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Method for Screening Gene Editing Products
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A technology for gene screening and gene editing, applied in biochemical equipment and methods, microbial assay/inspection, etc., can solve problems such as unfavorable practical applications, expensive instruments, and cumbersome identification of homozygous mutants, saving screening costs, simplifying The effect of the screening process
Active Publication Date: 2020-01-31
CHINA NAT RICE RES INST
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These methods have certain defects in practical application: PCR / RE detection method is only suitable for target sequences with restriction endonucleasecutting sites at editing sites, which limits the selection of target sequences; T7EI digestion method is suitable for identifying heterozygous Synzygous mutants, but the identification of homozygous mutants is relatively cumbersome, which is not conducive to practical application; the use of high-resolution melting curve analysis requires expensive instruments
These identification methods limit large-scale high-throughput screening of mutants generated after gene editing
At the same time, PCR / RE and T7EI enzymedigestion methods, which are mainly used to identify mixed cell lines obtained by gene editing methods, cannot accurately quantify the mutation efficiency in cell lines
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Embodiment 1-1
[0057] Embodiment 1-1, design and screen the ACT-PCR primer of OsPDS mutant
[0058] According to the principles of ACT-PCR primer design (such as figure 1 shown), and OsPDS-specific ACT-PCR primer sequences FA and RA were designed.
[0059] PDS-FA: 5'-TTGGTCTTTGCTCCTGCAGA-3'
[0060] PDS-RA: 5'-CTCCACTACAGACTGAGCACAAAGCTTC-3'
[0061] The source of OsPDS mutants can be found in published literature:
[0063] Example 1-2, determine the critical annealing temperature T of ACT-PCR by temperature gradient PCR amplification am
[0064] The primer pair PDS-FA and PDS-RA designed in Example 1-1 were used as ACT-PCR amplification primers, and rice Nipponbare genomic DNA was used as a template for PCR amplification. The reaction system is as follows:
[0065]
[0066] Reaction program: denaturation at 94°C for 2 minutes; then denaturation at 94°C for 30 seconds, annealing at a temperature gradient of 55°C-72°C for 30 seconds, extension at 72°C for 30 seconds, and 33 cycles of amplification; finally extension at 72°C for 2 minutes.
[0067] The results of electrophoresis of PCR products are shown in Figure 5 . according to Figure 5 , we know that: the maximum critical annealing temperature T am It is 64.8°C.
Embodiment 1-3
[0069] Embodiment 1-3, utilize ACT-PCR to screen OsPDS mutant
[0070] The genomic DNA of the sample to be tested was extracted according to the conventional CTAB method.
[0071] Using the primers designed in Example 1-1 as ACT-PCR amplification primers, transgenic T with OsPDS 0 Genomic DNA of strains from generation 1 to 22 was used as a template, and the critical temperature of 64.8° C. in Example 1-2 was used as annealing temperature for PCR amplification. The reaction system is as follows:
[0072]
[0073] Reaction program: denaturation at 94°C for 2 minutes; then denaturation at 94°C for 30 seconds, annealing at 64.8°C for 30 seconds, extension at 72°C for 30 seconds, and 33 cycles of amplification; finally extension at 72°C for 2 minutes.
[0074] The results of electrophoresis of PCR products are shown in the appendix Image 6 . Through ACT-PCR amplification, the OsPDS transgenic T 0 Mutants in generations 1-22 lines are: #1, #2, #3, #7, #8, #9, #10, #11, #12...
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Abstract
The invention discloses a method for screening gene editing products by ACT-PCR. The method comprises the following steps: (1) extracting genomeDNA of wild type to-be-detected sample; (2) design of primers: enabling a 3' terminal of a forward primer to stride across a gene editing quasi-cleavage site; taking a genome sequence of an objective gene as a reference of a reverse primer RA, and enabling the reverse primer RA to be positioned at a downstream of a complementary chain, wherein the Tm value of the reverse primer RA is not lower than that of the forward primer RA; (3) acquiring a maximum critical annealing temperature Tam and a minimum critical annealing temperature Tlm; (4) carrying out PCR amplification on FA / RA through a primer pair FA / RA by taking the genomeDNA as a template and setting annealing temperature to be Tlm< Tm< / = Tam; and (5) separating an amplified product through agarosegel electrophoresis. Samples corresponding to target strips which can be amplified are wild or hybridized mutants; and samples corresponding to target strips which cannot be amplified are homozygous mutants.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and application for screening gene editing products by using ACT-PCR. Background technique [0002] In recent years, gene editing systems such as ZFN, TALEN, CRISPR-Cas9, Cpf1, and Ago have been used to successfully realize genome editing in multiple species. These systems can finely modify specific DNA sequences in the genome, knock out or insert genes at specific positions. Genome editing using these systems generates a large number of mutants, and thus the effort to identify them is enormous. At present, there are mainly the following methods for screening gene editing mutants: PCR restriction (PCR / RE) detection method, T7EI restriction method, high-resolution melting curve analysis method, etc. These methods have certain defects in practical application: PCR / RE detection method is only suitable for target sequences with restriction endonucleasecutting site...
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