Application Of SSCP Technology In The Large-scale Screening Of Zebrafish Mutants

With the further study of genomics, the research of large-scale gene function becomes more and more popular, which gave birth to the rapid development of gene knock out. In recent years, a variety of gene knock out continue to emerge, from ZFNs to TALENs to CRISPR/Cas9 technology, makes the knock out easier and more efficient, especially the emergence of CRISPR/Cas9 technology and the rapid development of greatly simplifies the knock out operation process, reduce the difficulty of gene knock out.We use CRISPR/Cas9 technology to knock out the 10 genes of zebrafish on chromosome 1, although the CRISPR/Cas9 technology makes the knock out become easier, not all after microinjection into Cas9 mRNA and gRNA embryos could mutate. Therefore, we need to screen mutation carriers, heterozygous mutant and homozygous mutant from a large number of zebrafish. Sequencing is accurate but not economic. The purpose of this study is screening mutation from a large number of zebrafish by SSCP ((Single-Strand Conformation Polymorphism), then verify the mutation by sequencing furtherly.Firstly, we use the SSCP technology to screen the mutation carriers of 7 of 10 genes, the suspected mutation carriers which is detected by SSCP are real mutation carriers by sequencing. Then, we use SSCP technology to detect heterozygous mutants on 9 of 10 genes, we screened suspected heterozygous mutants by SSCP, which were verified by sequencing are real heterozygous mutants. Finally, we use the SSCP technology to screen homozygous mutants on 9 of 10 genes, except homozygous mutant of ZKO1130 gene not be screened, suspected homozygous mutants are detected on other genes, and they were verified by sequencing are real homozygous mutants. In the process of screening of homozygous mutants, we use the "twice-SSCP" technology to screen homozygous mutants furtherly which band type close WT’s, improving the rate of detecting of SSCP technology. At the same time, the traditional SSCP technology was improved, eliminating the steps of heating and ice cooling, simplify the experimental procedures. But we used the simplified SSCP technology to screen mutants of multiple genes, the result is the same as before.In summary, SSCP technology has a high accuracy rate and relevance ratio in screening of in mutation carriers, heterozygous mutants and homozygous mutants, which is suitable for large-scale screening of mutants. At the same time, simplify the experimental technology to further shorten the detection time, improve the detection efficiency.