Genetic Variation Detection Method Study Based On High-specific Taq Polymerase Variant

With the widespread use of CRISPR/Cas9 gene-editing technology,various technologies that can be used to detect gene editing efficiency have emerged,including Sanger sequencing,T7E1 nuclease/Surveyor,HRM,PCR-RFLP,ACT-PCR,and getPCR.Among them,getPCR technology can not only evaluate the efficiency of genome editing,but also can be used for the screening of genome editing single-cell clones.This technology uses detection primers that are complementary to the wild-type gene sequence and utilizes the sensitivity of Taq enzyme to primer/template complex mismatches to selectively amplify the wild-type sequence without amplifying the mutant gene template,based on fluorescence quantitative PCR to facilitate the detection and quantification of gene editing.It has the advantages of economic efficiency,simple and fast operation,high sensitivity and so on.In contrast,the existing commercial quantitative PCR products have low ability to distinguish gene variation,which limit the accuracy of gene editing detection.A Taq variant Taq388,which is highly sensitive to mismatch,was obtained by early member of the laboratory through molecular evolution strategy.On this basis,the application of Taq388 in genome editing efficiency detection and monoclonal screening,as well as its potential in single base gene mutation detection were evaluated,and a new genome editing detection method based on TaqMan probe was further developed.In getPCR analysis,both editing efficiency testing and screening of single cell clones containing indel,Taq388 showed excellent specificity,high ability to distinguish wild-type sequences from indel sequences,and is superior to a variety of existing commercial products.Furthermore,in order to improve the sensitivity of getPCR to indel sequence,we designed a special TaqMan probe,which is different from the conventional TaqMan probe and the distance above 2-4 bp between the same strand primer and the specially designed probe.The distance between the specially designed probe and the same strand primer is close to each other,and the distance is 0,so it is named aTaqMan probe.For DNA templates with indel,gene editing may not only cause mismatch between primers and templates,but also affect the annealing of TaqMan probes and template chains,and even cause spatial resistance effect between primers and probes,thus greatly improving the ability of getPCR to recognize genome editing.The results showed that aTaqMan could significantly improve the sensitivity of Taq to indel,and combined with Taq388 could further improve its ability to recognize genome editing.Furthermore,we evaluated the application potential of Taq388 in SNP genotyping and tumor mutation gene detection.At present,the techniques used for SNP genotyping include dideoxy chain termination,AS-PCR,NGS and so on.Digital PCR,BEAMing,Cold-PCR and other techniques are used for tumor mutation gene detection.These detection techniques are complex to operate,high cost,or are limited by the ability of PCR reaction to distinguish single bases.The results of genotyping analysis of five SNP loci indicated that Taq388 showed excellent ability to distinguish single base variation,whether it was PCR end point fluorescence method or quantitative calculation method based on Ct value,which greatly improved the accuracy and ease of operation of SNP genotyping analysis.In the tumor mutation gene detection,the detection of two common cancer-related gene mutations,TP53 c.818G>A and TP53 c.853G>A,shows that the sensitivity of Taq388 polymerase in detecting simulative ctDNA in PCR is close to 0.01%.