| β-thalassaemia is one of the autosomal genetic blood diseases characterized by absent or decreased production of normal beta hemoglobin. The incidence of β-thalassaemia is 0.65%, and the carriers account for 2%-3% in high incidence areas in South China. Over 170 types of mutations have been found in the whole world, among which, 21 are found in China. The most common mutations including CD41-42 (41.6%), CD17(18%), TATA-28 (8.0%), CD71-72(+A)(3.9%), TATA-29(1.2%) are at the same segment which is only 600 bps. Achondroplasia (ACH) is a common autosomal dominant inheritant dwarf caused by defection of cartilaginous ossification. The incidence of the neonate is 1-15/106. The pathogenic gene of ACH was located on 4p16.3 in 1994. Following researches revealed that it was caused by the mutation of the transmembrane dominant of fibroblast growth factor 3 (FGFR3) gene. 97% of the reported mutations are substitution of the 1138th base in the tenth exon. 95% is G-A and the others is G-C. Prenatal genetic diagnosis including preimplantation genetic diagnosis (PGD) has been proven to be an effective strategy for preventing the incidence of those diseases. Successful PGD on each single disease had been reported. But there was not any successful test which diagnosis more than two diseases at the same time for the restriction of detection technology and the sample content. Gene chip is a new technology which was developed by the recent years. This technology made it possible to diagnosis more than one genetic diseases with only one chip. In order to diagnosis β-thalassaemia and ACH at the same time, we successfully built a gene chip for gene mutation detection, which is simple, fast, accurate and reliable to the clinlic application after research on genetic diagnosis by single cell. The oligonucleotide probes with regard to the special mutations of these two diseases were designed by certain software and biotin labeling technology were used in our research. Methods and Results :According to the literature and the GENEBANK database, the mutant gene sequence were acquired and the primers were designed to amplify certain DNA segaments. Product 1 contained CD41-42, CD17 and TATA-28 of β-thalassaemia(accounting for 72.7% of the mutation frequency, 600bp); Product 2 contained the main mutation of ACH(accounting for 99% of the mutation frequency, 1264bp ). The reaction condition was optimized and two PCR reactions were processed in the same system.Four pairs of specific oligonucleotide probes based on the mutations were designed. The specificity and reliability of the probes were verified. The results indicated that difference of Tm between different probes. was less than 2℃. The probe parameters such as GC%, Hairpin dG, Dimer dG and repeated length met the demand of design. They could hybridize under the same conditions and could distinguish two target sequences whih single base differences.After checked on a nylon membrane, the selected detection probes and positive controls were used to make the glass chip. The results showed that the detection probes and positive controls could be seen while the negative controls, blank controls and other probes showed no signal when testing samples. The background was clear. To determine the optimal spotting concentration, the effect of probe concentration on its covalent immobilization and the hybridization efficiency were detected. The result showed that 1000-2000nmol/L of the probe could obtain relative good results. Thus we used the conentration of 1500nmol/L to make the DNA chip.In order to determine the optimal hybridization temperature, hybridization was performed under 40℃,45℃,50℃,55℃, respectively. The specificity of the probe was low when the hybridization temperature was below 45℃ and the sensitivity decreased significantly when the temperature was higher than 50℃. The hybridization was repeated 10 times under 48℃ and the results were stable. So 48℃ was chosen as our experimental hybridization temperature.Results of hybridization on the chip could be detec...
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