应用cDNA文库法制备HCV-1诊断芯片探针

DNA Microarray Probe Preparation Using cDNA Fragments Library from HCV Genotype 1

目的 探讨利用cDNA文库法制备丙型肝炎 (HCV -1)诊断基因芯片探针的可行性。方法 用限制性内切酶Sau3AⅠ消化HCV 1a及 1b全长cDNA ,所得的酶切片段 72℃补平加A ,AT克隆 ,PCR初步鉴定 ,并测序。结果 HCV两个亚型 1a、1b的全长cDNA得到 5 7个大小相对一致 ( 2 0 0 -10 0 0bp)的片段 ,平均每个亚型约 2 8个 ,PCR及序列分析表明 ,所扩增的片段均属于HCV -1的特异基因 ,可做为HCV -1诊断基因芯片探针。结论 利用cDNA文库法收集片段是一种快速。

Objective To investigate the feasibility of preparing HCV-1 diagnostic microarray probes using the technique of cDNA fragments library construction. Methods The full-length cDNAs of HCV of subtypes 1a and 1b were digested with restriction endonuclease Sau3A I, and the resulted fragments were cloned into the pMD18-T vector. Positive clones were isolated and identified by sequencing. Results A total of 57 different fragments were obtained, and sequence analysis showed that all the fragments ranging from 200 to 1000bp were specific gene fragments of HCV genotype 1, which can be efficiently used as probes in microarray prepapration. Conclusion The method of preparing microarray probes by construction of cDNA fragments library was effective, quick and simple.