Amplification Refractory Mutation System

All PCRs depend on the annealing of oligonucleotide primers to specific sites in target DNA, facilitating amplification of a unique sequence. For the polymerase to extend the primers, they must be perfectly annealed to the target sequence at their 3' ends. The amplification refractory mutation system (ARMS) exploits this by utilizing a primer that is designed such that its 3' end matches, for example, one of two alternatives at a mutant nucleotide (40). ARMS is ideally suited to the detection of point mutations and small insertions/deletions, and at its simplest, it involves two PCRs, one for each allele (Fig. 2). Thus, an ARMS PCR is allele-specific and earlier alternative names for the process have reflected this, including Allele-specific PCR (ASPCR) and PCR amplification of specific alleles (PASA) (41,42). A number of ARMS variants have been produced and these are discussed in the following subsections.

Additional mismatches near the 3' end of ARMS primers helps to increase the specificity of reactions, but too many mismatches can cause excessive destabilization. Differentiation of alleles is said to be problematic when the 3' mismatch is C: A or involves a T, but this is not our experience (43). The most discriminatory mismatches appear to be A:G/G:A, closely followed by Py: Py. It is wise to test the performance of a new ARMS PCR by varying reaction components and parameters, including primer concentration, buffer composition, magnesium concentration, annealing temperature, and PCR additives such as dimethyl sulfoxide (DMSO). Hot-start Taq is worth the extra cost. As always, novel applications require rigorous working up to test specificity, sensitivity, and other parameters of interest. It is essential to include a control PCR, that is not allele-specific, in the same tube as the ARMS reaction to avoid a null result. If a number of ARMS reactions are multiplexed, then this requirement is obviated.

3.1. DOUBLE ARMS Double ARMS utilizes a pair of allele-specific primers, instead of one specific and one common primer, as included in the standard ARMS test. The two polymorphic or mutant sites must be close enough to each other to allow a PCR to work, although given the distance that can be spanned by a long-range PCR (several kilobases), this is not the constraint it perhaps once was. Double ARMS enables the haplotyping of an individual in the absence of DNA from relatives. To do this, four ARMS PCRs must be set up: If locus 1 has two alleles (A and B) and locus 2 has two alleles (C and D), then these PCRs will be A x C, A x D, B x C, and B x D. Double ARMS can be useful for haplotyping doubly heterozygous individuals if two mutations are in cis or trans and/or to distinguish a carrier of a recessive disorder from an individual at risk of being affected. It has been used to haplotype alleles at closely linked loci, such as human leukocyte antigen (HLA) and rhesus blood groups (44).

3.2. MS-PCR In some diagnostic applications, it is possible to include both allele-specific primers, one for each alternative at the mutant or polymorphic site, which then together with the common primer allows each amplicon to act as a control for the other. In this instance, there must be a way of distinguishing the two amplicons, either by differential size or labeling (45-49). Advantages of MS-PCR include the fact that only a single PCR needs to be carried out and there is no need for a separate internal control. Sensitivity is improved because of the competitive nature of the PCR. Design of MS-PCR primers is somewhat more involved than simple ARMS assays, but this is not a significant negative factor.

3.3. MULTIPLEX ARMS In conditions where there are a restricted number of common mutations, then it might be efficacious to perform a test that simultaneously tests for as many of them as possible (multiplex ARMS). This is exemplified by commercially available kits to detect particular CFTR mutations that are prevalent in particular populations. For instance, the Elucigene CF20 kit detects 20 different mutations in the CFTR gene (44,49,50). It can be a difficult technical challenge to design primers that do not interfere with one another but still work in a multiplex ARMS. Amplicons are best distinguished by being either of differing sizes, or differentially labeled, or both.

3.4. ADVANTAGES AND LIMITATIONS OF ARMS The ARMS assays are generally quick, inexpensive, and simple to devise and work up. Inherently, they are not suited to screening for unknown mutations, but only mutations detected initially by some other technique. They do not require any unconventional PCR equipment, although the method used to detect amplicons can vary acording to that locally available or desired (from simple agarose gel electrophoresis to detection on an automated capillary fluorescent DNA analyzer). ARMS reactions are reasonably easily multiplexed and can also be used to determine haplotypes (51). In terms of sensitivity, they are generally able to detect 1 mutant allele in 40 normal alleles. As with all oligonucleotide-based tests, care must be taken at the design stage to avoid known polymorphisms that might affect primer binding (such as CFTR AF508); similarly, caution must be exercised in interpretation, bearing in mind that a hitherto unknown sequence variant could exist in a patient and it interferes with or appears to be a known mutation (52).

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