Previously, Sanger sequencing was used to detect mutations including unknown ones; however, it is time-consuming and has low sensitivity leading to false-negative results. Recently, ARMS-PCR was approved to be used in clinical practice for genotyping SNP (single nucleotide polymorphism) with the help of refractory primers. Designing primers for the mutant (with SNP) and normal (without SNP) alleles allows selective amplification which can be easily analyzed after electrophoresis. Modification of a single base occurs at the 3' end of the primer such that one primer matches the normal allele and the other matches the mutant allele. Both primer variants are combined in a single PCR reaction mixture so that the PCR is performed simultaneously. The presence of an allele variant depends on the ability of the primers to attach and amplify either the normal or the mutant allele. The mutant set of the primer is refractory or resistant to the normal PCR and vice versa; hence, the name, amplification refractory mutation system. ARMS-PCR may be more sensitive than Sanger Sequencing; however, calculating ΔCt based on fluorescent signals in ARMS-PCR may be highly subjective leading to inconclusive results.
Sanger sequencing and ARMS-PCR are the main technologies used to determine the genotype in the clinic. Recently, the mutation-selected amplification specific system PCR (MASS-PCR), a novel quantitative PCR (qPCR)-based assay, was developed to detect and amplify only mutations without sacrificing cost and time. The amplification of wild-type templates is suppressed therefore reducing subjectivity. After the cyclic amplification, only the mutant gene will emit a fluorescence peak which basically solved the defect of ARMS-PCR which amplifies both the normal and mutant alleles. MASS-PCR delivers a more straightforward result by using specific primers and probes that target the common mutations from disease panels. Upon sensitivity testing, MASS-PCR shows high consistency with next-generation sequencing and ARMS-PCR. Verification with direct sequencing for inconsistent results exhibits inclination with MASS-PCR results.
Amplification refractory mutation system PCR (ARMS-PCR) and mutation-selected amplification specific system PCR (MASS-PCR) are novel gene detection techniques with high specificity and sensitivity, in which normal and/ or mutant sequences are amplified by allele-specific primers. In a comparison study conducted by Cai, et al. (2019) in lung cancer tissues, MASS-PCR showed a greater consistency rate with Sanger Sequencing than ARMS-PCR. MASS-PCR detected more positive results than Sanger sequencing and ARMS-PCR. On the other hand, Sanger sequencing produced false-positive results. In disease panels, MASS-PCR showed higher accuracy and sensitivity than ARMS-PCR.
CD Genomics is one of the professional sequencing companies with extensive experience in detecting gene mutations. We can provide high-quality ARMS-PCR service to assess sequence variations and accelerate disease research.
Amplification refractory mutation system (ARMS) is the development of the application of PCR technology. ARMS-PCR can detect two alleles simultaneously by using a single PCR reaction system, which does not require restriction endonuclease and can distinguish whether the alleles are pure or not. ARMS technology has become one of the international personalized molecular detection techniques for cancer, as well as a good method for mutation detection in genetic diseases such as sickle cell anemia and thalassemia.
References:
