Preliminary Study On Allele Specificity Taqman Q-PCR For Human N-RAS

The epidermal growth factor receptor inhibitors cetuximab and panitumumab can effectively inhibit colorectal cancer,but the treatment of monoclonal antibodies is influenced by RAS gene,which is capable of activating EGFR signal pathway.EGFR monoclonal antibodies appear good effects on the treatment of patients with wild-type RAS gene.N-RAS mutations account for only about 2% of RAS mutations.The detection of N-RAS mutations was studied in this paper.Mutant and wild-type N-RAS plasmids were constructed by using 17 common mutation sites of N-RAS.After the plasmids were extracted,the band color was observed by electrophoresis and the color was bright.The plasmid concentration detected by ultraviolet-visible spectrophotometry was 40 ng/well.Ul.The sequencing confirmed that the template DNA sequence was consistent with the synthetic sequence.Next,primer Express 3.0 software was used to design wild-type,mutant primers and probes for the taqman quantitative PCR.After optimizing the PCR reaction system,the optimal reaction conditions were obtained.The final primer concentration was 0.4 μm/μL.The final concentration of the probe was 0.2 μmol/μL and the final concentration of the template DNA was 1 ng/μL,and a single-primer PCR system was established.After the single primer system was constructed,the specificity was verified by single primers.The Ct values of wild-type template fluorescence quantitative PCR of exon 12 of exon 2 were: 20.5456,20.0395,17.3253,and 17.5611.The Ct values of the wild-type template real-time PCR of the sub 13 codons were 26.2657,23.3040 and 26.7295.The Ct values of exon 61 wild-type template fluorescence quantitative PCR of codon 3 were 27.2140,26.9111,21.9652 and 20.6787.In the constructed multi-primer system,the Ct value of wild-type template fluorescence quantitative PCR of exon 12 and exon 2 of exon 2 was 18.027,and the Ct value of wild-type template fluorescence quantitative PCR of exon 3 of exon 61 For 30.4719.Using the wild-type Ct value as the standard,mutant fluorescence quantitative PCR can be performed to distinguish the difference of wild-type and mutant types Ct value.The multi-primer system was used to detect the sensitivity of 100%,10%,1%,0.1%,and 0 series concentration gradients of mutant DNA templates.The data showed that the detectable limit of mutation was less than 5%.Though a pair of primer real-time PCR detection system has been constructed,multi pairs of primers real-time PCR mutation detection system was further constructed.A pair of primer real-time fluorescence quantitative PCR system showed good results.The final primer concentration was 0.4 μmol/μL,a final probe concentration was 0.2 μmol/μL,and a final DNA concentration was 1 ng/μL.