Enzyme-Linked Electrochemical Assay For The Detection Of BRAF V600E Allele-specific PCR Amplicons

BackgroundCancer have become a prominent threat to human health.The assays of ear ly diagnosis and treatment of cancer has become a major research topic in the m ed-ical field and become a hot direction of basic and clinical research.There i s a huge difference between tumor cells and normal somatic cells in the genomic level, some of the important driver mutations can be acted as the new biomar kers for tumor diagnosis and prognosis.They can provide important information.T he clinical significance of measuring this type of driver mutations include:(1) Th e detection of tumor-derived mutant gene from theperipheral blood of cancer patien ts and otherbody fluids, or biopsies, to provide early diagnostic information;(2) Monitoring of tumor recurrence of patients (after surgery,radiotherapy or chemot herapy); molecular typing according to the mutation of the tumor tissue informatio n to provide a basis for the prognosis of the disease and choice of targeted dru g.BRAF is a member of the RAF kinase family which acts as a intermediary to transduce sinals from growth factor receptors on the cell membrane to the nucleus through MAPK signal-transduction pathway.An oncogenic somatic mutation(T→A) at nucleotide1799accounts for the most frequent of all BRAF genetic abnormalities.This transversion mutation results in the substitution of valine with glutamate acid in codon600(V600E) and converts BRAF into a dominant transforming protein.BRAF V600E protein constantly activates the MAPK pathway,which enhances the proliferative and metastatic potential of cancer cells. The BRAF V600E mutation has been found in diverse human cancers especialy melanoma, colorectal cancer, thyroid cancer, and hairy-cell leukemia.In the era of targeted treatments for colorectal cancer,BRAF genotyping is important not just in identifying patients with poor prognoses and distinguishing sporadic microsatellite instable colorectal cancer from hereditary non-polyposis colorectal cancer (Lynch syndrome),but also in predicting the therapeutic efficiency of monoclonal antibodies targeting epidermal growth factor receptor (EGFR).Given that BRAF V600E mutation is exclusively found in malignant cells,selective inhibitors of mutated BRAF,such as Vemurafenib,show powerful antitumor activity in patients with melanomas that harboring the BRAF V600E mutation.Thus, BRAF genotyping of tumor is now strongly requested to direct personalized therapies especialy in colorectal cancer and melanomas patients.Detection of minority somatic DNA mutations within an excess wild-type sample has been playing an important role in diagnosis during early cancer detection and tailoring therapy from various clinical specimens.Although methods to determine known mutations develop rapidly, few approaches to identify minority unknown mutations have emerged.ObjectiveTo establish an electrochemical method based on BRAF V600E gene mutation at a low level of colorectal cancer cells. The method combined with allele-specificamplification and enzymatic reaction of the electrochemical sensing principle,toachieve the detection of gene mutations of the BRAF V600E in a lower level.Compared with the method of Sanger sequencing and agarose gel electrophoresis, the sensitivity and specificity of them were estimated.In order to provide a new electrochemical assay for the detection of gene mutation in low-level cancer cells.Methods1. Allele specific amplification to detect colorectal cancer cell lines habor a BRAF gene V600E mutation1.1The genomic DNA of the two colorectal adenocarcinoma SW480and HT29cell lines were extracted. After that, a pair of primers were designed to amplify the exon15of the BRAF gene to observe whether they harbor a V600E mutation by sequencing.1.2Primer Premier5.0software was used to design allele-specific primers based on human GeneBank published sequences and the position of BRAF V600E mutation.With a optimal condition, allele-specific amplifications of the two cell lines were carried out, the results were confirmed by agarose gel electrophoresis.2. Enzyme-linked electrochemical assay for the detection of BRAF V600E allele-specific PCR amplicons.2.1Biotinylated dCTP was mix in the PCR reaction.To explore the effects of the amplification products by different proportions of biotin-dCTP.We optimize best condition to amplify according to the first part of experiment. Agarose gel electrophoresis were carried out for separation, identification the PCR products and select the ideal ratio with biotin-dCTP.2.2Ultrafiltration centrifuge tubes were used to purify the PCR products.2.3Characterization of gold magnetic nanoparticles was carried out by the scanning electron microscopy (SEM).2.4Repeatability experiment of the screen-printed carbon electrodes.2.5Mutant DNA template dilution experiment2.6After magnetic separation,alkaline phosphatase were labeled on the gold magnetic particles.Finally the current signal was detected on the screen-printed carbon electrode.2.7To compare the sensitivity and specificity,dilution of the V600E mutation template were used for Sanger sequencingafter amplification of the BRAF15exon, enzyme-linked electrochemical sensing method for detection of allele-specific amplification products and agarose gel electrophoresis detection of allele-specific amplification products.Results1. After PCR and sequencing, SW480was confirmed to harbor a wild-type BRAF while HT29harbor a BRAF V600E heterozygous mutation.2. With the increasing proportion of biotin-dCTP, the molecular weight of the amplified products were increases, while the brightness of the amplified products become gradient dark,indicating that the amplification efficiency along with the gradual decline.In order to get a higher amount of markup and less affection to amplification efficiency,we selected a50%proportion of biotin-dCTP to the following experiments.3.Characterization of Gold magnetic nanoparticles by the scanning electron microscopy show that, white particles were uniform and visible on the carbon electrode surface, about tens of nanometers in size.4. Four of the same batch of screen-printed electrodes were scaned in the [Fe(CN)6]4-/3-solution.The RSD%was4.8%(oxidation peak current), indicating that the reproducibility of the electrodes is ideal. 5. After labeling with enzymes to the allele-specific amplification products of different proportions of template harboring V600E mutation,differential pulse voltammetry was used to scaned.The results show that with the increase in the proportion of mutation templates, the enzymatic reaction products (ascorbic acid) oxidation peak current (0.25V) is gradually increased while the enzymatic reaction substrate (ascorbate-2-trisodium phosphate)oxidation peak current (0.75V) was gradually reduced.Indicating that there was a positive relationship between the enzymatic reaction and the amount of BRAFV600E mutation.A threshold value was calculated by this formula: Vthreshold=Vnegative+3VnegativeSD.The limit of detection of this assay was0.8%(0.024ng/ul). When the mutation ratio was between25%to3.1%,there was a linear relationship between oxidation peak current and mutation ratio, the correlation coefficient was0.978.6. Sequencing following conventional PCR showed that the detection limit of Sanger sequencing was25%(7.5ng/ul).7. Agarose gel electrophoresis detection of allele-specific amplification products showed that the detection limit was6.3%(1.89ng/ul)Conclusion1. An allele-specific amplification method was established for the detection of BRAF V600E mutation.The method can amplified HT29cells line templates that carrying the BRAF V600E mutation successfully,while not in SW480cells line.2. Based on the idea of associating PCR with electrochemical sensing, we linked the enzyme with the allele-specific PCR amplicons. After magnetic separation,the current signal of the enzyme reaction products were finally detected on the screen-printed carbon electrodes.Compare with Sanger sequencing ang agarose gel electrophoresis,this method showed good sensitivity and specificity.