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Tetra-Primer ARMS PCR Optimization for Detection of IVS-II-I (G-A) and FSC 8/9 InsG Mutations in β-Thalassemia Major Patients in Isfahan Population.

Hajihoseini S, Motovali-Bashi M, Honardoost MA, Alerasool N - Iran. J. Public Health (2015)

Bottom Line: Moreover, we have distinguished homozygous and heterozygous forms of these mutations successfully.The frequency of IVSII-1 (G-A) mutation from 30 patients in Isfahan was 86.6% (33.3% heterozygote, and 53.3% mutant homozygote) and for FS8-9 insG mutation was 16.6% (13.3% heterozygote, and 3.3% mutant homozygote).Tetra-primers ARMS PCR could be a reliable, accurate and simple technique for genotyping SNP and different mutations.

View Article: PubMed Central - PubMed

Affiliation: Genetics Division, Dept. of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.

ABSTRACT

Background: β-thalassemia, a monogenic autosomal recessive disorder, is prevalent in Middle East, particularly in Iran. In Iran, near to 20 mutations in the β-globin gene are introduced as common mutations with varying incidence frequencies in each city. Therefore, detection and screening for couples at high risk can help to solve the problems of this disease. In this study, optimized genotyping of two common mutations in Isfahan Province, IVSII-I (G-A) and FSC-8/9 insG, was performed using the T-ARMS method.

Methods: In this case-control study, 10 healthy individuals and 30 patients affected by β-thalassemia major with a mean 24.76 ± 4.5 years were selected from Omid Hospital in Isfahan Province. After designing tetra primers for two prevalent mutations IVSII-I (G-A) and FSC-8/9 insG, samples were genotyped using tetra-primers ARMS PCR technique.

Results: We have developed a sensitive single tube tetra-primers PCR assay to detect both IVSII-1 (G-A) and FS8-9 insG mutations. Moreover, we have distinguished homozygous and heterozygous forms of these mutations successfully. The frequency of IVSII-1 (G-A) mutation from 30 patients in Isfahan was 86.6% (33.3% heterozygote, and 53.3% mutant homozygote) and for FS8-9 insG mutation was 16.6% (13.3% heterozygote, and 3.3% mutant homozygote).

Conclusion: Tetra-primers ARMS PCR could be a reliable, accurate and simple technique for genotyping SNP and different mutations. So far, no study was done on optimization methods for genotyping mutations in β-thalassemia by T-ARMS. Here, we successfully adjusted and enhanced this method for recognizing two common mutations (FSC-8/9 insG and IVSII-I (G-A)) of β-thalassemia in Isfahan population.

No MeSH data available.


Related in: MedlinePlus

PCR amplification for detection of IVSII-I mutation in separate vials. In lanes 1 and 4, IVSII-I OF and IVSII-I OR primers are used, representing the control sequence. In lanes 2 and 5, IVSII-I OF and IVSII-I IR primers are used, representing the healthy sequence (allele G). Finally, in lanes 3 and 6, IVSII-I IF and IVSII-I OR) are used, representing the mutant sequence (allele A). M is a 100 bp ladder
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Figure 2: PCR amplification for detection of IVSII-I mutation in separate vials. In lanes 1 and 4, IVSII-I OF and IVSII-I OR primers are used, representing the control sequence. In lanes 2 and 5, IVSII-I OF and IVSII-I IR primers are used, representing the healthy sequence (allele G). Finally, in lanes 3 and 6, IVSII-I IF and IVSII-I OR) are used, representing the mutant sequence (allele A). M is a 100 bp ladder

Mentions: In order to optimize genotyping of IVSII-I mutation by using the T-ARMS technique, after designing the primers (Table 1), the temperature and concentration gradient of MgCl2 were performed for each pair of primers in separate vials using PCR technique. The temperature of 55°C with a concentration of 6mM of MgCl2 was chosen as optimum annealing temperature and concentration respectively. Control, wild type and mutant product of IVSII-I for patient and health control with optimal annealing temperature and Mg concentration are shown in Fig. 2. In the next step, polymerase chain reaction as a multiplex was performed in a vial with specific concentrations of each primer. Performing multiplex reaction with specific concentrations and different ratios of outer and inner primers showed the optimal concentration for IVSII-I should be 3 pM for outer primers (IVSII-I OF and IVSII-I OR) and 15 pM for inner primers (IVSII-I IR and IVSII-I IF; Fig. 3).


Tetra-Primer ARMS PCR Optimization for Detection of IVS-II-I (G-A) and FSC 8/9 InsG Mutations in β-Thalassemia Major Patients in Isfahan Population.

Hajihoseini S, Motovali-Bashi M, Honardoost MA, Alerasool N - Iran. J. Public Health (2015)

PCR amplification for detection of IVSII-I mutation in separate vials. In lanes 1 and 4, IVSII-I OF and IVSII-I OR primers are used, representing the control sequence. In lanes 2 and 5, IVSII-I OF and IVSII-I IR primers are used, representing the healthy sequence (allele G). Finally, in lanes 3 and 6, IVSII-I IF and IVSII-I OR) are used, representing the mutant sequence (allele A). M is a 100 bp ladder
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4402417&req=5

Figure 2: PCR amplification for detection of IVSII-I mutation in separate vials. In lanes 1 and 4, IVSII-I OF and IVSII-I OR primers are used, representing the control sequence. In lanes 2 and 5, IVSII-I OF and IVSII-I IR primers are used, representing the healthy sequence (allele G). Finally, in lanes 3 and 6, IVSII-I IF and IVSII-I OR) are used, representing the mutant sequence (allele A). M is a 100 bp ladder
Mentions: In order to optimize genotyping of IVSII-I mutation by using the T-ARMS technique, after designing the primers (Table 1), the temperature and concentration gradient of MgCl2 were performed for each pair of primers in separate vials using PCR technique. The temperature of 55°C with a concentration of 6mM of MgCl2 was chosen as optimum annealing temperature and concentration respectively. Control, wild type and mutant product of IVSII-I for patient and health control with optimal annealing temperature and Mg concentration are shown in Fig. 2. In the next step, polymerase chain reaction as a multiplex was performed in a vial with specific concentrations of each primer. Performing multiplex reaction with specific concentrations and different ratios of outer and inner primers showed the optimal concentration for IVSII-I should be 3 pM for outer primers (IVSII-I OF and IVSII-I OR) and 15 pM for inner primers (IVSII-I IR and IVSII-I IF; Fig. 3).

Bottom Line: Moreover, we have distinguished homozygous and heterozygous forms of these mutations successfully.The frequency of IVSII-1 (G-A) mutation from 30 patients in Isfahan was 86.6% (33.3% heterozygote, and 53.3% mutant homozygote) and for FS8-9 insG mutation was 16.6% (13.3% heterozygote, and 3.3% mutant homozygote).Tetra-primers ARMS PCR could be a reliable, accurate and simple technique for genotyping SNP and different mutations.

View Article: PubMed Central - PubMed

Affiliation: Genetics Division, Dept. of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.

ABSTRACT

Background: β-thalassemia, a monogenic autosomal recessive disorder, is prevalent in Middle East, particularly in Iran. In Iran, near to 20 mutations in the β-globin gene are introduced as common mutations with varying incidence frequencies in each city. Therefore, detection and screening for couples at high risk can help to solve the problems of this disease. In this study, optimized genotyping of two common mutations in Isfahan Province, IVSII-I (G-A) and FSC-8/9 insG, was performed using the T-ARMS method.

Methods: In this case-control study, 10 healthy individuals and 30 patients affected by β-thalassemia major with a mean 24.76 ± 4.5 years were selected from Omid Hospital in Isfahan Province. After designing tetra primers for two prevalent mutations IVSII-I (G-A) and FSC-8/9 insG, samples were genotyped using tetra-primers ARMS PCR technique.

Results: We have developed a sensitive single tube tetra-primers PCR assay to detect both IVSII-1 (G-A) and FS8-9 insG mutations. Moreover, we have distinguished homozygous and heterozygous forms of these mutations successfully. The frequency of IVSII-1 (G-A) mutation from 30 patients in Isfahan was 86.6% (33.3% heterozygote, and 53.3% mutant homozygote) and for FS8-9 insG mutation was 16.6% (13.3% heterozygote, and 3.3% mutant homozygote).

Conclusion: Tetra-primers ARMS PCR could be a reliable, accurate and simple technique for genotyping SNP and different mutations. So far, no study was done on optimization methods for genotyping mutations in β-thalassemia by T-ARMS. Here, we successfully adjusted and enhanced this method for recognizing two common mutations (FSC-8/9 insG and IVSII-I (G-A)) of β-thalassemia in Isfahan population.

No MeSH data available.


Related in: MedlinePlus