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Method for deriving mulriple DNA sequences from atom destructurizing

A technology of DNA sequence and diversity, applied in the field of gene mutagenesis research, can solve problems such as low degree of mutation, multiple types of mutation, and carcinogenic risk of chemical mutagens, achieving high biological safety, multiple types, and high tendency Effect

Inactive Publication Date: 2007-02-21
HEBEI ACADEMY OF AGRI & FORESTRY SCI INST OF GENETICS & PHYSIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of using chemical mutagens, mutagenic strains, and error-prone PCR for mutagenesis is that there are many types of mutations, but the disadvantage is that chemical mutagens have the risk of carcinogenicity, and the degree of mutation is not high when using mutagenic strains and error-prone PCR.
The use of genetic recombination is limited by human design and operational capabilities

Method used

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  • Method for deriving mulriple DNA sequences from atom destructurizing
  • Method for deriving mulriple DNA sequences from atom destructurizing
  • Method for deriving mulriple DNA sequences from atom destructurizing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Using the onion Ace-AMP1 gene (357bp) as a template to introduce α- 32 P-dATP

[0031] ①Primer design: The onion Ace-AMP1 gene was cloned on the pUC19 plasmid vector, and PCR primers were designed according to its sequence—the upstream primer was 5’-TAA GGT A CC ATG G TTCGC GTT GTA TC-3' (including restriction site NcoI), the downstream primer is 5'-TCC TGCCGC ATT GAA TTC TTG G-3' (including restriction enzyme site EcoRI), the theoretically amplified DNA fragment length is 357bp;

[0032] ②α- 32 The introduction of P-dATP (i.e. the first round of PCR): with the source DNA in step ① as a template, α- 32 P-dATP makes α- 32P-dATP: normal dATP=1:15, 35 cycles at 94°C for 1 min, 55°C for 0.5 min, and 72°C for 1 min to complete the first round of PCR reaction;

[0033] ③Radioactive decay: place the PCR amplification product in step ② at -20°C to decay for 7 half-lives;

[0034] ④The second round of PCR: use the decayed PCR product in step ③ as the template...

Embodiment 2

[0037] Embodiment 2: Using the specific DNA sequence (301bp) of wheat as a template, the introduction of α- 32 P-dATP

[0038] ①Primer design: clone a specific DNA sequence from wheat into the pGEM-T plasmid vector, and design PCR primers according to its sequence—forward primer E-AG is 5'-CCCGGG CAG GTA ATT CAG-3', complementary The strand primer M-CG is 5'-TCG CGG CCG AGG TTAACA-3', the theoretically amplified DNA fragment length is 301bp;

[0039] ②α- 32 The introduction of P-dATP (i.e. the first round of PCR): with the source DNA in step ① as a template, α- 32 P-dATP makes α- 32 P-dATP: normal dATP=1:15, 35 cycles at 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min to complete the first round of PCR reaction;

[0040] ③Radioactive decay: place the PCR amplification product in step ② at -20°C to decay for 7 half-lives;

[0041] ④The second round of PCR: use the decayed PCR product in step ③ as the template, and use the normal dNTP as the reaction substrate to perform...

Embodiment 3

[0044] Example 3: Using the specific DNA sequence (301bp) of wheat as a template to introduce α- 32 P-dCTP

[0045] What is incorporated in the reaction substrate dNTP in step ② is α- 32 For P-dCTP, other steps are the same as in Example 2; step ⑥ sequencing and analysis: 10 clones in step ⑤ are randomly selected for sequencing analysis by Songbao Biological Company. The results showed that: 9 of the 10 clones had mismatches at expected positions, of which: 1 had mismatches at 5 positions; 1 had mismatches at 4 positions; 2 clones had mismatches at 2 different positions Mismatches and deletions of 1 base; 5 had 1 mismatch at a different position. Controls have no mismatches at any position. The positions where replication mismatch occurs are bases C and G, and the rule of mismatch is that C is mismatched to T, and G is mismatched to A, that is, base conversions of G→A and C→T occur (see Sequence Table 3).

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Abstract

This invention relates to a method for deriving polymorphorous DNA sequences through atomic allosterism. The method comprises: designing primers, performing PCR to introduce dNTP having 32P or 35S, performing radioactive decay, performing second PCR, conducting DNA cloning, sequencing and analyzing. The method can derive large numbers of novel DNA sequences with multiple sites mutation. The mutated sites are controllable through adjusting the category of alpha site-specific dNTP, by which the contents of guanine and cytosine can also be controllably changed. The method has such advantages as high mutagenic effect, high directional tendency, high biosafety and no environmental damage. The method can be used to mutate any gene.

Description

technical field [0001] The invention relates to a method for deriving DNA sequence diversity by using atomic allosterism, which belongs to the field of gene mutagenesis research. Background technique [0002] Why "If you sow melons, you will reap melons, and if you sow beans, you will reap beans"? The scientific discoveries of the last century have given a relatively satisfactory answer - the traits of organisms are controlled by genes; a gene is a specific sequence of DNA; this DNA is composed of four nucleotides (ie A, T, G , C) arranged in a certain order; as long as the order of A, T, G, and C in the DNA is changed, the gene can be changed, so that the melons grown are no longer the original melons, and the beans grown are no longer the original ones. beans. [0003] Over the past decade, genes have become a highly commercially competitive resource. In the 1990s, Monsanto's cotton seeds entered the Chinese market, relying on a specific DNA sequence isolated from Bacil...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12N15/11C12N15/10
Inventor 王海波赵和吕孟雨柴建芳吴志明马民强张春义
Owner HEBEI ACADEMY OF AGRI & FORESTRY SCI INST OF GENETICS & PHYSIOLOGY
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