GENOMIC SEQUENCING AND METHYLATION ANALYSIS BY LIGATION MEDIATED PCR
GENOMIC SEQUENCING AND METHYLATION ANALYSIS BY LIGATION MEDIATED PCR
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DOI：
10.1126/science.2814502
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发表时间：
1989-11-10
期刊：
影响因子：
56.9
通讯作者：
RIGGS, AD
中科院分区：
文献类型：
--
作者：
PFEIFER, GP;STEIGERWALD, SD;RIGGS, AD
基因组测序允许研究体内DNA甲基化和蛋白质-DNA相互作用，但由于哺乳动物基因组的复杂性，其使用受到限制。一种新开发的基因组测序程序，其中使用连接介导的聚合酶链反应（PCR），产生高质量的，可重复的序列梯，开始只有1微克的未克隆的哺乳动物DNA每个反应。通过包含多个引物可以同时产生不同的序列梯，并通过再杂交分别可视化。杂交所需的放射性相对较少，暴露时间也较短。基因组DNA中的甲基化模式很容易检测;例如，发现人X连锁PGK-1（磷酸甘油酸激酶1）5“区域中的17个CpG二核苷酸在无活性的人X染色体上甲基化，但在活性X染色体上未甲基化。
Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5''region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.