Protocol:Amplification refractory mutation system (ARMS)

Principle
The ARMS technique for detecting known point mutations was first described by Newton et al [26]. It has been developed for the diagnosis of all the common &beta;-thalassaemia mutations found in all the main ethnic groups [10]. The technique is based on the principle of allele-specific priming of the PCR process, i.e. a specific primer will only permit amplification to take place when its 3' terminal nucleotide matches with its target sequence. Thus to detect the &beta;-thalassaemia mutation IVSI-5 (G->C), the 3' nucleotide of the ARMS primer is G in order to base pair with the substituted C in the mutant DNA. The primer forms a G-G mismatch with normal DNA, but this is a weak mismatch and will not prohibit extension of the primer by itself. Only strong mismatches (C-C, G-A and A-A) were found to reduce priming efficiency to zero or below-5%, and to prevent amplification, a further mismatch with the target sequence had to be introduced at the second, third or fourth nucleotide from the 3' end of the primer (27).

As a general rule for ARMS primer design, if the 3' terminal mismatch is a weak one, a strong secondary mismatch is engineered. If it is a strong one, a weak secondary mismatch is introduced. Try putting the mismatch at the second nucleotide in the first instance and test the primer for specificity and generation of product. The position of the mismatch can be altered if the primer does not work, or the strength of the mismatch increased if non-specific bands are observed. According to Little in Current Protocols in Human Genetics (28), the strength of mismatch pairings are; maximum, GA, CT, TT; strong, CC; medium, AA, GG; Weak, CA, GT; none, AT, GC.

The mutation-specific ARMS primers used in the Oxford laboratory to diagnose the 25 most common &beta;-thalassaemia mutations, plus the hemoglobin variants HbS, HbC and HbE, are listed in Table 3. All are 30 bases long so that they can all be used at a single high annealing temperature (65 oC).

A typical ARMS test for a single mutation consists of two amplifications in the same reaction mixture using the same genomic DNA as substrate. One amplification product results from the specific ARMS primer and its primer pair (when the mutation is present in the genomic DNA) and the other amplification results from two primers that generate a control fragment in all cases. The generation of control product indicates the reaction mixture and thermal cycler is working optimally. Figure 5.7 shows the screening of a DNA sample for a &beta;-thalassaemia mutation. The strategy is to screen for the common mutations expected in the country of the ethnic origin of the patient first and then to screen for the rarer mutations. After which, uncharacterised mutations are identified by genomic sequencing.

Primers for the diagnosis of the normal alleles for many of these mutations are listed in Table 5.5. These are required when both partners of a couple requesting prenatal diagnosis of &beta;-thalassaemia carry the same mutation. An example of prenatal diagnosis using mutant and normal ARMS primers is shown in Figure 5.8.

Method

 * 1) Prepare a reaction mixture (4 ml) comprising of: 0.5 ml of 10x ARMS PCR buffer;1.25 ml of 1.35 mM dNTP mixture; 2.65 ml of sterile distilled water
 * 2) Pipette 20 &mu;l of PCR reaction mixture into a 0.5 &mu;l tube.
 * 3) Add 1 &mu;l of each primer (1 OD unit/ml).
 * 4) Add 0.05 &mu;l of Taq DNA polymerase (5u/&mu;l).
 * 5) When more than one test is being performed, a primer and the enzyme can be mixed together in a separate tube before addition to the reaction mix. This decreases pipetting errors as larger quantities are used.
 * 6) Add 1 &mu;l of genomic DNA (100 ng/&mu;l).
 * 7) Overlay with 25 &mu;l of mineral oil.
 * 8) Mix, centrifuge and place in thermal cycler.
 * 9) Amplify for 25 cycles as follows: 1 min at 94 oC/1 min at 65 oC/1.5 min at 72 oC with a final extension period of 3 min at 72 oC following the 25th cycle.
 * 10) Remove tubes from thermal cycler and add 5 &mu;l of blue dye. Mix and centrifuge.
 * 11) Load a 20 &mu;l aliquot onto a 3% agarose gel and run at 100 V for approx. 45 min in TBE
 * 12) Stain gel in ethidium bromide solution (0.5 &mu;g/ml) for 15-30 minutes, visualise bands on a UV light box (312 nm) and photograph with an electronic camera system or a Polaroid CU-5 camera fitted with an orange filter (e.g. Wratten 22A).

Materials

 * 1) DNTPs: Add together 50 &mu;l of a 100 mM solution of each dNTP (as purchased) and 3.8 ml of distilled water. The 1.25 mM dNTP stock solution should be stored in frozen aliquots.
 * 2) ARMS PCR buffer: 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at room temperature), 1.5 mM MgCl2, 100 &mu;g/ml gelatin. A 10x stock buffer can be prepared by adding together 0.5 ml of 1 M Tris-HCl (pH 8.3 at room temperature), 1.25 ml of 2 M KCl, 75 &mu;l of 1 M MgCl2, 5 mg gelatin, and 3.275 ml of distilled water. The stock buffer is heated at 37 0C until the gelatin dissolves and then frozen in aliquots.
 * 3) Taq polymerases: suggested ones are as follows, AmpliTaq Gold (PE Biosystems) works best for ARMS-PCR/RE digestion assays and Platinum Taq (Gibco Life Technologies) for gap-PCR (see Methods). (Tris-borate -EDTA (TBE) buffer: 89 mM Tris-borate, 89 mM boric acid,10 mM EDTA PH 8.0).

Table 5.4 Primer sequences used for the detection of the common &beta;-thalassaemia mutations by ARMS-PCR Legend: The above primers are coupled as indicated with either primer A: CCCCTTCCTATGACATGAACTTAA, B: ACCTCACCCTGTGGAGCCAC;C: TTCGTCTGTTTCCCATTCTAAACT; or D: GAGTCAAGGCTGAGAGATGCAGGA. The control primers used were primers D plus E: CAATGTATCATGCCTCTTTGCACC (which yield a 861 bp product as shown in Figure 1) for all the above mutation specific ARMS primers except the two marked *. Control primers used with these two are the G&gamma;-Hind III RFLP primers[8].

Table 5.5. Primer sequences used for the detection of normal DNA sequences by ARMS-PCR.

See Table 5.4 legend for details of primers A-D and control primers.