**8.3 ARMS-PCR for β<sup>E</sup>**

**Procedure**: Two 25-μL reactions are performed; M-reaction and N-reaction. Both M and N-reactions contain 150 ng genomic DNA, 200 μM of each dNTP; 0.6 units Taq DNA polymerase, 0.2 μM of "S-primer"; 5′-ACC TCA CCC TGT GGA GCC AC-3′, 0.15 μM of "HbE-M primer"; 5′-TAA CCT TGA TAC CAA CCT GCC CAG GGC GTT-3′ (for M-reaction only), 0.15 μM of "HbE-N primer"; 5′-TAA CCT TGA TAC CAA CCT GCC CAG GGC GTC-3′ (for N-reaction only), 0.2 μM of "P1 primer"; 5′-GCG ATC TGG GCT CTG TGT TCT-3′, 0.2 μM of "P2 primer"; 5′-GTT CCC TGA GCC CCG ACA CG-3′, 10 mM Tris pH 8.8; 50 mM KCl and 1.5 mM of MgCl2.

**Thermal cycles**: A total of 35 thermal cycles was carried out with each cycle comprising DNA denaturation at 95°C for 1 min, primer annealing at 65°C for 1 min and primer extension at 72°C for 1 min; the initial denaturation was extended to 5 min while the final extension was prolonged to 5 min.

**Detection of amplified products**: The amplified products were separated in 2.5% agarose gel electrophoresis at 120 V for 15–20 min before visualizing with a UV-transilluminator. The fragments sizing 260 bp is the specific amplified products, and the PCR products sizing 314 bp are the control products (**Figure 27**).

#### **Figure 27.**

*ARMS-PCR for identifying β<sup>E</sup> mutation. The 314-bp amplified products are internal control. The 260-bp amplified products are the β<sup>E</sup> specific products. Sample #3 is homozygote for β<sup>E</sup> mutation as the 260-pb amplified products are seen in only "M-reaction." Sample #1 is negative for the β<sup>E</sup> mutation since the 260-bp amplified products are seen in only "N-reaction." Sample #2 is heterozygote for the β<sup>E</sup> mutation since the 260-bp amplified products are seen in both "M-reaction" and "N-reaction."*

**Interpretation**: Both M and N-reactions must have the 314-bp control products and the results can be read. If both M- and N-reactions have 260-bp PCR products, the samples are heterozygote for β<sup>E</sup> with genotype of either β<sup>E</sup> /βA or β<sup>E</sup> /β<sup>T</sup> (A represents HbA; T represents other types of β-globin gene mutation). If the 260-bp PCR products are seen in only M-reaction, the samples are homozygote for β<sup>E</sup> with genotype β<sup>E</sup> /β<sup>E</sup> . If samples have the 260-bp PCR products in only N-reaction, the samples are negative for the β<sup>E</sup> with genotypes of either βA/βA or β<sup>T</sup> /β<sup>T</sup> (A represents HbA; T represents other types of β-globin gene mutation) (**Figure 27**).
