AFLP-based detection of DNA methylation
AFLP-based detection of DNA methylation
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DOI：
10.1007/bf02825064
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发表时间：
2000-01-01
影响因子：
2.1
通讯作者：
Korban, SS
中科院分区：
文献类型：
--
作者：
Xu, ML;Li, XQ;Korban, SS
利用Hpa Ⅱ和Msp Ⅰ对胞嘧啶甲基化反应的敏感性差异，建立了一种改进的扩增片段长度多态性（AFLP）技术，用于真核生物DNA甲基化的研究。基因组DNA用EcoR I和一种异构体的混合物消化，并连接到寡核苷酸接头上。在两轮选择性PCR扩增后，接着在Long Ranger凝胶电泳上进行DNA分离，限制性片段的子集可以展示在X射线胶片上。比较Hpa Ⅱ和Msp Ⅰ的AFLP带型，揭示了DNA甲基化的程度。该技术已成功地应用于苹果（Malus critica cv. Gala）从田间生长的成年树的叶子和体外生长的芽培养物中提取的基因组DNA。结果表明，25%以上的AFLP条带来自甲基化序列，其中少数为成年树或试管苗特有的条带。这些结果表明，该方案在鉴定甲基化DNA谱中是有效的。
By using the isoschizomers Hpa II and Msp I which display differential sensitivity to cytosine methylation, a modified amplified fragment length polymorphism (AFLP) technique has been developed to investigate DNA methylation profiles in eukaryotic organ isms. Genomic DNA was digested with a mixture of EcoR I and one of the isoschizomers, and ligated to oligonucleotide adapters. After two rounds of selective PCR amplification, followed by DNA separation on a Long Ranger gel electrophoresis, a subset of restriction fragments can be displayed on an X-ray film. Comparison of AFLP banding patterns between Hpa II and Msp I revealed the extent of DNA methylation. The technique has been successfully applied in this study to investigate DNA methylation profiles of apple (Malus domestica cv. Gala) genomic DNA extracted from leaves of field-grown adult trees and in vitro-grown shoot cultures. The results showed that up to 25 percent of AFLP bands were derived from methylated sequences, and among those, a few bands unique to either adult trees or in vitro shoots were observed. These results demonstrated that this protocol is effective in identifying methylated DNA profiles.