High speed analysis of methylation pattern of genomic DNA with the improved bisulfite method
| Project/Area Number |
17590060
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Nihon Pharmaceutical University (2006)
Okayama University (2005) |
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Principal Investigator |
NEGISHI Kazuo Nihon Pharmaceutical University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (70116490)
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| Co-Investigator(Kenkyū-buntansha) |
YAHATSU Hikoya Shujitu University, Faculty of Pharmacy, Professor, 薬学部, 教授 (10012593)
OYAMA Hiroshi Nihon Pharmaceutical University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 助教授 (50221700)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | 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 |
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| Research Abstract |
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
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Report
(3 results)
Research Products
(15 results)
