Approaches to Detecting DNA Base Modification in the Brain

X. Li1, W. Wei2

1University of California Irvine, Irvine, CA, United States; 2The University of Queensland, St Lucia, QLD, Australia


Chromatography and mass spectrometry

The first approach developed for detecting DNA modifications was reported more than 90 years ago when Johnson & Coghill (1925) showed the presence of the pyrimidine 5-methylcytosine (5mC) in a nucleic acid by observing the optical properties of the crystalline picrate. However, the accuracy of their report has been called into question, and this technique has not been widely recognized.

In 1947, a chromatography experiment was conducted to separate amino acids by migration with organic solvents in filter paper (Consden, Gordon, & Martin, 1947). Hotchkiss (1948) then optimized this technology with a butyl alcohol system, which allows isolated nucleic acids to be recovered under favorable conditions. This, in turn, allowed purine and pyrimidine to be identified, and their quantities can be determined by UV spectrophotometry. Wyatt successfully used paper chromatography to measure the global amount of 5mC by UV spectrophotometry. Paper chromatography has thus been considered as the first method that can be used to determine DNA modifications (Wyatt & Cohen, 1952). This approach is still widely used.

After the development of improved technologies, high-performance liquid chromatography (HPLC) methods have been used to accurately assay genome-wide DNA methylation. However, HPLC methods require a large quantity (5-50 |tg) of genomic DNA and synthesis of 32P-labeled deoxyribonucleosides, as well as a relatively long running time (Gama-Sosa et al., 1983; Wagner & Capesius, 1981). The development of electrospray ionization (ESI) enabled liquid chromatography-mass spectrometry (LC/ MS) to be used for the quantitative determination and structural characterization of polar and ionic molecules, such as nucleic acids. In 2002, a new online LC/MS method for the measurement of methylated cytosine was developed and named LC/ESI-MS. In this approach DNA is enzymatically hydrolyzed and DNA hydrolyzates subsequently are separated by reverse-phase HPLC. By coupling UV spectra analysis and the mass spectra

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DNA Modifications in the Brain ISBN 978-0-12-801596-4

of chromatographic peaks, the system can rapidly, precisely, and selectively quantify global DNA methylation. Moreover, this approach has been successfully performed on as little as 1 pg of DNA (Friso, Choi, Dolnikowski, & Selhub, 2002).

To date, merging chromatography and MS has become a common approach, allowing very sensitive and reliable quantitative detection of different DNA modifications at the whole-genome level. This approach is capable of detecting both common and rare modifications, such as 5mC, 5-hydroxymethylcytosine (5hmC), N6-methyldeoxy- adenosine (m6dA), 8-oxo-7,8-dihydroguanine, and N7-methylguanine (Chao, Wang, Yang, Chang, & Hu, 2005; Friso et al., 2002; Greer et al., 2015; Hu, Chen, Hsu, Yen, & Chao, 2015). A major limitation of this approach is that, in general, it is not informative about the sequence context in which the DNA modifications occur. It is thus of limited utility in studying the function of any specific DNA modification.

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