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ARMS PCR 323
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Multiplex Amplification Refractory
Mutation System for the Detection
of Prothrombotic Polymorphisms
David Stirling
1. Introduction
First described by Newton and colleagues in 1989 (1), amplification refractory mutations
system (ARMS) has become a standard technique that allows the discrimination
of alleles that differ by as little as 1 bp. The system is simple, reliable, and nonisotopic.
It clearly distinguishes heterozygotes at a locus from homozygotes for either allele. The
system requires neither restriction enzyme digestion, nor allele-specific oligonucleotides
as conventionally applied, nor the sequence analysis of polymerase chain reaction
(PCR) products. The basis of the system is that oligonucleotides with a mismatched
3′-residue will not function as primers in the PCR under appropriate conditions.
A standard ARMS PCR consists of two complementary reactions (two tubes) and
uses 3 primers. One primer is constant and complementary to the template in both reactions,
and the other primers differ at their 3′ terminal residues and are specific to either
the wild-type DNA sequence or the mutated sequence at a given base—only one of these
primers is used per tube. If the sample is homozygous mutant or homozygous wildtype
amplification will only occur in one of the tubes, if the sample is heterozygous
amplification will be seen in both tubes.
Here, we report our protocol for the multiplex ARMS detection of the polymorphisms
in clotting factor V and II associated with increased risk of thrombosis (2,3). A portion
of the factor IX gene is amplified as an internal positive control.
2. Materials
1. Thermal cycler.
2. Plate mixer.
3. Vertical PAGE system.
4. Electrophoresis Power Pack.
5. Thermowell 96-well plate and covers.
6. Round tips (200 µL).
7. Flat-cap PCR tubes (0.5 mL).
8. Thermostable DNA Taq polymerase.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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