摘要
目的建立简便可靠的非同位素杂交筛选方法，筛选家族性急性髓系白血病cDNA消减文库中的差异表达基因。方法用地高辛（DIG）标记消减文库cDNA作为探针，通过差异筛选技术进行差异表达片段的筛选，杂交阳性结果经RT-PCR再度验证。结果DIG标记探针浓度为250～500pg／μL，效率为48％～96％，并且杂交信号清晰，重现性好。应用DIG标记探针改良的差异筛选技术所得到的阳性结果经RT-PCR验证符合率达到86％。结论采用DIG标记探针具有高度的灵敏度和良好的特异性，可避免放射性污染．简化实验操作，可作为替代同位素^32P-dCTP标记探针的方法。
Objective To screen differentially expressed genes in cDNA subtracted library of a familial acute myelogenous leukemia by hybridization DIG labeled probe. Methods cDNA subtracted library was constructed from Fujian province an acute myelocyte leukemia family, one patient serve the tester and another normal familial person serve as driver. The differential expressed fragments of cDNA subtracted library were screened by reformed differential screening technique which the probes were labeled with DIG, and identified by one-step RT-PCR. Results The concentration of the probes labeled with DIG was between 250 and 500 pg/μL, the efficiency was 48%～96%. The hybridization signals were distinct and the results can be repeated again, The positive results used the improved technique was 86 % identically with RT-PCR test. Conclusion DIG labeling probes substituted the conventional ^32P-dCTP isotope probes. This technique was not only maintaining the high sensitivity and specificity but also averting isotope pollution and simplifying experimental procedures.
出处
《福建医科大学学报》
2007年第4期296-299,共4页
Journal of Fujian Medical University
基金
福建省科技厅重大科研项目(2003F003)
