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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

Schematic drawing of tetra-primers ARMS-PCR method. Genetic variation shown is a single nucleotide substitution. By using two pairs of primers, two different and specific segments are amplified for each nucleotide. Specificity of each of these two primers is because of a mispairing at the 3′ ends of the inner primers. However, to make the bindings more specific, a second mispairing is introduced at the second position at the 3′ end of each inner primer (17)
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Figure 1: Schematic drawing of tetra-primers ARMS-PCR method. Genetic variation shown is a single nucleotide substitution. By using two pairs of primers, two different and specific segments are amplified for each nucleotide. Specificity of each of these two primers is because of a mispairing at the 3′ ends of the inner primers. However, to make the bindings more specific, a second mispairing is introduced at the second position at the 3′ end of each inner primer (17)

Mentions: RFLP (Restriction Fragment Length Polymorphism), ASO-PCR (Allele Specific Oligo nucleotide-Polymerization Chain Reaction) and sequencing are used as a routine tests for genotyping and identifying β-thalassemia mutations in distinct populations, but each method still suffers from specific weaknesses and therefore, finding a simple, sensitive, accurate and inexpensive method for screening purposes is still one of the major problems in genetic diagnostic laboratories (14–16). In 2001, Shu Ye et al. introduced a simple and cost-effective method for genotyping single nucleotide polymorphisms called tetra-primers ARMS PCR (tetra primer amplification refractory mutation system) (17). This method includes a PCR reaction in a vial with two sets of primers followed by an electrophoresis analysis on agarose gel. One set of primers are specific for variety or polymorphism (inner primers) and the others one are outer primers that create control bond in PCR reaction. Inner primers are specific for mutant and wild type alleles and they are designed opposite of each other. PCR reaction using outer and inner primers were done in one vial, so two outer primers and inner-outer primers can react with each other and create different product with different length that they could be separated on gel electrophoresis. In Fig. 1, a schematic drawing for detecting single nucleotide polymorphisms by using the T-ARMS technique is presented. Unlike ASOPCR, in T-ARMS, for much specificity, two inner primers have another mismatch at second nucleotide near 3′ end.


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)

Schematic drawing of tetra-primers ARMS-PCR method. Genetic variation shown is a single nucleotide substitution. By using two pairs of primers, two different and specific segments are amplified for each nucleotide. Specificity of each of these two primers is because of a mispairing at the 3′ ends of the inner primers. However, to make the bindings more specific, a second mispairing is introduced at the second position at the 3′ end of each inner primer (17)
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Schematic drawing of tetra-primers ARMS-PCR method. Genetic variation shown is a single nucleotide substitution. By using two pairs of primers, two different and specific segments are amplified for each nucleotide. Specificity of each of these two primers is because of a mispairing at the 3′ ends of the inner primers. However, to make the bindings more specific, a second mispairing is introduced at the second position at the 3′ end of each inner primer (17)
Mentions: RFLP (Restriction Fragment Length Polymorphism), ASO-PCR (Allele Specific Oligo nucleotide-Polymerization Chain Reaction) and sequencing are used as a routine tests for genotyping and identifying β-thalassemia mutations in distinct populations, but each method still suffers from specific weaknesses and therefore, finding a simple, sensitive, accurate and inexpensive method for screening purposes is still one of the major problems in genetic diagnostic laboratories (14–16). In 2001, Shu Ye et al. introduced a simple and cost-effective method for genotyping single nucleotide polymorphisms called tetra-primers ARMS PCR (tetra primer amplification refractory mutation system) (17). This method includes a PCR reaction in a vial with two sets of primers followed by an electrophoresis analysis on agarose gel. One set of primers are specific for variety or polymorphism (inner primers) and the others one are outer primers that create control bond in PCR reaction. Inner primers are specific for mutant and wild type alleles and they are designed opposite of each other. PCR reaction using outer and inner primers were done in one vial, so two outer primers and inner-outer primers can react with each other and create different product with different length that they could be separated on gel electrophoresis. In Fig. 1, a schematic drawing for detecting single nucleotide polymorphisms by using the T-ARMS technique is presented. Unlike ASOPCR, in T-ARMS, for much specificity, two inner primers have another mismatch at second nucleotide near 3′ end.

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