Rapid quantification of total genomic DNA methylation degree by short-end injection capillary zone electrophoresis

https://doi.org/10.1016/j.chroma.2008.01.032Get rights and content

Abstract

We propose a new capillary zone electrophoresis method applying short-end injection technique for the fast evaluation of methylcystosine/total cytosine ratio after acidic DNA hydrolysis. By short-end injection and by using a 100 mmol/l Tris solution titrated with 1 mol/l phosphoric acid to pH 3.75 as background electrolyte, cytosine and methylcytosine were separated with a good resolution in less than 1.5 min. Stepwise multiple linear regression with DNA methylation degree as the dependent variable and age, cysteine, homocysteine and methionine as independent variables, showed a negative association with age and that total cysteine is the most important determinant of DNA methylation.

Introduction

Although the term epigenetics was introduced by Waddington since 1942 to refer to “the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being” [1], never as in these last years it has become newsworthy. The original definition, however, has been refined. Nowadays, epigenetic word indicates those reversible heritable traits that violate the conventional notion of inheritance and that are due to the alteration in genes expression that do not entail changes to the underlying DNA nucleotides sequence. Several evidences have been collected indicating the existence of a system with gene-regulating activity accountable for numerous events involving gene-expression, that are not explainable solely by the DNA code itself or by the classical control mechanisms. Processes such as X-chromosome inactivation, genomic imprinting and embryonic development are under epigenetic control and an epigenetic regulation has been also recognized as an important issue in carcinogenesis [2]. It is thought, however, that the list of disorders to which epigenetic abnormalities might contribute is very long [3], [4], [5], [6]. Molecular processes underlying epigenetic phenomena encompass a large collection of mechanisms such as DNA methylation, histone modifications or chromatin remodelling. Among these, DNA methylation is the most studied epigenetic post-synthesis modification of DNA polymer. In this reaction, a hydrogen atom is replaced with a methyl group at the level of the carbon 5 position of the pyrimidine ring of cytosine (C) to form 5-methylcytosine (mC). This modified cytosine is designed as the fifth base of human DNA and it constitutes approximately 1% [7] of the bases in mammalian genomes. A specific class of DNA methyltransferases (DNTMs) catalyzes this transformation by using S-adenosyl-l-methionine (SAM) as methyl group donor. This covalent modification of DNA occurs in the context of the palindromic dinucleotide 5′-CpG-3′ [8], [9], [10] and mainly involves the CpGs in bulk DNA. Conversely, the CG-rich regions upstream of the promoter area of 50–60% of all genes, the so-called CpG islands, usually are not methylated. To this regard, there are evidences that when changes in the methylation status of this 0.5–4 kilobase (kb) sequence occurs, also the activity of the CpG islands-linked genes is affected. When this process is framed in a pathological context, the dysregulation in the expression of the genes involved can lead directly to many different disease states. Therefore, due to the impact that an aberrant either hypo- or hyper-methylation of cystosine may have on human health, techniques to reliably detect and measure DNA methylation are important tools for diagnostic [11] and biological investigations. For this purpose, extremely various methods have been proposed [12] and, although an initial delay [13], also capillary electrophoresis (CE) has been proven to be a powerful technique for the determination of the extent of DNA methylation [14], [15]. In this light, we propose a new method applying short-end injection technique for the fast evaluation of total genomic DNA methylation degree. Moreover, to find links between DNA methylation and some potential epigenetic biohumoral mediators, whose levels may be responsive to the environmental factors, we have also measured the plasma levels of some biochemical parameters such as homocysteine, cysteine, methionine, glycaemia, total cholesterol, HDL cholesterol, creatinine and triglycerides as well as the correlation between age and DNA methylation status of 71 apparently healthy individuals.

Section snippets

Standards

Cytidine (Cy), 5-methylcytidine (mCy), adenosine (Ad), guanosine (Gu), thymidine (Ty), H3PO4, HCl, formic acid, Tris, and deoxyribonucleic acid from calf thymus were purchased from Sigma–Aldrich, Italia (Milan, Italy). All the nucleosides were prepared as 1 mmol/l stock solutions in Milli-Q grade water (Millipore, Milford, MA, USA) and stored at −80 °C until use. Fresh working solutions from 0.25 to 200 μmol/l (calibrators) were obtained by serial dilution in ultrapure water of stock solutions.

Participants to the study and sample collection

Background electrolyte optimization

In our previous methods [17], [18], we found that Tris phosphate run buffer allowed good efficiency peaks and baseline analytes separation when run was performed in acidic condition and normal injection mode. Starting from this knowledge, in an attempt to set up a new throughput capillary electrophoresis method for the fast separation and detection of cytosine and methylcytosine, we decided to use a Tris phosphate acidic run buffer in a short-end injection configuration. When an acidic running

Discussion

Among the analytical approaches so far developed for the assessment of global DNA methylation [19], [20], [21], the acidic hydrolysis of the nucleic acid by means of formic acid followed by detection and quantification of the products of DNA hydrolysis [22], provides significant advantages in terms of time and effort expended. After acidic treatment, DNA is reduced to free positively charged bases which can be separated by capillary electrophoresis. Due to its great efficiency, CE allows to

Acknowledgements

The work described in this paper is based in part on the Master's Thesis of Ms. Elisabetta Pisanu conducted at University of Sassari; the authors wishes to express appreciation for her technical advice. We also thank Ms. Maria Antonietta Meloni for her help in editing the English and grammar of the manuscript.

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