应用McMSP分析Rassfla启动子区域甲基化

Analysis Rassfla Methylation Patterns by McMSP

目的应用McMSP分析抑癌基因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SYBRGreenⅠ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、1：1000、1：5000、1：10000稀释后，分别行MSP和McMSP。结果（1）不应用商业试剂盒同时将SYBRGreenⅠ浓度降低，仍能获得较好结果；（2）Q—PCRMCA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1：10000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—PCRMCA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。

Objects 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） MeMSP 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.