全基因组扩增技术对DNA甲基化保真性的影响

doi:10.3969/j.issn.1004-616x.2013.04.001

癌变·畸变·突变

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全基因组扩增技术对DNA甲基化保真性的影响

王二曼,何志妮,李道传,张标,陈雯,曾晓雯*

中山大学公共卫生学院预防医学系，广东广州 510080

收稿日期:2013-01-10 修回日期:2013-05-28 出版日期:2013-07-30 发布日期:2013-07-30
通讯作者: 曾晓雯，E-mail：zengxw123 @ 163.com
作者简介:王二曼 (1984- )，女，河南省开封市人，硕士研究生，研究方向：分子毒理学。
基金资助:
国家杰出青年基金 (30925029)，中国博士后科学基金特别资助项目 (201104373)

The effect of whole genomic amplification on DNA methylation fidelity

WANG Er-man，HE Zhi-ni，LI Dao-chuan，ZHANG Biao，CHEN Wen，ZENG Xiao-wen*

Faculty of Preventive Medicine, School of Public Health, Sun Yat-sen University, Guangzhou 510080, Guangdong, China

Received:2013-01-10 Revised:2013-05-28 Online:2013-07-30 Published:2013-07-30
Contact: ZENG Xiao-wen，E-mail：zengxw123 @ 163.com

摘要/Abstract

摘要：

目的：采用焦磷酸测序技术对全基因组扩增后DNA甲基化保真性进行研究。方法：采用全基因组扩增试剂盒对L02、A549和HCT116细胞进行全基因组亚硫酸氢盐修饰后的DNA扩增，选择3个特异基因 (MGMT、GSTP1和P16)和反映基因组总甲基化水平的LINE1序列，利用焦磷酸测序技术检验扩增前后甲基化的保真性。结果：DNA平均扩增量为246倍，片段长度>1 000 bp，符合甲基化检测的基本要求。反映总甲基化水平的LINE1在扩增前后无明显变化；扩增后3个特异基因甲基化的变化幅度与扩增前基因甲基化水平相关。当基因的甲基化水平≥60%时，其扩增前后变化不大；当基因甲基化水平在≥20%~60%之间时，扩增前后的变化较大，且无一致的变化趋势；而当基因甲基化水平低于<20%时，扩增后基因甲基化水平下降显著。此外，焦磷酸测序结果表明全基因组扩增对各特异基因多个CpG甲基化位点的影响与对该基因平均甲基化水平的影响一致。结论：全基因组亚硫酸氢盐修饰后的DNA扩增适用于基因整体甲基化水平和甲基化率≥60%的特异基因的甲基化研究，但会影响低DNA甲基化率基因 (<60%)的DNA甲基化的保真性。

关键词: DNA 甲基化, 全基因组扩增, 焦磷酸测序, 保真性

Abstract:

OBJECTIVE: Pyrosequencing was applied to study the effect of whole genome amplification (WGA) on the fidelity of DNA methylation. METHODS：EpiTect Whole Bisulfitome Kit was used to perform DNA amplification after whole genome bisulfite modification of L02，A549 and HCT116 cells. The DNA methylation of three specific genes (MGMT，GSTP1，P16) and LINE 1 sequence were analyzed by pyrosequencing. RESULTS：The DNA amplification obtained was 246 folds higher in DNA content and > 1 000 bp fragment length which met the basic requirements of DNA methylation detection. The DNA methylation level of LINE1 did not change after WGA，indicating that WGA had no effect on the whole genome methylation. However，the change of specific DNA methylation after WGA correlated with their original methylation level. When the original methylation rate was higher than 60%，the methylation condition of three genes remained stable after WGA. There was significant change with irregular trend of DNA methylation level after WGA when the initial DNA methylation ranged between ≥20%-60%. DNA methylation rate of three genes declined significantly in all cells when the initial DNA methylation level was lower than 20%. In addition，pyrosequencing result showed that the effect of WGA on genes CpG sites methylation level was similar to that on its mean methylation rate. CONCLUSION：WGA technology was suitable for the study of whole genome methylation and specific gene with higher methylation rate (≥60%). However，we must take caution for using the WGA technology when the rate of DNA methylation was relative low (< 60%).

Key words: DNA methylation, whole genome amplification, pyrosequencing, fidelity

王二曼,何志妮,李道传,张标,陈雯,曾晓雯. 全基因组扩增技术对DNA甲基化保真性的影响[J]. 癌变·畸变·突变, doi: 10.3969/j.issn.1004-616x.2013.04.001.

WANG Er-man，HE Zhi-ni，LI Dao-chuan，ZHANG Biao，CHEN Wen，ZENG Xiao-wen. The effect of whole genomic amplification on DNA methylation fidelity[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2013.04.001.

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全基因组扩增技术对DNA甲基化保真性的影响

The effect of whole genomic amplification on DNA methylation fidelity

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