克隆外显子探针反向斑点杂交检测DMD基因缺失

目的 :制备检测 DMD基因常见易缺失外显子的核苷酸探针 ,通过反向斑点杂交试验验证其特异性 ,为初步研制 DMD基因诊断芯片作准备。方法 :从健康人外周静脉血白细胞提取基因组 DNA,应用经典 18对引物对 DMD基因常见易缺失外显子进行 PCR扩增。将扩增产物与 p GEM@- T Easy载体连接 ,转化 E.coli JM10 9感受态细胞。克隆目的片段 ,并作测序鉴定。以此为探针进行反向斑点杂交试验。结果 :序列分析表明 ,克隆片段代表 DMD基因 18个常见易缺失外显子 ;以这些片段为探针进行反向斑点杂交 ,其结果与 PCR相符。结论 :克隆的基因片段用作探针在反向斑点杂交试验中显示出较好的特异性 ,可用于 DMD基因常见缺失的检测

Detection of deletions of the DMD gene by reverse dot-blotting hybridization with the cloned exon probes

Qbjective: Preparation of probes used to detect the major deletion prone exons of DMD gene, and doing a feasibility study by reverse dot blotting with them. Methods: Human genomic DNA was extracted from peripheral venose leukocytes of healthy people. Using PCR technique, 18 nucleic acid fragments were amplified. The fragments was extracted from 2% agarose gel, and then connected with pGEM T Easy vector. The recombinants were transformed into E. coli JM 109 competent cells, followed by being planted in Amp LB culture medium and being cultured. The positive clones were selected. The fragments were completely released by Not I digestion of recombinants. The specificity of these fragments used as probes was detected by reverse dot blotting hybridization assay with a patient of DMD and a healthy people. Results: The cloned fragments were high consistent with several exons of the DND gene. The hybridization results were coincidental with the results of PCR identification. Conclusion: The cloned exon probes are specific and suitable for the identification of clinical DMD gene deletion diagnoses.