Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Henri Dunant Rd, Bangkok 10330, Thailand.
Acta Vet Scand. 2010 Mar 5;52(1):18. doi: 10.1186/1751-0147-52-18.
The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls.
Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII). The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates.
Approximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90%) were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8%) and the lowest in fibroblastic DNA (92.3%, P < or = 0.05). Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P < or = 0.05).
The powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the HpaII locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic cells.
本研究旨在应用一种任意引物甲基化敏感聚合酶链反应（PCR）检测方法（称为扩增甲基化多态性聚合酶链反应（AMP PCR）），以研究来自荷斯坦公牛的体细胞核和生殖细胞的甲基化谱。
从三头公牛的精子、白细胞和成纤维细胞中提取基因组 DNA，并使用甲基化敏感内切酶（HpaII）进行消化。将天然基因组和酶处理的 DNA 样品用作任意引物 PCR 检测的模板，使用 30 组单短寡核苷酸引物。PCR 产物在银染变性聚丙烯酰胺凝胶上分离。根据两个模板之间标记物的存在/缺失多态性，分析三种类型的 PCR 标记物；消化抗性标记物、消化敏感标记物和消化依赖标记物。
每个样品从 27 组引物中产生约 1000 个 PCR 标记物，其中大多数（>90%）为消化抗性标记物。白细胞 DNA 中的消化抗性标记物比例最高（94.8%），成纤维细胞 DNA 中的消化抗性标记物比例最低（92.3%，P < 0.05）。与其他细胞类型相比，精子中含有更多的消化敏感标记物（3.6%，白细胞和成纤维细胞分别为 2.2%和 2.6%，P < 0.05）。
AMP PCR 检测方法的强大之处在于产生与甲基化相关的标记物，而无需事先了解基因组序列。使用不同引物获得的数据提供了不同细胞类型中全基因组 DNA 甲基化含量的概述。通过使用该技术，我们发现 DNA 甲基化谱是组织特异性的。与体细胞相比，雄性生殖细胞在 HpaII 位置的甲基化程度较低，而具有良好特征的体细胞的染色质与多功能体细胞的染色质相比，甲基化程度较高。
