PCR引物法及随机引物法标记cDNA探针杂交效率的研究

目的 :为提高原位杂交所用cDNA探针的杂交效率。方法 :采用正义引物PCR法及正义、反义引物PCR法制备地高辛 (Dig)标记的TGF_βcDNA探针。同时采用常规的随机引物法标记上述探针。用斑点杂交及原位杂交法比较三者的效率。结果 :在斑点杂交中 ,正义引物PCR法所标记的单链探针的杂交效率最高 ,高于随机引物法一个数量级。在探针浓度相同条件下 ,正义引物PCR法标记探针的原位杂交效果优于随机引物法标记的探针。正义和反义引物PCR法标记的双链探针的杂交效率次于正义引物PCR法所标记的单链探针

The Study on Hybridizing Efficiencies of cDNA Probes Labeled with PCR Priming and Random Priming

To improve the hybridizing efficiency of cDNA probe used in situ hybridization the sense primer PCR(SPCR) as well as both sense and antisense primers PCR(SASPCR) were adopted to generate the Dig labeled TGF_β cDNA hybridizing probe.For the sake of contrast,the random priming was used.The dot blot and in situ hybridization were applied to compare the efficiencies of the three methods.The results showed that:In dot blot,the hybridizing efficiency of probe labeled with the method of SPCR was the highest in the three methods,and 10 times higher than that of probe labeled with random priming.Under the same concentration of probe,the in situ hybridizing reaction of using the SPCR probe was more distinct than that of using random priming probe.The hybridizing efficiency of probe labeled with SASPCR was lower than that of probe labeled with SPCR.