【摘要】 目的:建立基于Rotor-Gene的链融解曲线法的甲基化标记筛选技术,分析各种影响因素,评估其实用性。方法:克隆Hela细胞雄激素受体基因外显子-1的Bisulfite-PCR产物,挑出经测序证实的可以代表不同甲基化程度的一组质粒,并以之为模板,在Rotor-Gene3000实时定量PCR仪进行扩增和链融解,分析融链温度、链融解曲线与甲基化程度、甲基化模式的相关性及各种影响因素。结果:基于Rotor-Gene的链融解曲线法,不仅可以区分甲基化与非甲基化,而且能够鉴别片段内细微的甲基化差异;主要的影响因素有升温幅度、缓冲液离子强度、产物浓度等。结论:基于Rotor-Gene的链融解曲线法,可以集扩增和链融解于一体,是一种经济简便的甲基化分析技术,可用于样品的甲基化筛选。更多还原

【Abstract】 Aim:To develop a simple methylation scanning technique using methylation specific melting curve analysis (MS-MCA) with Rotor-Gene.Methods:Plasmid clones containing androgen receptor(AR) exon-1 bisulfite fragment which represented different methylation patterns were prepared. Melting curve of the PCR products amplified from these plasmids was analyzed with a Rotor-Gene 3 000 real time thermal cycler.Results: PCR products representing different methylation patterns could be distinguished clearly. Main factors that affected the melting curves included salt concentration, products concentration and temperature changing speed.The relationship between melting temperature, melting curve and methylation degree, methylation pattern and the influencing factors were analyzed.Conclusion: The methylation-specific melting curve analysis with Rotor-Gene simplifies methylation scanning of genomic DNA.更多还原