Development Of An Assay For Carcinogenic K-ras Hot Spot Mutations And Its Application In Circulating DNA

Objective:To establish a PCR method for the detection of seven hot spot mutations in K-rasgene by exonuclease polymerase mediated mutation sensitive on/off switch combinedwith real time PCR. With the use the multiplex PCR and single PCR, seven hot spotmutations can been dectected by melting curve and amplification curve. Whether thesemutations exist or not can be used to predict the tumor occurrence, to guide the rationaltherapy of colorectal cancer or other cancer patients, and analysis of prognostic.Methods:The PCR products of wild K-ras gene were inserted into the PGEM-T vector fortransformation into E.coli JM109competent cells for preparing wild type mtDNAvector, By overlapping extension site-directed mutagenesis techniques, seven types ofPCR products harboring different muataions were obtained. These PCR products werethen inserted into the PGEM-T vector for transformation into E.coli JM109competentscells for preparing the relevent seven types of mutation templates These newlyconstructed vectors were confirmed by DNA sequencing. Sequence-specific forwardprimers targeting wild type and mutation type templates respectively were designedwith3’ terminal phosphorothioate modification, which constitute amplicons with apublic reverse primers that is not phosphorothioate modified.Two-directional primerextension was performed using Pfu polymerases combining with quantitative PCRtechnique to analyze the results. Singlet PCR and multiplex PCR reaction conditionswere optimized. This assay was applied to the detection for tumor tissues or bloodsamples collected from healthy individuals and patients.Results：Efficient primer extension was observed while the point mutation specific primersmatch their specific corresponding templates either in single PCR or in multiplex PCRamplification usig the exo+polymerase. However, wild type primers were not extended and on products were bserved. DNA samples extracted from blood of20normalindividuals and one gene pool sample of200normal persons were tested using thenewly developed assay. The specific mutation detection primers failed to amplifyproducts in these smples while products were amplified by specific wild detectionprimers. These results demonstrated that no point mutations in these samples. Allsamples were amplified with sequencing primers and sent for sequencing. Sequencingresults confirmed that all of these samples harbored no point mutations in the K-rasgene. DNAs extracted from tissue of15lung cancer samples were tested using thenewly developed assay, and products were amplified in5cancer sample by specificmutation primers, suggesting the presence of point mutations in these samples. Fromcell free DNA extracted from the plasma of14cancer patients,3products wereamplified, suggesting the presence of point mutations in these3samples. All tumorsamples yielding mutation products were amplified with sequencing primers and sentfor sequencing. Sequencing results confirmed that all of these samples harbored at leastone point mutations in the K-ras gene, indicating the usefulness of the newly developedmultiplex PCR in genetic diagnostics. This new assay may have wide application inclinical genetic diagnostics, especially in somatic mutations.Conclusions:This work successfully eatablished PCR method with specific primers for detectionof the seven hot spots mutations in K-ras gene of somatic mutation by the mutationsensitive “molecular switch”, consisting of high fidelity DNA polymerase andphosphorothioate modified primers. This method can detect the micro-mutations in cellfree DNA. This technology has a greater potential for K-ras gene screening, earlywarning of the tumor, to guide Clinical treatment.