Selective Enrichment Of PIK3CA H1047R Fragments By PCR With Repeated Restriction Enzyme Digestion

Objective: To develop a sensitive assay for PIK3 CA H1047R mutation detection.Method: Wild-type DNA template and mutant DNA template were constructed using routing subcloning technique. Wild-type and mutant templates were mixed with the ratios of 100:1, 300:1 and 1000:1, respectively. The PCR products amplified using the high-fidelity DNA polymerase were purified and digested by the restriction enzyme BseJI for 1h. 2 μl of the restriction enzyme digested products were used as the template three rounds of PCR with each 10 cycles following 1h restriction digestion. DNA sequencing was used to evaluate the efficiency of this assay coupled with PCR and restriction digestion.Result: Plasmid templates harboring PIK3 CA H1047R mutation and its corresponding wild type fragments were constructed. The combination of PCR and repeated restriction enzyme digestion during PCR was able to selectively enrich the mutant fragments from the mixing templates with both wild type and mutant DNA sequence. As compared to the sensitivity of 5:1 to 10:1 of previous detection methods, the present study showed an assay which enhancing the sensitivity up to 1000:1 by Sanger’s sequencing in identifying the PIK3 CA H1047R mutation.Conclusion: A rapid and highly sensitive assay to detect PIK3 CA H1047R mutation was developed. The high sensitivity of this assay is mainly attributed to the repeated application of restriction enzyme digestion. This strategy may suggest a new direction in assay development using restriction enzymes in order to providing restriction in each cycle of PCR amplification.