Rapid Diagnosis Of Nondeletion α-thalassemia By Reverse Dot Blot

Objective To establish a rapid and convenient reverse dot blot (RDB) assay for detecting six types of nondeletion a-thalassemia in Chinese. This method can be used as a tool for rapid diagnosing Chinese nondeletion a-thalassemia defects.Methods1. Preparation of genomic DNA samples and construction of rare gene mutations: Genomic DNA was extracted from peripheral blood using standard methods. The rare point mutations were prepared from the wild-type a2-globin gene using the PCR-based site-directed mutagenesis (SDM). These modified genes were cloned into the pGEM-T vector and sequenced.2. Preparation of membrane strips: 12 oligonucleotide probes were designed for six mutations of a-thalassemia and labeled with -NHs to the 5'-end. The membrane strips were prepared in our own Lab.3. Selective amplify human a2 globin gene by using PCR: Biotin-labled primers, Bio-Cl and Bio-C3, were designed for amplification of human a2 globin gene selectively. A 1,085-bp fragment obtained by PCR encompasses all six a gene mutations. These PCR products can be used directly for hybridizing to strips with fixed oligonucleotide probes.4. Hybridization: The PCR products were hybridized to strips immobilized a set of oligonucleotide probes followed by colour development to detect nondeletion a-thalassemia defects.Results The PCR system incorporating biotin-primer Cl and C3 specifically amplify a 1,085-bp fragment of the a2 gene which encompasses all six a-globin gene mutations. After hybridization to sequence-specific oligonucleotide probes and colour development, all of the six types of nondeletion a-thalassemia commonly encountered in Chinese can be identified.Conclusions The preparation of membrane strips used in this RDB detection system is simple and quick. The method dose not need semi-nested PCR and the products of PCR system in the method specifically amplify a 1085 bp fragment of the a2 gene which encompasses all six a gene mutationscan be hybridized directly to the membrane strips. The biotin is labeled directly to primers and the hybridization conditions are identical. So, it is a sensitive, specific and rapid method for screening nondeletion a-thalassemia defects. This established reverse dot blot technique could be used as a tool in rapid diagnosing for Chinese nondeletion a-thalassemia defects.