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Journal of Chemistry
Volume 2014, Article ID 536134, 8 pages
http://dx.doi.org/10.1155/2014/536134
Research Article

Construction of Differential-Methylation Subtractive Library

1School of Life Sciences, Lanzhou University, Lanzhou 730000, China
2Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Nangang District, Harbin 150081, China

Received 16 May 2014; Accepted 23 June 2014; Published 9 July 2014

Academic Editor: Wang Zhenhua

Copyright © 2014 Wei Hu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Stress-induced ROS changes DNA methylation patterns. A protocol combining methylation-sensitive restriction endonuclease (MS-RE) digestion with suppression subtractive hybridization (SSH) to construct the differential-methylation subtractive library was developed for finding genes regulated by methylation mechanism under cold stress. The total efficiency of target fragment detection was 74.64%. DNA methylation analysis demonstrated the methylation status of target fragments changed after low temperature or DNA methyltransferase inhibitor treatment. Transcription level analysis indicated that demethylation of DNA promotes gene expression level. The results proved that our protocol was reliable and efficient to obtain gene fragments in differential-methylation status.