A New Technique of Detecting β-thalassaemia Mutati
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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.
Key words: β-thalassaemia; preamplification; reverse dot blot (RDB)
β-thalassaemia is one of the autosomal genetic blood diseases characterized by absent or decreased production of normal β-hemoglobin. It is caused by the mutations within the β-globin gene. Prenatal diagnosis for β-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. The amounts of DNA for preimplantation genetic diagnosis (PGD) and non-invasive prenatal diagnosis are very limited. The sensitivity of nested PCR is great enough to allow the analysis of DNA in a single cell. Successful PGD protocols for β-thalassaemia have been introduced by using nested PCR followed by restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and direct sequencing [1-3]. However, a single cell can be analyzed only once and it is difficult to detect multiple mutations in any one cell by nested PCR. Most have not addressed the problem of diagnosing the wide spectrum of genotypes encountered in most populations in which β-thalassemia is common ......
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.
Key words: β-thalassaemia; preamplification; reverse dot blot (RDB)
β-thalassaemia is one of the autosomal genetic blood diseases characterized by absent or decreased production of normal β-hemoglobin. It is caused by the mutations within the β-globin gene. Prenatal diagnosis for β-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. The amounts of DNA for preimplantation genetic diagnosis (PGD) and non-invasive prenatal diagnosis are very limited. The sensitivity of nested PCR is great enough to allow the analysis of DNA in a single cell. Successful PGD protocols for β-thalassaemia have been introduced by using nested PCR followed by restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and direct sequencing [1-3]. However, a single cell can be analyzed only once and it is difficult to detect multiple mutations in any one cell by nested PCR. Most have not addressed the problem of diagnosing the wide spectrum of genotypes encountered in most populations in which β-thalassemia is common ......
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