Establishment Of Detecting BRAF Mutation By Peptide Nucleic Acid Clamping PCR

BackgroundThe occurrence of colorectal carcimoma is booming gradually. Patients in late stage or people with recurrence after operation need treatment of biological targeted agents.The guidelines of therapy on chemicals in colorectal tumor both in United States and in China indicate that the targeted agents(cetuximab and panitumumab),which belong to resisting EGFR should be used in primary or secondary chemical therapy.Based on appearance of the ascending demands,it is necessary for patients to implement the tests of drug sensitivity related genotypes.The findings of large samples from international multicenter shows that conduct tests of type of gene on KRAS and BRAF simultaneously,diagnose in terms of the outcomes,which is already discussed,which can maximize the precision of forseeing the sensitivity of targeted drugs for sufferers.The major screening methods of mutated gene include:Direct sequencing,Taqman correlative ARMS,q PCR related to High Resolution Melting and Scorpion guided ARMS.Although direct sequencing is considered as "Golden Standard" in consensus by international academy,it is poor in sensitivity(10%),which do harms to senior clinical usage itself.Scorpion-ARMS and Taqman-ARMS belong to patents abroad,which have comparatively high sensitivity(0.1%) and are mainly applied in detecting different kinds of tissues,such as tissue from operation,needle biopsy or microscopy.The procedures of these tests will be finished in 2 hours.Those technologies have been successfully made into commercial kits and widely confirmed by clinical performance.There are several kits which have got the permissions of clinical application from CFDA in China.q PCR-HRM needs advanced Q-PCR facilities,which confined its usage only to research,whose sensitivity is average(1%). PNA is a kind of imitated DNA analogs.(2-aminoethyl)-glycine units is the substitute of phosphodiester backbone.The most appropriate matched PNA/DNA duble chains are usually more steady than the DNA-DNA counterparts.In PCR,PNA can not be used as substrate of Taq-enzyme or other enzymes.Even with a single mismatch,the Tm of PNA will drop 9-10℃.Nowadays,PNA has been considered as a very helpful modifying tool in the realm of medical area.This research will utilize the core technology(PNA-Clamping) to detect BRAF mutation in tissues of colorectal cancer,which uses specific designed PNA to hinder amplification of BRAF wildtype in order to elevate its sensitivity and decrease its lower limit of detection.ObjectiveTo establish detection of BRAF mutation by PNA-Clamping PCR(PNA-PCR/BRAF) and determine lower limit of detectionMethodsPlasmids/cell line DNA harbouring BRAF mutation and tissue DNA with BRAF wildtype were mixed and serially diluted into reference samples(mutation/total:50%,25%,12.5%,6.25%,3.1%,1.6%,0.8%,0.4%,0)for 10 independent tests by PNA-PCR/BRAF method.Mutation CT and total CT which calculate ΔCT(mutation CT-total CT) originated from tests.The perfect Cut-off values of ΔCT for judging were identified by Receiver Operating Characteristic(ROC) curve,which also determined the positive judgement standard(the value of Cut-off) and lower limits of detection(LLOD) on PNA-PCR/BRAF method.ResultsComparing positive reference samples with negative reference samples on values of CT and ΔCT showed significant discrepancy(P<0.05).However,there is no difference between positive reference samples and negative reference samples on values of total CT.The Area Under Curve(AUC) of ROC curve in COLO205 cell line was 0.9,whose Cut-off value was 11.8.The corresponding sensitivity, specificity were 78.6% and 100%,respectively.Its LLOD was 3.1%.Likewise,the AUC of ROC curve in plasmid M1 was 0.974,whose value of Cut-off was 12,whose sensitivity even reached to 92.9% and which maintained specificity in 100%.The relevant LLOD was 1.6%.Similarly, the AUC of ROC curve in plasmid M2 was 0.921,along with 12 as Cut-off. Finding the value of sensitivity was 87.1%, with precondition for 100% specificity.The corresponding LLOD was 1.6%.ConclusionThis is a preliminary research of determing the values of Cut-off and LLOD on PNA-PCR/BRAF method.These values provides further clinical applications with important parameters.