083] Preparation of HPV L1 gene fragments by PCR: same as in Example 1.

[0084] Preparation of capture microspheres: same as Example 1.

[0085] Using dCas9 / sgRNA to combine HPV L1 gene fragments: recombinant dCas9 protein was purchased from GenScript (GeneScript, Nanjing). The dCas9 reaction (30 μL) consists of 1 × dCas9 reaction buffer (the components are the same as NEB Cas9 nuclease reaction buffer), 1 μM dCas9 (GeneScript), 300 nM sgRNA1 (HPV18L1-1; Table 2), 300 nM sgRNA (HPV18L1-2; Table 2 ), and 0.5 μL RNase inhibitor (Thermo). Incubate at room temperature for 5 minutes (this process is hereinafter referred to as pre-assembly). The above solution was mixed (32 μL) with d...

Embodiment 3

[0089] Example 3 Detection of target DNA fragments with different sgRNA target spacing by PABE method

[0090] experimental method:

[0091] Preparation of sgRNA by in vitro transcription method: sgRNA1 and sgRNA2 were prepared according to the operation method of Example 1. When preparing sgRNA1 against target DNA, use F1, R, F2, F3, and sgR1 oligonucleotides to perform three rounds of PCR amplification to prepare sgRNA1 transcription templates. When preparing sgRNA2 against target DNA, use F1, R, F2, F3, and sgR2 oligonucleotides for three rounds of PCR amplification to prepare sgRNA2 transcription templates. The PCR reaction system and reaction procedure are the same as in Example 1.

[0092] Target DNA fragment preparation: synthesize two free bases with two sgRNA target sequences and 10bp respectively. Among the primers used, the upstream primer is a fixed sequence with HPV18-E6-E7-1 at the 5′ end Target sequence (Ladder-F; Table 1); the downstream primer has the targe...

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Abstract

The invention discloses a sequence-specific nuclear acid binding protein based nucleic acid detecting and ribotyping method and application thereof. The method is characterized in that nucleic acid tobe detected is mixed with microspheres of which the surfaces are provided with sequence-specific nuclear acid binding protein, sequence-specific nuclear acid binding protein and microspheres, and then incubation is performed under room temperature; the microspheres are observed through a microscope tool, to realize the nucleic acid detecting and ribotyping. With the adoption of the method, even fm-level DNA can be quickly and simply without the complex, time-consumption and high-cost processes such as nucleic acid amplification and nucleic acid hybridization in traditional nucleic acid detection. According to the method, the properties of sequence-specific nuclear acid binding protein on specific recognizing and binding of nucleic acid molecules are utilized, so that the key bottleneck problems such as nucleic acid hybridization and amplification in the current fields of nucleic acid detecting and ribotyping are successfully avoided; the visual, digital and super-sensitive quick detection of nucleic acid is realized; and the method is extremely high in value of wide application in the field of nucleic acid detection.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for nucleic acid detection and typing based on a sequence-specific nucleic acid binding protein and an application thereof. 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. To sum up, there are three main types 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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2522/101C12Q2521/507C12Q2521/327C12Q2563/149
Inventor 王进科徐新慧
Owner SOUTHEAST UNIV
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