Abstract
In plant cells, genomic DNA exists in three organelles: the nucleus, chloroplast, and mitochondrion. Genomic DNA can be damaged by endogenous and exogenous factors, but the damaged DNA can be repaired by DNA repair systems. To quantify the extent of their repair activity of on individual genomic DNA, a PCR-based assay utilizing long amplicons is valuable for evaluable. This assay is based on the inhibitory effects of methyl methanesulfonate (MMS)-induced DNA damage on the amplicons. This assay is useful for assessing DNA double-strand repair pathways, such as homologous recombination repair, as it detects DNA double-strand breaks produced by MMS in vivo.
Keywords: Chloroplast, Genome stability, PCR, DNA damage, Physcomitrella patens
Background
The quantification of genomic DNA damage is useful for analyzing DNA repair mechanisms. This assay utilizes real-time PCR to quantify the nuclear, chloroplast, and mitochondrial DNA copy number for the normalization of long PCR products, providing more accurate quantification compared with that by the previous protocol by Hunter et al. (2010).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
The long amplicon PCR assay is based on Hunter et al. (2010) with slight modifications. The genomic DNA extraction protocol is based on PHYSCObase (http://moss.nibb.ac.jp/). This work was supported by the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Funding Program for Next-Generation World-Leading Researchers (NEXT Program).
References
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