High speed analysis of methylation pattern of genomic DNA with the improved bisulfite method

采用改进的亚硫酸氢盐法高速分析基因组 DNA 甲基化模式

• 批准号: 17590060
• 负责人: NEGISHI Kazuo
• 金额: $ 2.24万
• 依托单位: Nihon Pharmaceutical University (2006)Okayama University (2005)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590060/
• 关键词: Ammonium bisulfite; Urea; Epigenetics; 5-Methyldeoxyuridine; Deamination; Cytosine; DNA; Transamination; 亜硫酸水素アンモニウム; 5-メチルシトシン; チミン; 重亜硫酸付加体; エピジェネティック; 発がん; 尿素; キトサン

Bisulfite genomic sequencing' involves treatment of a given DNA sample with bisulfite followed by PCR amplification and sequencing, through which C residues in the original DNA are found as T and mC as C. In this procedure, a treatment with 3-5 M sodium bisulfite for 12-16 hr at 55℃ has been conventionally used.Recently, we were able to improve the efficiency of this procedure by introducing a highly concentrated (10 M) bisulfite solution. The deamination can be finished within 5 min. Therefore, the procedure can be done much faster than by conventional methods. We had expected that the shorter incubation time could improve the degradation of DNA, the most serious shortcoming of the bisulfite genomic sequencing. However, we found that the degradation was still extensive as the conventional method. Aiming at further improvement of the procedure, we explored the effect of adding urea in this bisulfite treatment, as urea was reported to improve the deamination efficiency. The results indi … More cate that urea did not enhance the deamination efficiency. In the PCR, we did not observe significant improvements regarding the amounts of DNA necessary to obtain adequate amplification. We conclude that urea gave no significant effect in the bisulfite genomic sequencing of the DNA used. We are screening the chemicals that reduce DNA degradation during the bisulfite treatment.We have also examined the treatment at the near neutral pH, where degradation of DNA is expected to be slower than at acidic pH. We have succeeded to prepare a highly concentrated bisulfite solution at pH 6. We confirmed deamination of cytosines in DNA with this reagent. We are currently analyzing efficacy of this reagent.Bisulfite ion catalyzes transamination as well as deamination of cytosines. With use of strong nucleophilic amines, transamination is even faster than deamination. In the course of this study, we synthesized several transaminated cytidine analogues. We have demonstrated that they can be highly mutagenic in viral reverse transcription. Less

“亚硫酸氢盐基因组测序”涉及用亚硫酸氢盐处理给定的DNA样品，然后进行PCR扩增和测序，通过该测序，原始DNA中的C残基被发现为T，mC被发现为C。在此过程中，通常使用3-5 M亚硫酸氢钠在55℃下处理12-16小时。最近，我们能够通过引入高浓度（10 M）亚硫酸氢盐溶液来提高此过程的效率。脱氨可在5分钟内完成。因此，该过程可以比常规方法快得多。我们曾期望较短的孵育时间可以改善DNA的降解，这是亚硫酸氢盐基因组测序最严重的缺点。然而，我们发现，降解仍然是广泛的传统方法。为了进一步改进工艺，我们探索了在亚硫酸氢盐处理中添加尿素的效果，因为据报道尿素可以提高脱氨效率。结果表明， ...更多信息 添加尿素不能提高脱氨效率。在PCR中，我们没有观察到获得足够扩增所需的DNA量的显著改善。我们得出结论，尿素在所使用的DNA的亚硫酸氢盐基因组测序中没有显著影响。我们正在筛选在亚硫酸氢盐处理过程中减少DNA降解的化学物质。我们还研究了在接近中性pH的条件下的处理，在接近中性pH的条件下，DNA的降解预计会比在酸性pH下慢。我们成功地制备了pH为6的高浓度亚硫酸氢盐溶液。我们用该试剂证实了DNA中胞嘧啶的脱氨基作用。亚硫酸氢根离子催化胞嘧啶的转氨作用和脱氨作用。在使用强亲核胺的情况下，转氨作用甚至比脱氨作用更快。在本研究中，我们合成了几种转氨基胞苷类似物。我们已经证明，它们可以在病毒逆转录中高度致突变。少