小中大1、对于重亚硫酸氢盐脱氨基处理的效率,怎么评估?
单从实验来讲,体外甲基化一份DNA,做PCR,转克隆,应该至少挑10个克隆,对该序列10个以上的CpG岛做测序统计,来评估重亚硫酸氢盐脱氨基处理的效率!(我曾做了30个CpG岛的评估,转化效率达98%,因此判定此处理是有效的可以进行基于重亚硫酸氢盐处理的其它实验如MSP或COBRA)
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以下问题,请教:
1, 体外甲基化反应是否按设计发生了,如何确认?
2, 所谓做PCR,应该是指BSP,而BSP引物针对处理转化的DNA序列设计,本身只扩增处理转化的。也就是只把转化了的分子挑出了测序,那自然是转化效率很高了。是否存在此问题,请讨论。
3, 如何克服克隆偏性Cloning bias等假象?参见 Methods. Volume 27, Issue 2, June 2002, Pages 101-107. Identification and resolution of artifacts in bisulfite sequencing
4, 挑够了10个克隆,结果的可信度如何呢?The limited number of clones examined per sample drastically reduces the statistical power of bisulfite sequencing data. If, for example, 5 out of 10 clones are methylated at any given site, the 95% confidence interval for the true proportion of DNA methylation at that site is between 18.4 and 81.6%. Also, if a difference in DNA methylation of 20% between two samples (from 50 to 70%), is to be statistically validated, 100 clones for each sample would have to be sequenced and analyzed. Brena RM, Auer H, Kornacker K, Hackanson B, Raval A, Byrd JC, Plass C. Accurate quantiAccurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform.Nucleic Acids Res. 2006 Feb 7;34(3):e17 Nucleic Acids Research 2006 34(3):e17
5, “对该序列10个以上的CpG岛做测序统计,来评估重亚硫酸氢盐脱氨基处理的效率!(我曾做了30个CpG岛的评估,转化效率达98%,------”该句内一个CpG岛是指一个CpG二核苷酸?还是一个包含很多CpG二核苷酸的CpG Island.
6, 无论MSP、BSP,引物可以自行设计,也可以参考文献。对文献应该学会甄别,区分好坏。文献已经有的,并且符合研究目的的,尽量采用之,可少走弯路,节省国家经费。即使自行设计,肯定也是要参考文献的。我们都是要站在巨人肩膀上的吗!!!本人也做了N多基因,全部是自行设计的引物系统,但不得不看大量的文献。吾辈怎敢拿着国家的钱,去撞运气,总是要严密论证的吗。