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Method for absolutely quantifying number of DNA double-strand fractures in cells and application

A double-strand break, absolute quantitative technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as the inability to detect the number of DSBs, the restriction of the detection efficiency of DSBs, and the inability to get rid of the sequence dependence of the detection site, etc. Achieve high sensitivity and cost-effective, low cost, low time cost effect

Pending Publication Date: 2022-02-11
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, most of the existing studies using digital droplet PCR to detect DNA double-strand breaks are aimed at the gene editing efficiency of specific gene loci, but they cannot detect all the DSB numbers existing in the genome, nor can they get rid of the sequence dependence of the detection sites. This greatly restricts the detection efficiency of DSB

Method used

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  • Method for absolutely quantifying number of DNA double-strand fractures in cells and application
  • Method for absolutely quantifying number of DNA double-strand fractures in cells and application
  • Method for absolutely quantifying number of DNA double-strand fractures in cells and application

Examples

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Effect test

Embodiment 1

[0036] Example 1. TD-PCR Absolute Quantification of DNA Double-Strand Breaks Produced by the Chemical Drug Etoposide

[0037] (1) Materials

[0038] 1. Cell line: human myeloma cell line U2OS

[0039] 2. Exogenous nucleotide fragments: The nucleotide fragments used in the present invention are designed by the inventor, the forward strand and the reverse complementary strand are chemically synthesized by Qingke Company, and the inventors prepare them by annealing into double strands. Its detailed sequence information is as follows:

[0040] Sense strand sequence:

[0041] 5'-PHO-GCTCGCGTTTAATTGAGTTGTCATATGTTAATAACGGTATACGCGA-PHO-3' (SEQ ID No: 1, wherein "PHO" represents a phosphorylation modification);

[0042] Reverse Complementary Sequence:

[0043] 5'-TCGCGTATACCGTTATTAACATATGACAACTCAATTAAACGCGAGC*T-3' (SEQ ID No: 2, where "*" represents a phosphorothioate bond).

[0044] 3. Adapter: the adapter used in the present invention was designed by the inventor, the forward st...

Embodiment 2

[0080] Example 2. TD-PCR Absolute Quantification of Off-target Efficiency of Gene Editing Nuclease Cas9 and Its Variants

[0081] In the process of gene editing, nuclease Cas9 and its variants rely on it to cut DNA double strands to generate gaps, and rely on intracellular NHEJ and HR repair pathways to repair DNA double strand breaks, thereby editing the genome. During this process, the off-target effect of Cas9 nuclease will non-specifically cut genomic DNA, resulting in DNA double-strand breaks with unknown sequence information. Therefore, we quantify the off-target efficiency of different Cas9 nuclease variants through the sequence independence of TD-PCR.

[0082] (1) Materials

[0083] 1. Cell line: human kidney epithelial cell 293T

[0084] 2. Plasmids containing sequences of different Cas9 nuclease variants

[0085] The carrier is pX330 vector, the nuclease Cas9, espCas9, XCas9 are provided by the laboratory, the target gene locus is EMX1 (NC_000002.12), the sgRNA seq...

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Abstract

The invention discloses a method for absolutely quantifying the number of DNA double-strand fractures in cells and application. The method comprises the following steps: inserting an exogenous nucleotide fragment with terminal modification into a fixed cell genome DNA (Deoxyribonucleic Acid) double-chain fracture position, then fragmenting the genome and connecting the genome with an adapter, designing a specific PCR (Polymerase Chain Reaction) primer and a specific fluorescent probe aiming at the exogenous nucleotide fragment and the adapter, and constructing a digital droplet PCR system by combining a cell internal reference gene, and quantifying the number of DNA double-strand fractures in the cells. According to the invention, the detection of DNA double-strand fractures gets rid of sequence dependence, the method is widely suitable for absolutely and accurately quantifying the number of DNA double-strand fractures caused by various reasons such as animal and plant endogenous diseases, radiation, drugs or gene editing, and can be used as an effective tool for detecting problems such as ray damage, chemical drug damage, gene editing off-target efficiency and the like; the method has a very wide application prospect in molecular biology and clinical diagnosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting the number of DNA double-strand breaks (DSB) in cells based on digital droplet PCR and an application thereof. Background technique [0002] DNA double-strand break (DSB) is a common DNA damage existing in intracellular chromatin, which is characterized by the simultaneous breakage of two complementary strands in the DNA double helix structure at the same pair of complementary deoxynucleotides. The causes of DSB damage mainly come from three aspects: physical factors, chemical factors, and biological factors. Physical rays generated during tumor radiotherapy can directly break DNA double strands; certain biological metabolites and chemical reagents can also induce DSBs; biological gene editing technologies such as CRISPR / Cas9 can also non-specifically cut DNA to cause DSBs. Excessive accumulation of DSBs in the body will destroy the stability of genetic materi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6855
CPCC12Q1/6851C12Q1/6855C12Q2523/301C12Q2525/191C12Q2531/113C12Q2563/159C12Q2545/101C12Q2563/107
Inventor 牛佳浩王尧孙育杰
Owner PEKING UNIV
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