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Thanks to the ultrahigh sensitivity of single-molecule imaging, this method only requires 50 pg of DNA or less for each measurement, representing orders of magnitude less DNA than is required by previous quantification methods such as the HPLC-MS or other fluorescence-based methods (19, 21–23).

Besides being a general detection and quantification method, single-molecule imaging provides a unique opportunity to study the colocalization states of 5m C and 5hm C, which has been unknown because no previous method could perform an integrated analysis of 5m C and 5hm C in the same DNA molecule.

This identifies the previously undetectable and unappreciated 5hm C/5m Cp Gs as one of the major states for 5hm C in the mammalian genome and suggest that they could function in promoting gene expression.

Epigenetic modifications of DNA contribute critical regulatory functions to the underlining genetic sequence.

DNA fragments are end-labeled with biotin, 5hm C is labeled with Cy5, and 5m C is labeled with Cy3.

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