Establishment Of A Genotype Switch Mutation Enrichment Technique For The Detection Of BRAF V600E In CtDNA

Background:Liquid biopsy has become an attractive non-invasive strategy that was widely used in early diagnosis,therapeutic effect evaluation,and recurrence monitoring of tumors.The detection targets are circulating tumor cells and cell-free circulating nucleic acids released from the primary tumor and / or metastatic deposits into peripheral blood,especially circulating tumor DNA(ctDNA)and exosomes.ctDNA is the most promising biomarkers for early diagnosis of tumor,individualized treatment,and prognosis detection of tumors.It carries tumor gene mutation information,methylation information,etc.These information helps tumor diagnosis,location,and drug resistance analysis,etc.The detection methods of ctDNA is mainly divided into two categories: PCR technology and sequencing technology.The detection sensitivity of ddPCR is high,but its high cost and low throughput restrict its wide clinical application.Traditional PCR and qPCR are the most widely used methods of genetic mutation detection,but the PCR method has great limitations for the detection of ctDNA or rare mutations.Although the improved rare mutation detection technology based on PCR,such as Amplification Refractory Mutation System-PCR(AMRS-PCR),Coamplification at Lower Denaturation Temperature PCR(COLD-PCR),etc.,can detect the rare mutation with sensitivity increased to 1%,or even0.1%,these methods are insensitive to ctDNA detection,especially to early tumor ctDNA detection.BRAF V600 E is a common mutation type in papillary thyroid cancer and colorectal cancer patients.It can be used as a biomarker of liquid biopsy for early diagnosis of tumor,tracking of curative effect,and individualized treatment.However,at present the detection technology based on PCR for detecting BRAF V600 E is can not meet the requirements of ctDNA detection.Therefore,this study intends to develop a novel technology based on PCR to improve the detection sensitivity of ctDNA.Objective:This study aimed to establish a simple,low-cost,high-sensitivity ctDNA detection technology based on the PCR platform by combining PCR methods and thermostable restriction enzymes.BRAF V600 E,the most common mutation type of BRAF gene,was used as a model to evaluate the clinical application prospect of this technology.Methods:1.Construct the wild-type and the mutant type plasmids of BRAF V600 E by site-directed mutagenesis for the method establishment and the following detection.2.Optimize and select the reaction buffer that can satisfy both DNA polymerase and thermostable restriction enzyme TscAI.3.Establish Genotype Switch Mutation Enrichment Technique,and use templates with various mutation ratios to evaluate the sensitivity of the technology to rare mutation detection.4.Collect blood and tissue specimens from patients with clinical papillary thyroid carcinoma,extract cfDNA from blood specimens,and use the constructed gene conversion mutation enrichment technology to detect and compare the results with tissue mutation analysis to evaluatethe technology’s ability to detect clinical samples.Results:1.The Genotype Switch Mutation Enrichment Technique established in this study used the thermostable restriction endonuclease TscAI to specifically recognize and cleave the wild-type template of BRAF V600 E.When a mutation occurs,the sequence change leads great reduction of the digestion activity.Combining PCR and thermostable restriction enzymes can achieve digestion while PCR,so that wild-type templates or PCR products can be digested by TscAI,while mutant templates cannot be digested,and can be amplified and enriched.2.This technology selected Qiagen’s HotStarTaq Plus DNA polymerase and thermostable restriction enzyme TscAI as a dual enzyme combination,and performs PCR and digestion reactions in FastDigest Buffer.3.The Genotype Switch Mutation Enrichment Technique can detect the ratio of mutant/wildtype as low as 1/100000,and its detection sensitivity reaches 0.001%.4.The results of clinical cfDNA tests were consistent with that of tissue mutation analysis.Conclusions:Combining the PCR methods and the thermostable restriction enzyme TscAI,a genotype conversion mutation enrichment technique was established for the detection of BRAF V600 E in ctDNA in plasma.