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RESEARCH ARTICELS
A New Technique of Detecting β-thalassaemia Mutations from a Single Cell
Ping Yi 1*, Li Li1, Hong Yao 1, Bing Deng 3, Zhuqin Chen 1, Yuanguo Zhou 2
Abstract: To explore a technique for detecting β-thalassaemia multipoint mutations from a single cell simultaneously, a set of allele specific oligonucleotide (ASO) probes used for detecting the eight familiar β-thalassaemia mutations (CD41-42, IVS-II-654, CD17, TATA box nt-28, CD71-72, TATA box nt-29, CD26, IVS-I-5) were immobilized on a strip of nylon membrane. The genome of an individual cell was amplified by Primer Extension Preamplification (PEP) with the mixture of 15-base random oligonucleotides. The aliquots (5μl) from PEP were used to amplify the objective gene fractions of β-thalassaemia gene by nested or semi-nested PCR. The membrane was hybridized with the final amplified products and then treated with Streptavidin - HRP and color development. 3 lymphocytes were picked up from blood samples of 1 healthy female and 4 patients who had β-thalassaemia mutations respectively. Each single lymphocyte was lysed in the proteinase K buffer. The amplifIcation efficacy was 93.3% and the rate of dropping out allele was 6.7%. Reverse dot blot (RDB) was applied to the final amplified products from the five participants. The results of diagnosis were same to the expected ones, and their genotypes were N/N, CD17(A→T)/N, IVS-II-654(C→T)/CD17(A→T), CD41-42(-CTTT)/N and TATA box nt-28(A→G)/N, respectively. So we believe that, PEP and RDB could detect multiple β-thalassaemia mutations from a single cell simultaneously and be applied to preimplantation genetic diagnosis and non-invasive prenatal diagnosis for β-thalassaemia. The research provides experimental evidences for the feasibility of applying PEP and DNA array technology to screening multiple genetic mutations from a single cell.
A New Technique of Detecting β-thalassaemia Mutations from a Single Cell
Ping Yi 1*, Li Li1, Hong Yao 1, Bing Deng 3, Zhuqin Chen 1, Yuanguo Zhou 2
Abstract: To explore a technique for detecting β-thalassaemia multipoint mutations from a single cell simultaneously, a set of allele specific oligonucleotide (ASO) probes used for detecting the eight familiar β-thalassaemia mutations (CD41-42, IVS-II-654, CD17, TATA box nt-28, CD71-72, TATA box nt-29, CD26, IVS-I-5) were immobilized on a strip of nylon membrane. The genome of an individual cell was amplified by Primer Extension Preamplification (PEP) with the mixture of 15-base random oligonucleotides. The aliquots (5μl) from PEP were used to amplify the objective gene fractions of β-thalassaemia gene by nested or semi-nested PCR. The membrane was hybridized with the final amplified products and then treated with Streptavidin - HRP and color development. 3 lymphocytes were picked up from blood samples of 1 healthy female and 4 patients who had β-thalassaemia mutations respectively. Each single lymphocyte was lysed in the proteinase K buffer. The amplifIcation efficacy was 93.3% and the rate of dropping out allele was 6.7%. Reverse dot blot (RDB) was applied to the final amplified products from the five participants. The results of diagnosis were same to the expected ones, and their genotypes were N/N, CD17(A→T)/N, IVS-II-654(C→T)/CD17(A→T), CD41-42(-CTTT)/N and TATA box nt-28(A→G)/N, respectively. So we believe that, PEP and RDB could detect multiple β-thalassaemia mutations from a single cell simultaneously and be applied to preimplantation genetic diagnosis and non-invasive prenatal diagnosis for β-thalassaemia. The research provides experimental evidences for the feasibility of applying PEP and DNA array technology to screening multiple genetic mutations from a single cell.