![]() Key words: Reversed dot blot hybridization primer-specific amplification beta-thalassemia * Correspondence: Mohammad Al-Haggar, MD, Mansoura University Childrens Hospital, Mansoura, Egypt, Tel. We can conclude that common â-thalassaemia mutations can be simply and reliably detected using ARMS technique rather than by using the ready made strips applied for RDBH. Using both techniques, mutations of highest frequencies were intronic within IVS I-110, IVS I-6 and IVS I-1 and on the other hand, the least frequent ones were exonic and promoter mutations. Considering ARMS results were our gold standard ones, RDBH was found of average sensitivity and specificity in detection of the top three common mutations but when analyzing other mutations it was associated with relatively higher false positive rates. Sensitivity and specificity of RDBH against ARMS method was measured using ROC curve. Mutations were estimated and were compared to that determined via the use of ARMS technique through amplification using sequence specific primers (SSP) one for mutant and another for normal allele with internal control primers. Every DNA sample was tested for 8 common beta thalassaemia mutations using RDBH method (hybridization to 8 chemically-labelled probes fixed on membrane strips, one for mutant and another for normal probe sets) followed by colour detection and interpretation. They were subjected to a thorough history stressing on consanguinity and family history, blood sampling for standard haematological testing and DNA extraction. They comprised 23 males and 17 females with age range 2.4-18 years (mean 7.8☐.6). ![]() Forty patients were enrolled from those presented to Genetic Laboratories in Mansoura University Childrens Hospital, Egypt, for molecular diagnosis of â-thalassaemia. We aim to evaluate the newly developed Reversed Dot Blot Hybridization (RDBH) technique against the established Amplification Refractory Mutation System (ARMS) in detection of most common Mediterranean mutations of â-thalassaemia in Egyptian cases from Nile Delta Regions. So, it is a specific and multiplex detection assay for screening non-deletion alpha-thalassemia defects in Chinese.Abstract. This method does not need semi-nested PCR, and the products amplified by biotinylated primers can be used directly to hybridize with the probes on strips under the identical conditions of hybridization. Molecular analysis of the -globin gene was first carried out to confirm the hemoglobinopathy status in the patient samples by covalent reverse dot blot hybridization (CRDB), amplification. After being hybridized with sequence-specific oligonucleotide probes and colour development, it could simultaneously identify all six types of non-deletion alpha-thalassemias encountered in Chinese. The PCR system using biotin-labeled primers could specifically amplify a 1085 bp fragment of human alpha2 globin gene which encompasses all six alpha-thalassemia mutations. ![]() Label biotin to primers and amplify human alpha2 globin gene selectively, then hybridize PCR products with a set of oligonucleotide probes immobilized on strips, and develop colour to detect non-deletion alpha-thalassemia defects. To establish a rapid and convenient method of reverse dot blot (RDB) analysis for detecting the point mutations of non-deletion alpha-thalassemia in Chinese.
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