Han Weiguo, Cauchi Stephane, Herman James G, Spivack Simon D
Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, NYS Department of Health, Albany, NY 12201, USA.
Anal Biochem. 2006 Aug 1;355(1):50-61. doi: 10.1016/j.ab.2006.05.010. Epub 2006 Jun 2.
A tag-modified bisulfite genomic sequencing (tBGS) method employing direct cycle sequencing of polymerase chain reaction (PCR) products at kilobase scale, without conventional DNA fragment cloning, was developed for simplified evaluation of DNA methylation sites. The method entails subjecting bisulfite-modified genomic DNA to a second-round PCR amplification employing GC-tagged primers. Qualitative results from tBGS closely correlated with those from conventional BGS (R=0.935, p=0.002). In application, the intertissue and interindividual CpG methylation differences in promoter sequence for two genes, CYP1B1 and GSTP1, were then explored across four human tissue types (peripheral blood cells, exfoliated buccal cells, paired nontumor-tumor lung tissues), and two lung cell types in culture (normal NHBE and malignant A549). Predominantly conserved methylation maps for the two gene promoters were apparent across donors and tissues. At any given CpG site, variation in the degree of methylation could be determined by the relative height of C and T peaks in the sequencing trace. Methylation maps for the GSTP1 promoter diverged between NHBE (unmethylated) and A549 (completely methylated) cells in a previously unexplored upstream region, correlating with a 2.7-fold difference in GSTP1 mRNA expression (p<0.01). The tBGS method simplifies detailed methylation scanning of kilobase-scale genomic DNA, facilitating more ambitious genomic methylation mapping studies.
我们开发了一种标签修饰的亚硫酸氢盐基因组测序（tBGS）方法，该方法可对千碱基规模的聚合酶链反应（PCR）产物进行直接循环测序，无需进行传统的DNA片段克隆，从而简化了DNA甲基化位点的评估。该方法需要使用带有GC标签的引物对经过亚硫酸氢盐修饰的基因组DNA进行第二轮PCR扩增。tBGS的定性结果与传统BGS的结果密切相关（R = 0.935，p = 0.002）。在应用中，我们随后在四种人类组织类型（外周血细胞、脱落的颊细胞、配对的非肿瘤-肿瘤肺组织）以及两种培养的肺细胞类型（正常NHBE和恶性A549）中，探索了两个基因CYP1B1和GSTP1启动子序列中的组织间和个体间CpG甲基化差异。在不同供体和组织中，两个基因启动子的甲基化图谱主要是保守的。在任何给定的CpG位点，甲基化程度的变化可以通过测序图谱中C和T峰的相对高度来确定。在一个先前未探索的上游区域，GSTP1启动子的甲基化图谱在NHBE（未甲基化）和A549（完全甲基化）细胞之间存在差异，这与GSTP1 mRNA表达的2.7倍差异相关（p < 0.01）。tBGS方法简化了对千碱基规模基因组DNA的详细甲基化扫描，有助于开展更具挑战性的基因组甲基化图谱研究。
