摘要目的 应用McMSP分析抑癌基冈Rassfla启动子区域甲基化,同时比较MSP与McMSP之间的异同.方法 (1)对常规甲基特异性PCR(MSP)扩增后的产物进行熔解曲线分析(MCA);(2)将SYBR Green I直接加入PCR反应前体系进行扩增;(3)探索McMSP分析的灵敏度,将标本按1:10、1:100、1:500、1:1 000、1:5 000、1:10 000稀释后,分别行MSP和MeMSP.结果 (1)不应用商业试剂盒同时将SYBR Green I浓度降低,仍能获得较好结果;(2)Q-PCR MCA能将完全甲基化和完全未甲基化DNA标本区分;(3)在将DNA标本1:10 000稀释后(DNA量＜2pg)仍可以检测到所需要的目的产物,而常规MSP在1:100稀释(DNA量＞100pg)时就已不能检测到产物;(4)McMSP区分正常癌旁组织和癌组织的能力较常规MSP大大增强.结论 利用Q-PCR MCA分析Rassfla启动子区域甲基化迅速准确,操作简便,结果以曲线形式给出,具有较常规MSP更高的灵敏度和区分能力.

abstractsObjects Detect TSG Rassfla promoter region methylation pattern based on Q-PCR,and compare McMSP and MSP.Methods Firstly,run MCA to the MSP products;secondly,add SYBR Green I to the pre-PCR mix; Finally, dilute the original DNA templates and compare the sensitivity between MSP and McMSP.Results (1)The results are satisfied even without commercial kits and lower SYBR Green I concentration;(2)McMSP can distinguish the samples which are fully methylated and fully unmethylated; (3)Q-PCR can perform perfectly even the original (DNA templates < 2pg),while,MSP become introducible even the original (DNA templates > 100pg); (4) McMSP can distinguish normal and cancer tissue more efficiently than MSP.Conclusions Analyzing TSG promoter region methylation pattern by Q-PCR MCA is quick, sensitive and advantage, as well as its better distinguish ability.