Development of a two-tube multiplex PCR method for common deletional determinants of [alpha]-thalassemia using induced fluorescent resonance energy transfer (iFRET) technology

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Development of a two-tube multiplex PCR method for common deletional determinants of [alpha]-thalassemia using induced fluorescent resonance energy transfer (iFRET) technology

 

Author: Lau, Ming-tat
Title: Development of a two-tube multiplex PCR method for common deletional determinants of [alpha]-thalassemia using induced fluorescent resonance energy transfer (iFRET) technology
Degree: M.Sc.
Year: 2008
Subject: Hong Kong Polytechnic University -- Dissertations.
Thalassemia.
Polymerase chain reaction.
Energy transfer.
Department: Dept. of Health Technology and Informatics
Pages: xi, 58 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2198991
URI: http://theses.lib.polyu.edu.hk/handle/200/592
Abstract: Alpha-thalassemia is a common hereditary condition caused by the deletion of one or more o-globin genes leading to decreased or absence of a-globin chain production. In Hong Kong, the prevalence of a-thalassemia carrier status is about 4%. The severity depends on the number of a-globin genes deleted. The clinical manifestation can be asymptomatic, moderate microcytic hypochromic anaemia or even severe anaemia. Both parents having two-gene deletion in one of their chromosome 16's (a "-thalassemia in heterozygous state) can give birth to hydrop fetalis. Therefore, a rapid and simple test should be used for screening for a-thalassemia because the routine method cannot determine the genotype. A conventional two-tube multiplex PCR was first designed in this study. This assay used four different pairs of primers targeting wild type alleles ( a 2) and three a -thalassemia deletional alleles (-a37, -a42 and ~OSEA) common in Southeast Asia. One pair of primers amplifying the APOB gene was also included in each tube to serve as an internal control. One hundred samples were collected from patients diagnosed to have o-thalassemia by routine haematological tests, and analyzed by the multiplex PCR methods. After tested by the newly established assay, 81% were found to have two-gene deletion and 5% three-gene deletion. The latter were consistent with result of numerous haemoglobin H inclusion bodies from the routine method: three samples had a genotype of —SEA/-or3 7 and two samples had a genotype of —SEA/-Q!4 2. In the second stage, attempt was made to adapt the conventional two-tube multiplex PCR to a real-time system. One of each pair of primers was labeled and PCR was monitor real time in LC480. The PCR products were also checked by agarose gel electrophoresis. Although non-specific signals were still be found during melting curve analysis by optimizing the concentration of SYBR Green I, initial denaturation step and ramping rate of LC 480, the bands for deletional alleles (-a3 7, -a42 and ~OSEA) could be found on the gel except the band for the wild-type. In conclusion, the conventional two-rube multiplex PCR was established and can be used as a reliable and simple method for determining the genotype of a-thalassemia in the laboratory. But the assay using real-time system still needs further investigation.

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