Construction Of The Gene Mutation Library Of Chinese β-thalassemia

AbstractObjective To construct a complete gene library containing all mutation types of Chinese ~-thalassemia. This gene library in which the DNA sequences are awareness can be used as standard samples to evaluate the specificity and reliability of a new diagnostic approaches including the gene chip for detecting Chinese ~3-thalassemia mutations.Methods 1. Construction of gene mutation library involving 13 common types of Chinese j3-thalassemia: Firstly, the target genes are obtained directly from human genome DNA sample with a given 13-thalassemia mutation using PCR method. These genes are then cloned into pGEM~-T vector using classical gene engineering techniques. The host bacterial strain is E. coli JM1O9. Each of inserted DNA fragment in these recombinants was sequenced to verify the DNA sequence of mutants cloned.2. Construction of gene mutation library involving 16 rare types of Chinese ~-thalassemia: The genes of interest containing the desired point muation were prepared from the wild-type ~3-globin gene using the PCR-based sitedrected mutagenesis(SDM). These modified genes were cloned into the pGEM-T vector and sequenced.3. Establishment of two improved megaprimer PCR methods for SDM.Method 1: First, a plasmid DNA template containing full length wild-type f~globin gene was constructed. The megaprimer containing SDM residues was then synthesized using the internal mutagenic primer and the forward flanking primer in the first round PCR. The final full-length mutation product was procuced by PCR using a gel-purified megaprimer and a reverse flanking primer in the second round of PCR.Method 2: This protocol is based on the design of two different plasmid [)NA templates. Template I lacks the binding site for reverse flanking primer, and template 2 lacks the binding site for forward flanking primer. In this way, two wild-type plasmid DNA templates should not be amplified by the two flanking primers simultaneously. A megaprimer was synthesized in the first PCR reaction (PCRI) using template I, forward primer and mutagenic primer. The megaprimer obtained without the cumbersome gel purification step wasdirectly added to the second PCR reaction system. The second PCR reaction (PCR2) has two stages and was performed using template 2, megaprimer, forward and reverse flanking primer. During the first stage of PCR 2, the megaprimer was extended to form the frill-length mutation product. In the second stage of PCR 2, the extended megaprimer containing 5DM residues was subsequently amplified with the two flanking primers. So all of the final PCR products contained the desired mutation.Results 16 types of rare j3-thalassemia mutations in Chinese were obtained successfully using the PCR-based mutagenesis, and the success rate of mutagenesis could reach 100%. A complete gene mutation library involving all 29 Chinese f~-tha1assemia mutants was constructed.Conclusions Two PCR-based site-directed mutagenesis methods have been established. These simple and convenient assays should be useful, as a routine molecular cloning tool, in the mutagenesis research and in construction of the rare genes causing genetic diseases. By using 5DM and direct PCR techniques, we have completed a cloning library covering all known 13-thalassemia mutants in Chinese. Our library will be used as a standard samples to evaluate the new molecular diagnostic approaches including the gene 梒hip for typing Chinese f3-thalassemia mutations.