Y Zhao1, M Zhong, Z Liu, X Xu. 1. Department of Cell Biology and Medical Genetics, the First Military Medical University, Guangzhou, Guangdong 510515 P. R. China. gzxuxm@public.guangzhou.gd.cn
Abstract
OBJECTIVE: Most frequent molecular lesions of alpha-thalassemia-2 determinants are deletions of one alpha-globin gene. Satisfactory PCR methodologies for detecting the deletions are required for molecular diagnosis and genetic screening since there was no internal control in most published PCR-based strategies. The purpose of this study is to develop a reliable PCR protocol specific for the two common alpha-thalassemia-2 determinants with internal control. METHODS: The multiple repeat elements and the high GC-content of the alpha-globin locus impose severe limitations on designing suitable primers and optimizing stable conditions for PCR. In this study, two multiplex PCR systems were successfully set up. One was designed to detect the rightward deletion (-alpha(3.7)/) with two pairs of primers including one newly optimized pair for amplification of the internal standard to indicate the success of failure of PCR amplification. The other, to the leftward deletion(-alpha(4.2)/) with three primers, which were designed according to the newly sequenced data of the -alpha(4.2) and HbQ-alpha(4.2) deletions in this lab(Genbank Accession No. AF221717). In the PCR system, one is used as a common upstream primer and the other two are used as specific downstream primers for typing the normal allele and the deletion one, respectively. RESULTS: Easily interpretable, unambiguous amplifications were observed by using the multiplex PCR systems for the detection of the two common alpha-thalassemia-2 determinants. The three or four primers were run in the same tube under the same condition and both of these two systems could be used at the same thermal cycle parameters. For typing the rightward deletion, a mutant-specific amplification of 1.7 kb and a 1140 bp amplified band as a normal and system control were produced. For typing the leftward deletion, two PCR-amplified bands, a 956 bp fragment specific for a -alpha(4.2) gene and a 1140 bp one for a normal allele were found. CONCLUSION: Two sets of PCR systems with internal controls for detecting the most common two alpha-thalassemia-2 determinants have been established and may be suitable for molecular diagnosis and population screening.
OBJECTIVE: Most frequent molecular lesions of alpha-thalassemia-2 determinants are deletions of one alpha-globin gene. Satisfactory PCR methodologies for detecting the deletions are required for molecular diagnosis and genetic screening since there was no internal control in most published PCR-based strategies. The purpose of this study is to develop a reliable PCR protocol specific for the two common alpha-thalassemia-2 determinants with internal control. METHODS: The multiple repeat elements and the high GC-content of the alpha-globin locus impose severe limitations on designing suitable primers and optimizing stable conditions for PCR. In this study, two multiplex PCR systems were successfully set up. One was designed to detect the rightward deletion (-alpha(3.7)/) with two pairs of primers including one newly optimized pair for amplification of the internal standard to indicate the success of failure of PCR amplification. The other, to the leftward deletion(-alpha(4.2)/) with three primers, which were designed according to the newly sequenced data of the -alpha(4.2) and HbQ-alpha(4.2) deletions in this lab(Genbank Accession No. AF221717). In the PCR system, one is used as a common upstream primer and the other two are used as specific downstream primers for typing the normal allele and the deletion one, respectively. RESULTS: Easily interpretable, unambiguous amplifications were observed by using the multiplex PCR systems for the detection of the two common alpha-thalassemia-2 determinants. The three or four primers were run in the same tube under the same condition and both of these two systems could be used at the same thermal cycle parameters. For typing the rightward deletion, a mutant-specific amplification of 1.7 kb and a 1140 bp amplified band as a normal and system control were produced. For typing the leftward deletion, two PCR-amplified bands, a 956 bp fragment specific for a -alpha(4.2) gene and a 1140 bp one for a normal allele were found. CONCLUSION: Two sets of PCR systems with internal controls for detecting the most common two alpha-thalassemia-2 determinants have been established and may be suitable for molecular diagnosis and population screening.
Authors: X M Xu; Y Q Zhou; G X Luo; C Liao; M Zhou; P Y Chen; J P Lu; S Q Jia; G F Xiao; X Shen; J Li; H P Chen; Y Y Xia; Y X Wen; Q H Mo; W D Li; Y Y Li; L W Zhuo; Z Q Wang; Y J Chen; C H Qin; M Zhong Journal: J Clin Pathol Date: 2004-05 Impact factor: 3.411