**8.1 ARMS-PCR for β41/42**

**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 "M41/42 primer"; 5′-GAG TGG ACA GAT CCC CAA AGG ACT CAA CCT−3′ (for M-reaction only), 0.15 μM of "N41/42 primer"; 5′-GAG TGG ACA GAT CCC CAA AGG ACT CAA AGA-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 439 bp is the specific amplified products, and the PCR products sizing 314 bp are the control products (**Figure 25**).

#### **Figure 25.**

*ARMS-PCR for identifying the β41/42 mutation. The 314-bp amplified products are internal control. The 439-bp amplified products are the β41/42 specific products. Sample #1 is homozygote for β41/42 mutation as the 439-pb amplified products are seen in only "M-reaction." Sample #3 is negative for the β41/42 mutation since the 439-bp amplified products are seen in only "N-reaction." Sample #2 is heterozygote for the β41/42 mutation since the 439-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. It both M and N-reactions have 439-bp PCR products, the samples are heterozygote for β41/42 with genotype of either β41/42/βA or β41/42/β<sup>T</sup> (A represents HbA; T represents other types of β-globin gene mutation). If the 439-bp PCR products are seen in only M-reaction, the samples are homozygote for β41/42 with genotype β41/42/β41/42. If samples have the 439-bp PCR products in only N-reaction, the samples are negative for the β41/42 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 25**).

### **8.2 ARMS-PCR for β17**

**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 "M17 primer"; 5′-CTC ACC ACC AAC TCA GCC ACG TTC AGC ATA-3′ (for M-reaction only), 0.15 μM of "N17 primer"; 5′-CTC ACC ACC AAC TTC ATC CAC GTT CAC ATT-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 239 bp is the specific amplified products, and the PCR products sizing 314 bp are the control products (**Figure 26**).

**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 239-bp PCR products, the samples are heterozygote for β 17 with genotype of either β 17/βA or β 17/β<sup>T</sup> (A represents HbA; T represents other types of β-globin gene mutation). If the 239-bp PCR products are seen in only M-reaction, the samples are homozygote for β 17 with genotype β 17/β 17. If samples have the 239-bp PCR products in only N-reaction, the samples are negative for the β 17 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 26**).

**151**

**Figure 27.**

*ARMS-PCR for identifying β<sup>E</sup>*

*amplified products are the β<sup>E</sup>*

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

**8.3 ARMS-PCR for β<sup>E</sup>**

MgCl2.

**Figure 26.**

**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

*ARMS-PCR for identifying the β17 mutation. The 314-bp amplified products are internal control. The 239-bp amplified products are the β17 specific products. Sample #1 is homozygote for β17 mutation as the 239-pb amplified products are seen in only "M-reaction." Sample #3 is negative for the β17 mutation since the 239-bp amplified products are seen in only "N-reaction." Sample #2 is heterozygote for the β17 mutation since the 239-bp* 

**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

**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**).

 *mutation. The 314-bp amplified products are internal control. The 260-bp* 

 *mutation as the 260-pb amplified* 

 *mutation since the 260-bp amplified* 

 *mutation since the 260-bp* 

 *specific products. Sample #3 is homozygote for β<sup>E</sup>*

5 min while the final extension was prolonged to 5 min.

*products are seen in only "M-reaction." Sample #1 is negative for the β<sup>E</sup>*

*amplified products are seen in both "M-reaction" and "N-reaction."*

*products are seen in only "N-reaction." Sample #2 is heterozygote for the β<sup>E</sup>*

*amplified products are seen in both "M-reaction" and "N-reaction."*

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

#### **Figure 26.**

*Beta Thalassemia*

**Figure 25.**

**8.2 ARMS-PCR for β17**

of MgCl2.

**Interpretation**: Both M and N-reactions must have the 314-bp control products and the results can be read. It both M and N-reactions have 439-bp PCR products, the samples are heterozygote for β41/42 with genotype of either β41/42/βA or β41/42/β<sup>T</sup>

*ARMS-PCR for identifying the β41/42 mutation. The 314-bp amplified products are internal control. The 439-bp amplified products are the β41/42 specific products. Sample #1 is homozygote for β41/42 mutation as the 439-pb amplified products are seen in only "M-reaction." Sample #3 is negative for the β41/42 mutation since the 439-bp amplified products are seen in only "N-reaction." Sample #2 is heterozygote for the β41/42 mutation* 

represents HbA; T represents other types of β-globin gene mutation). If the 439-bp PCR products are seen in only M-reaction, the samples are homozygote for β41/42 with genotype β41/42/β41/42. If samples have the 439-bp PCR products in only N-reaction,

represents HbA; T represents other types of β-globin gene mutation) (**Figure 25**).

**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 "M17 primer"; 5′-CTC ACC ACC AAC TCA GCC ACG TTC AGC ATA-3′ (for M-reaction only), 0.15 μM of "N17 primer"; 5′-CTC ACC ACC AAC TTC ATC CAC GTT CAC ATT-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

**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

**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 239 bp is the specific amplified products, and the PCR products sizing 314 bp are the control products (**Figure 26**).

**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 239-bp PCR products,

represents HbA; T represents other types of β-globin gene mutation). If the 239-bp PCR products are seen in only M-reaction, the samples are homozygote for β

HbA; T represents other types of β-globin gene mutation) (**Figure 26**).

17 with genotype of either β

17. If samples have the 239-bp PCR products in only N-reaction, the

17 with genotypes of either βA/βA or β<sup>T</sup>

17/βA or β

/β<sup>T</sup>

17/β<sup>T</sup> (A

17 with

(A represents

5 min while the final extension was prolonged to 5 min.

the samples are heterozygote for β

17/β

samples are negative for the β

the samples are negative for the β41/42 with genotypes of either βA/βA or β<sup>T</sup>

*since the 439-bp amplified products are seen in both "M-reaction" and "N-reaction."*

(A

/β<sup>T</sup> (A

**150**

genotype β

*ARMS-PCR for identifying the β17 mutation. The 314-bp amplified products are internal control. The 239-bp amplified products are the β17 specific products. Sample #1 is homozygote for β17 mutation as the 239-pb amplified products are seen in only "M-reaction." Sample #3 is negative for the β17 mutation since the 239-bp amplified products are seen in only "N-reaction." Sample #2 is heterozygote for the β17 mutation since the 239-bp amplified products are seen in both "M-reaction" and "N-reaction."*
