A method for rapid identification of genome-wide gene function in microorganisms
A genome-wide, gene-function technology, applied in the field of rapid identification of microbial genome-wide gene functions, can solve problems such as inability to effectively answer, inability to identify at the genome-wide level, and low efficiency.
Active Publication Date: 2016-05-04
SHANGHAI HANYU BIOLOGICAL SCI & TECH CO LTD
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Problems solved by technology
The disadvantage of this method is that only one mutant gene can be found at a time, and it cannot be identified at the whole genome level, which consumes manpower, material resources and time, and the efficiency is low
But even pathogenic microorganisms encode at least thousands of genes. What are the functions of these genes and which genes are necessary for the reproduction, growth, transmission and infection of microorganisms? These questions cannot be effectively answered only by analyzing the genome structure and sequence
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preparation example Construction
[0028] (1) Preparation steps of mixed DNA
[0029] Using pathogenic microbial strains as carriers, using the characteristics of Tn transposons that can be randomly inserted into the microbial genome, cultivate a batch of bacterial mutants with different insertion densities, and release the DNA of all mutant strains after reaching a certain growth curve. At this time , the released DNA is the mixed DNA of various mutant strains;
[0030] (2) Synthetic steps of specific primers
[0031] The mixed DNA obtained in step (1) is fragmented, and the fragmentation process is to take a DNA fragment with a length of 300-800bp protruding from the end; then connect all the fragmented DNA to a DNA sticky-end adapter, and the DNA sticky-end The 5' end of the linker is a Y-shaped bifurcation, and its sequence formulas are:
[0032] 5-ACACTCTATCGCTACACGACGCTCTTCCCTAC*T-3;
[0033] 5-GTAGGGAAGAGCTCGTATGCCCTCTTCTGATTG-3;
[0034] After different DNA sticky-end adapters are connected to both ...
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The invention discloses a method for quickly identifying the gene function of microbial whole genome, which comprises the following steps: (1) preparation of mixed DNA (deoxyribonucleic acid); (2) synthesis of a specific primer; (3) amplification of the specific primer; (4) insertion of a sequencing primer; (5) sequencing according to the follow-up steps of conventional high-throughput sequencing. According to bioinformation analysis, the density of the obtained mutants is that one transposon is inserted into every 17 basic groups on average, and the insertion density can cover all coding genes in the whole genome, thereby proving that the method disclosed by the invention can be used for identifying the gene function from the aspect of whole genome very quickly and effectively.
Description
technical field [0001] The invention relates to a method for identifying the gene function of the whole genome in the field of biotechnology, in particular to a method for rapidly identifying the gene function of the whole genome of microorganisms. Background technique [0002] The traditional method of identifying gene function is to find a mutant with loss of function of a single gene through forward genetics, and then use molecular genetics to find the region where the mutant gene is located. The disadvantage of this method is that only one mutant gene can be found at a time, and it cannot be identified at the genome-wide level. It is relatively labor-intensive, material and time-consuming, and the efficiency is low. [0003] At present, with the advancement of high-throughput sequencing technology, the industry can already use high-throughput sequencing technology to completely analyze the structure of genomes, especially small genomes, such as pathogenic microorganism g...
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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2525/191C12Q2531/113
Inventor 何玮柴燕群
Owner SHANGHAI HANYU BIOLOGICAL SCI & TECH CO LTD