SEARCH NCBI gene FOR 你的基因,找到你的基因,打开,可以看到 Genomic regions, transcripts, and products, 点击线条示意图上面的NC-XXXXXX.X,连接到你的基因序列,可以清楚的看到其每个外显子在基因组序列中的位置,第一个外显子的起点即转录起始点,围绕该点上下游500bp可以认为即所谓的核心启动子区,最小启动子区。扩大一点范围,可以远至转录起始点上游1000,乃至2000,都可归属于启动子区。其中有无启动子元件,可以用专门软件预测分析。
我一般就是在打开的基因序列页面上,通过修改上面的range: from XXXXX to XXXXXXX,来调出围绕基因转录起始点的序列,进行分析,设计合适的引物,进行MSP、BSP或克隆启动子。
当然,常见基因的启动子有的本身在数据库里就有记录,不用这么麻烦,直接调出就行了。但上述方法适应范围更广,别人没有研究过的,你也可以依据人类基因组计划成果进行研究。
该页面底部显示的序列不是该基因的转录序列。而是基因组序列,包括外显子和内含子的序列。改为Range: from 67326696 to 67329196 ,点后面的更新按纽,显示的就是我贴给你的序列。就是基因的启动子区。
对于该基因适用。对于其它基因,可能序列走向和HGP测序序列是互补的,这在注释条目里会有说明。此时,Range: from XXXXXXX to XXXXXXX,后边的数字会大于前边的。此时,应以后边的那个数字为中心修改,而不是前边的。否则你调出的是基因的尾巴,而不是基因的龙头,启动子区。作者: bs4665 时间: 2015-7-2 09:23
看了aifuhong博士发表的言论,深有感触,aifuhong在甲基化领域的研究确实化了不少心思,有自己的很多经验和心得,值得学习!我认真读了您的每一篇留言,很有收获。以下几个问题和您探讨(这里权且交流):
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)(关于CpG岛的定义我就不熬述了)
2、MSP难吗?
不难,96年建立该方法发了NAR上,现在是10年后了!
3、引物设计难一定参考文献吗?第一次做可以参考。我做了n多基因没有一个参考文献的,尽信书不如无书!我更相信事实依据。可以参考文献:Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter
4、如何知道MSP不是假阳性?或者,对自己设计的引物不自信?
因为定性PCR电泳条带是不足以说明任何问题的,只有测序比对序列才是“金”标准!
5、Bisulfite sequecing PCR(BSP),它的引物一般不含CpG岛,这样以保证能同等扩增甲基化和非甲基化的模板,然后转克隆测序,一般来说,至少是10个克隆;如果你只有1%的模板发生了甲基化,那么你最少得挑100个克隆才统计到!而MSP的灵敏度可以达到0.1%(参考:Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands)
5、如何找一个基因的启动子区?
先搞清楚定义,然后在DXY上查一下,有个blog专门论述此问题!
6、如何定量评估差异甲基化?
见附件!
如果有什么问题,或者对表观遗传的一些问题感兴趣,可以点击进入一起讨论一起进步,
cuturl('http://www.dxy.cn/bbs/post/view?bid=64&id=10553215&tpg=1&ppg=1&sty=1&age=0')
有一个好的氛围,一个好的平台,是我们飞翔的基础!作者: qianqin1977 时间: 2015-7-2 10:34
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。作者: 园丁## 时间: 2015-7-2 10:43
Accuracy and precision are conventional criteria in evaluating the performance or uncertainty of an analytical method. Accuracy is practically represented by difference of the result from a method and that from a reference one while precision is represented by the degree of variations of the results as expressed in CV. For quantitative analysis of DNA methylation, no reference method with which other methods could be compared is available yet. Bisulfite-mediated sequencing is currently accepted as one to produce most detailed and quantitative information on DNA methylation. Nonetheless, the bisulfite sequencing itself is not an absolute or acceptable reference method because it employs several manipulation processes through which distortion of information could be accompanied.
Incompleteness of bisulfite-mediated conversion of bases as well as biases in PCR, sub-cloning and sequencing processes are major potential sources of analytical uncertainties. Owing to unavailability of a reference method, the overall accuracy of our method for quantification of DNA methylation could not be directly assessed. Instead, only comparisons with the result of bisulfite-mediated sequencing are presented as indirect indicators while the shortcoming of the approach is fully acknowledged. Compared with bisulfite sequencing, our method does not include sub-cloning and sequencing procedures albeit the same bisulfite conversion and PCR procedures are included. Therefore, our method is expected to yield a quantification performance with a smaller or similar level of measurement uncertainty compared with that from bisulfite sequencing.
Nucleic Acids Res. 2006 May 5;34Musical Note:e61. Yang I, Park IY, Jang SM, Shi LH, Ku HK, Park SR. Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR.作者: 园丁## 时间: 2015-7-2 10:44
以下问题,请前辈解释:
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(注意,与是否甲基化无关)。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?作者: 园丁## 时间: 2015-7-2 11:07
Bisulfite sequecing PCR(BSP),它的引物一般不含CpG岛,这样以保证能同等扩增甲基化和非甲基化的模板,然后转克隆测序,一般来说,至少是10个克隆;如果你只有1%的模板发生了甲基化,那么你最少得挑100个克隆才统计到!而MSP的灵敏度可以达到0.1%(参考:Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands)
最近有点忙!
1、0.1%而不是1%;
2、1个CpG是CpG Island,多个是CpG Islands(CGIs);
3、关于CpG岛的定义(我们母语一个和多个CpG是没有复数区别的,确实经常产生这样的口误或者歧异!):参考文献:An evaluation of new criteria for CpG islands in
the human genome as gene markers(注意这里是复数)
BIOINFORMATICS Vol. 20 no. 7 2004, pages 1170–1177
DOI: 10.1093/bioinformatics/bth059
忙完这些天再来讨论,期待中!
平安夜平安幸福!NEW YEAR! My friends!作者: vvmmoy 时间: 2015-7-2 11:15