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Year : 2017  |  Volume : 1  |  Issue : 1  |  Page : 2-8

Development of hydrolysis probe real-time polymerase chain reaction and high-resolution melting analysis protocols for screening of e280k and c.1055del.g mutations in phenylalanine hydroxylase gene

1 Department of Biochemistry, Faculty of Medicine, University of Tripoli, Tripoli, Libya
2 National Centre for Disease Control, Tripoli, Libya
3 Faculty of Medical Technology, University of Tripoli, Tripoli, Libya

Correspondence Address:
Abdulla Bashein
Department of Biochemistry, Faculty of Medicine, University of Tripoli, P. O. Box 70733, Tripoli
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/LJMS.LJMS_2_17

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Background: Phenylketonuria (PKU) is one of the most common inborn errors of amino acids metabolism. It is an autosomal recessive disease that is caused by mutations in phenylalanine hydroxylase (PAH) gene. In the North Africa and Eastern Mediterranean region, E280K missense mutation and c.1055del.G frameshift mutation in PAH gene are one of the most common pathogenic mutations seen in PKU patients. Materials and Methods: In this study, we developed molecular protocols for rapid screening of the PKU patients for these two mutations. These protocols are based on hydrolysis probe real-time polymerase chain reaction technique using allele-specific probes labeled with 6-carboxyfluorescein (FAM) for wild-type (WT) and hexachloro-6-carboxyfluorescein (HEX) for mutant genotypes and Black Hole Quencher 1 as a quencher and high-resolution melting analysis using EvaGreen saturating dye. Results: There was complete accordance between the developed protocols in differentiating genotypes and they proved to be rapid, sensitive, and efficient for the detection and differentiation between WT, mutant, and heterozygous genotypes of the E280K and c.1055del.G mutations. Conclusions: These protocols allow easy molecular screening of the mutations studied among the families of affected people, especially for premarital screening.

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