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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 FSC 8/9 mutation in one vial using T-ARMS technique (multiplex PCR). From left to right, M is a 100 bp marker; lane 1 is a sample from patient; lane 2 is a sample from a healthy individual. The sample from patient is confirmed as heterozygous in multiplex conditions, as demonstrated previously in separate vials
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Figure 5: PCR amplification for detection of FSC 8/9 mutation in one vial using T-ARMS technique (multiplex PCR). From left to right, M is a 100 bp marker; lane 1 is a sample from patient; lane 2 is a sample from a healthy individual. The sample from patient is confirmed as heterozygous in multiplex conditions, as demonstrated previously in separate vials

Mentions: For FSC-8/9 mutation, optimum temperature and concentration gradients of MgCl2 50mM was 68°C and 1.5 μl which obtained by amplification of each fragment in separate vials (Fig. 4). In the next step, polymerase chain reaction as a multiplex was performed in a vial with specific concentration of each primers and it became evident that 5 pM of each primers is suitable for identifying the FSC-8/9 mutation. Results of optimal annealing temperature, primers and MgCl2 concentration for multiplex PCR have been shown in Fig. 5 and 6.


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 FSC 8/9 mutation in one vial using T-ARMS technique (multiplex PCR). From left to right, M is a 100 bp marker; lane 1 is a sample from patient; lane 2 is a sample from a healthy individual. The sample from patient is confirmed as heterozygous in multiplex conditions, as demonstrated previously in separate vials
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: PCR amplification for detection of FSC 8/9 mutation in one vial using T-ARMS technique (multiplex PCR). From left to right, M is a 100 bp marker; lane 1 is a sample from patient; lane 2 is a sample from a healthy individual. The sample from patient is confirmed as heterozygous in multiplex conditions, as demonstrated previously in separate vials
Mentions: For FSC-8/9 mutation, optimum temperature and concentration gradients of MgCl2 50mM was 68°C and 1.5 μl which obtained by amplification of each fragment in separate vials (Fig. 4). In the next step, polymerase chain reaction as a multiplex was performed in a vial with specific concentration of each primers and it became evident that 5 pM of each primers is suitable for identifying the FSC-8/9 mutation. Results of optimal annealing temperature, primers and MgCl2 concentration for multiplex PCR have been shown in Fig. 5 and 6.

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