Introduction
With the rapid advancement of precision medicine and targeted therapies, the ability to accurately detect and identify gene mutations has become a cornerstone of clinical diagnostics. ARMS-PCR (Amplification Refractory Mutation System PCR) is a highly sensitive, highly specific, and cost-effective molecular technique. By designing allele-specific primers, ARMS-PCR enables rapid and accurate detection of genetic mutations, playing a vital role in cancer screening, genetic disorder diagnostics, and evaluation of targeted drug responses.
What Is ARMS-PCR?
ARMS-PCR, or Amplification Refractory Mutation System PCR, is based on allele-specific primer extension. It relies on the principle that Taq DNA polymerase can only initiate extension when the 3’ end of a primer is perfectly complementary to the target base at a mutation site.
To achieve this, the 3’ end of the forward primer is designed to be a perfect match for the mutant sequence and to contain a single base mismatch with the wild-type sequence. This allows only the mutant DNA to be amplified.By comparing the amplification patterns observed via qPCR or gel electrophoresis, the presence of mutant or wild-type alleles can be distinguished, allowing for accurate SNP genotyping.
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Figure 1: Principle of ARMS-PCR
Limitations of Traditional ARMS-PCR
Despite its advantages, ARMS-PCR faces some limitations in practice:
1. Primer Design Is Mutation-Specific ARMS primers must be specifically designed for known mutations and are not suitable for broad-range mutation screening.
2. Limited Specificity Due to Polymerase Tolerance Taq polymerase may still extend primers with mismatched 3’ ends on the wild-type template, though with lower efficiency. This results in substantially increased Ct values for wild-type amplification, leading to poor specificity and potential false positives.
Conventional Workarounds
To improve specificity, most strategies aim to reduce non-target amplification while preserving target signal, including:
1. Introducing additional mismatches at the primer's 3' end
2. Designing complex primer structures such as looped (hairpin) primers
3. Using blockers to inhibit wild-type amplification
While these methods provide some improvement, they often suffer from complex design processes, low generalizability, and suboptimal specificity. Frequent optimization is required to determine the number and position of mismatches, increasing time and cost.
★★★ BiOligo's Solution ★★★
To overcome these limitations, BiOligo has developed a proprietary solution: the BiOligo ARMS Probe.
Core Principle
BiOligo's ARMS primers incorporate a unique 5'AS modification at the primer containing the SNP site. This chemical modification alters the hybridization conformation between the primer and the template, thermodynamically blocking mismatched extension on wild-type templates. This results in either no amplification or substantially increased Ct values for wild-type DNA, enabling clear discrimination of point mutations.
Figure 2. Schematic diagram of BiOligo ARMS modification.
Product Highlights
Easy Primer Design – No Complex Optimization Required
There is no need to introduce artificial mismatches or complicated structures like looped primers. Simply incorporate the 5'AS modification for optimal experimental performance. Primer design is streamlined and efficient.
BiOligo Tip:ARMS probe performance is closely related to primer Tm; it is recommended that the Tm exceed the annealing temperature by 3–5°C.
High Specificity — Accurate SNP Genotyping
BiOligo ARMS Probes utilize thermodynamic blocking of mismatched base extension, significantly outperforming traditional chemical kinetic-based designs. This results in precise SNP genotyping and greatly reduces false positives.
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Using EGFR-L858R as a target, mutant DNA at 1 ng/μL and gDNA at 10 ng/μL were used as templates. Comparative testing with conventional and BiOligo ARMS-modified primers revealed that the ΔCt value of gDNA increased dramatically with BiOligo's modification, indicating superior specificity.
