Establish A New Method To Detect Early Stage Mutation Of TP53(R175H) Gene Based On A New Gene Transfer System

Objective: To establish a method to detect early stage mutation of TP53 gene(R175H)with a new gene transfer system and to provide a basis for early tumor screening.Method: Wild-type DNA template(W, including Nar I restriction site) and mutant DNA template(T) of TP53 gene were constructed. Both gene templates were mixed with the proportions of(W:T) 1:0, 100:1, 300:1, 1000:1, the mixture were PCR amplified with the high fidelity DNA polymerase and digested with the restriction endonuclease in vitro, then the products were again PCR amplified and sequenced.Result: Agarose gel electrophoresis showed that the PCR products of the wild-type gene fragments could be digested by NarI restriction endonuclease, while the three PCR products with other mixing rates could not be completely digested by the enzyme due to the presence of mutation fragments, and the mutant gene was amplified, the result was confirmed by sequencing.Conclusions: The detection of early stage mutation of TP53 gene(R175H) using the gene transfer system composed by combined specific primers based on high fidelity DNA polymerase and low temperature restriction endonuclease is feasible. And the gene transfer system probably has great application value on early stage gene screening.