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Preparation method of high-purity ssDNA

A high-purity, s1 enzyme technology, applied in the field of molecular biology, can solve problems such as mixing dsDNA, and achieve the effect of overcoming the problem of a large amount of residual dsDNA

Inactive Publication Date: 2021-06-01
通用生物(安徽)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods still have a large amount of dsDNA mixed in, and the present invention provides a method for removing a large amount of dsDNA. First, the ssDNA is obtained by digesting the phosphorylated single strand with lambda exonuclease, and then passing the double-strand specific enzyme. Remove residual double-stranded DNA, obtain high-purity ssDNA without dsDNA residue, and overcome the problem of a large amount of dsDNA residue

Method used

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  • Preparation method of high-purity ssDNA
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  • Preparation method of high-purity ssDNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] see Figure 1-3 Shown, a kind of high-purity ssDNA preparation method comprises the following steps:

[0031] Step 1: Synthesize the desired target gene into the puc57 vector;

[0032] The second step: synthesis of phosphorylated double-stranded DNA;

[0033] Step 3: Lambda exonuclease digests phosphorylated double-stranded DNA to obtain ssDNA;

[0034] Step 4: Remove residual double-stranded DNA by double-strand-specific enzymes to obtain high-purity ssDNA.

[0035] Preferably, the synthesis of the desired gene of interest specifically includes the following steps in the puc57 carrier:

[0036] Step 1: According to the required ssDNA sequence, the required DNA sequence is amplified by the principle of overlapping PCR to obtain the PCR product;

[0037] The second step: the puc57 vector plasmid is linearized by ECORV enzyme, and the pcr product is connected with the linearized vector to obtain the recombinant product;

[0038] Step 3: Transfer 9ul of the recombinan...

Embodiment 2

[0047] A method for preparing high-purity ssDNA, comprising the following steps:

[0048] Step 1: Synthesize the desired target gene into the puc57 vector;

[0049] The second step: synthesis of phosphorylated double-stranded DNA;

[0050] Step 3: Lambda exonuclease digests phosphorylated double-stranded DNA to obtain ssDNA;

[0051] Step 4: Remove residual double-stranded DNA by double-strand-specific enzymes to obtain high-purity ssDNA.

[0052] Preferably, the synthesis of the desired gene of interest specifically includes the following steps in the puc57 carrier:

[0053] Step 1: According to the required ssDNA sequence, the required DNA sequence is amplified by the principle of overlapping PCR to obtain the PCR product;

[0054] The second step: the puc57 vector plasmid is linearized by ECORV enzyme, and the pcr product is connected with the linearized vector to obtain the recombinant product;

[0055] Step 3: Transfer 10ul of the recombinant product to T1 competent c...

Embodiment 3

[0064] A method for preparing high-purity ssDNA, comprising the following steps:

[0065] Step 1: Synthesize the desired target gene into the puc57 vector;

[0066] The second step: synthesis of phosphorylated double-stranded DNA;

[0067] Step 3: Lambda exonuclease digests phosphorylated double-stranded DNA to obtain ssDNA;

[0068] Step 4: Remove residual double-stranded DNA by double-strand-specific enzymes to obtain high-purity ssDNA.

[0069] Preferably, the synthesis of the desired gene of interest specifically includes the following steps in the puc57 carrier:

[0070] Step 1: According to the required ssDNA sequence, the required DNA sequence is amplified by the principle of overlapping PCR to obtain the PCR product;

[0071] The second step: the puc57 vector plasmid is linearized by ECORV enzyme, and the pcr product is connected with the linearized vector to obtain the recombinant product;

[0072] Step 3: Transfer 11ul of the recombinant product to T1 competent c...

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Abstract

The invention discloses a preparation method of high-purity ssDNA. The preparation method comprises the following steps: 1, synthesizing a required target gene into a puc57 vector; 2, synthesizing phosphorylated double-stranded DNA; 3, digesting the phosphorylated double-stranded DNA by using l ambda exonuclease, so as to obtain ssDNA; and 4, removing the residual double-stranded DNA by using a double-stranded specific enzyme to obtain the high-purity ssDNA. The method comprises the following steps: firstly, obtaining ssDNA by using a method of digesting a phosphorylated single strand by using l ambda exonuclease, and then removing residual double-strand DNA by using a double-strand specific enzyme to obtain high-purity ssDNA without dsDNA residue, so that the problem of residue of a large amount of dsDNA is solved; and the method has the advantages of simplicity, convenience, high efficiency, high purity and no double-stranded DNA residue.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a method for preparing ssDNA, in particular to a method for preparing high-purity ssDNA. Background technique [0002] Gene editing is an emerging and more precise genetic engineering technology that can modify specific target genes in the genome of organisms. Recent developments in gene editing technology have transformed our ability to manipulate genomes. Editable site-specific nucleases, specifically the CRISPR / Cas system, introduce double-strand breaks at targeted genomic locations that can then be repaired by engineered mechanisms that select endogenous DNA. [0003] The CRISPR / Cas9 gene editing tool has been successfully applied to obtain gene knockout mutations (knockout), but many facts have proved that it is very difficult to apply to gene knockin. The main difficulty in gene knock-in is the preparation of the repair template and how to co-introduce it into cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/70
CPCC12N15/11C12N15/70
Inventor 雍金贵崔康乐王维坤骆晓雯张庆
Owner 通用生物(安徽)股份有限公司