PCR法制备地高辛标记探针改良PCR-Select Differential Screening试剂盒

目的:探讨采用地高辛(digoxin,DIG)标记探针替代PCR-Select differential screening试剂盒中同位素标记探针的方法.方法:抑制性消减杂交(suppression subtractive hybridization,SSH)所分离的差异表达序列转移至硝酸纤维素膜,以PCR法掺入DIG-11-dUTP制备探针,按DIG标记探针常规操作进行杂交显色,杂交阳性结果采用reverseNorthern blot再度杂交验证.结果:应用DIG标记探针改良PCR-Select Differential Screening试剂盒所得到的阳性结果经reverse Northern blot验证符合率达到90%.结论:DIG标记探针改良PCR-Select differential screening试剂盒在避免了放射污染同时具备很高的特异性,完全可以替代同位素标记探针.

Improve PCR-select differential screening kit with digoxin labelled probe

Aim: To explore a method to replace the isotope probe in the PCR-Select differential screening Kit with DIG labeled probe. Methods: Differential expressed sequences isolated from Suppression Subtractive Hybridization(SSH) was translocated onto nitric fibrin film. Probes were prepared with DIG-11-dUTP mixed in by PCR. Hybridization and coloration followed routine operation procedures of DIG lableled probe. Positive hybridization results were verified with reverse Northern blot. Results: The positive results from the PCR-Select differential screening Kit improved with DIG labeled probe achieved 90% correspondence verified by reverse Nor- thern blot. Conclusion: The PCR-Select differential screening Kit improved with DIG labeled probe maintaines high specificity while avoiding isotope pollution. It can replace isotope probe completely.