G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.
G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.
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DOI：
10.1371/journal.pone.0113955
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Kennedy MA
中科院分区：
文献类型：
--
作者：
Stevens AJ;Stuffrein-Roberts S;Cree SL;Gibb A;Miller AL;Doudney K;Aitchison A;Eccles MR;Joyce PR;Filichev VV;Kennedy MA
我们观察到当对母系印记的人类MEST亚型5‘端的CpG岛上的一个区域进行基因分型时，等位基因的明显非孟德尔行为。该区域包含三个总连锁不平衡的单核苷酸多态(SNPs)，使得在人类群体中只出现两种单倍型。尽管使用了多个引物和几种基因分型方法，但每个受试者只检测到一种单倍型，而不是两种都检测到。我们观察到该区域包含能够形成几个G-四链结构的基序。圆二色谱和天然聚丙烯酰胺凝胶电泳法证实，在钾离子存在下，至少有三个G-四链体在体外形成，其中一个结构在聚合酶链式反应(PCR)缓冲液中的Tm大于99℃。我们证明，在PCR扩增过程中，甲基化的母体等位基因总是丢失的，而G-四链体的形成和甲基化胞嘧啶的存在都导致了这种现象。这种观察到的亲本特异性等位基因缺失对研究和诊断环境中印记基因的分析具有重要意义。
We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a T m of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.
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DOI：
10.1073/pnas.93.18.9821
发表时间：
1996-09-03
影响因子：
11.1
作者：
Herman, JG;Graff, JR;Baylin, SB
通讯作者：
Baylin, SB
影响因子：
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影响因子：
3.8
作者：
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通讯作者：
Tycko, B
影响因子：
14.9
作者：
Kikin O;D'Antonio L;Bagga PS
通讯作者：
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