Application Of Mutation-sensitive Molecular Switch In The Rapid Screening Of Breast Cancer PIK3CA Gene Mutations

Objective: To investigate the effects of the high-fidelity DNA polymerase mediatedmutation-sensitive molecular switch on the rapid screening the breast cancer PIK3CA genemutation, and analysis the relationship between the PIK3CA mutation and the breastcancer.Methods: Tumor tissue samples were obtained from90primary breast cancerpatients who underwent mastectomy surgery during the period from2008to2011at thesecond affiliated hospital of Soochow University, another10breast fibroadenoma and10adjacent breast tissue around lesions(>5cm) were selected as a control. The G1633A（E545K）mutation of the exon9and the A3140G(H1047R) mutation of the exon20weredetected with the action of mutation-sensitive molecular switch. First, the A3140G(H1047R) mutation was selected as the detection target to verify the value ofmutation-sensitive molecular switch in the mutation detection. The experimental design isas the follows:①Genomic DNA was extracted from healthy people and breast tissue andquantitatived;②With normal DNA as template, site-directed mutagenesis is accomplishedto obtain a fragment of DNA contained3140mutation points of the PIK3CA gene(base isG);③With normal DNA as template, PCR amplification is accomplished to obtain afragment of DNA contained3140wild points of the PIK3CA gene(base is A);④mutatedand wild DNA fragment would be inserted into an expression plasmid, and the plasmidwas transformed into competent bacteria to obtain the mutation and wild plasmid astemplate which was quantitatived and verified;⑤a llelic specific primerstargetingmutation type and wild type were designed with the primers’3’terminal phosphorothioatemodification and PCR amplification was done separately to test the high-fidelity DNApolymerase and3’phosphorothioate modified primers’ sensitivity and specificity;⑥Thesame PCR reaction volume and condition were used to detect the tissue genomic DNA.The amplified PCR products were confirmed by agarose gel Electrophoresis. Second, the G1633A (E545K) mutation was detected using the verified mutation-sensitive molecularswitch. Last, we analysis the relationship between the PIK3CA mutation and the breastcancer.Results: The quantitatived mutation template and wild type template were dilutedfrom10~9、10~8、10~7、10~6、10~5to104copies/μl to test the high-fidelity DNA polymerase and3’phosphorothioate modified primers’ sensitivity and specificity. The results revealed thatthe mutation-sensitive molecular switch had better sensitivity and specificity when theconcentration of the template lower than10~6copies/μl,so it can be used to detected thetissue genomic DNA(104to105copies/μl). The PCR results were consistent with theresults of DNA sequencing, which revealed that the molecular switch can be used for thedetection of the somatic gene mutations.With the action of mutation-sensitive molecular switch,18(20％)PIK3CA genemutations were found in these90cases of breast cancer, which contained14cases ofA3140G mutation(15.6％),3cases of G1633A mutation(3.3％) and2cases of G1624Amutation found additionally(2.2％). One breast cancers possessed two distinct PIK3CAmutations (A3140G with G1624A).The mutation of PIK3CA gene significantly associateswith c-erbB-2negativity,but it had no significant correlation with the age, tumor size,lymph node status, ER/PR status and EGFR expression. No mutation was found in thebreast fibroadenoma and adjacent breast tissue.Conclusion: The mutation-sensitive molecular switch can screen the PIK3CA genemutation rapidly and accurately.18(20％)PIK3CA gene mutations were found in these90cases of breast cancer and no mutation was found in the breast fibroadenoma and adjacentbreast tissue. The mutation of PIK3CA gene significantly associates with c-erbB-2negativity, but it had no significant correlation with the age, tumor size, lymph node status,ER/PR status and EGFR expression.