PERSONAL DOCENTE DE LA INSTITUCION EDUCATIVA MANUEL VERAMENDI E HIDALGO

There are a number of techniques which rely on the chemical modification of DNA in order to detect methylation. The Sss I acceptance assay was one of the first techniques developed for the measurement of methylated DNA. Sss I DNA methyltransferase is an orphan DNA methyltransferase, which recognises any CpG and uses S-adenosylmethionine (SAM) as a methyl donor in CpG methylation. Using tritium labelled SAM the number of unmethylated CpGs can be directly assessed by scintillation measurement of the pooled sample of DNA. However this technique does not provide an accurate measurement of individual CpGs (Wu et al, 1993).

-C-C-G-G- -G-G-C-C-

Hpa II cuts only if central Cytosine is unmethylated

Methylated site

The Sss I acceptance assay was followed by the development of chloroacetaldehyde assay, which was an assay for genome wide methylation, which relied on the detection of the intensely fluorescent ethenocytosine derivative of 5-methylCytosine, after bisulfite conversion of DNA. Methylation was quantified by fluorimeter readings. However, the chloroacetaldehyde assay, still lacked the sensitivity to assess individual CpGs (Dahl and Guldberg, 2003).

These methods have been superseded by bisulfite conversion of DNA. Treatment of genomic DNA with sodium bisulfite effectively deaminates unmethylated Cytosine residues to Uracil under specific conditions, where 5-methylCytosines are deaminated at a very slow rate (Frommer et al, 1992). Bisulfite converted DNA differs from genomic DNA, as unmethylated Cytosine is converted to Uracil, whereas methylated Cytosines are retained; this causes the 2 strands of bisulfite DNA to be non-complementary.

The bisulfite conversion of DNA is based on the deamination of Cytosine to Uracil. Genomic DNA is prepared by sonication, shearing, or digestion with restriction enzymes. The DNA is then denatured with sodium hydroxide and treated with a concentrated solution of sodium bisulfite at pH 5. The DNA is then desalted and desulfonated with sodium hydroxide, before being neutralised, desalted, and finally dissolved in water for use (figure 1.23) (Grunau et al, 2001).

Bisulfite converted DNA can then be amplified by bisulfite-specific PCR primers for (BS-PCR); or by methylation or unmethylation-specific PCR (MS-PCR or US-PCR). Bisulfite-specific primers (BSPs) hybridise with bisulfite converted DNA, the primers do not include CpG sites, and the assumption is made that single Cytosines are transformed to Uracil and incorporated as Thymine during PCR (Li and Dahiya 2002).

Methylation specific primers (MSPs) hybridise with methylated bisulfite converted DNA; primers include CpG sites which are assumed to retain their Cytosines. Unmethylation-specific primers (USPs) hybridise with unmethylated bisulfite converted DNA (i.e. the transformed DNA); primers include CpG sites in which the

Cytosines are assumed converted to Thymine. This is because after PCR with Taq DNA polymerase Uracil is incorporated into the PCR product as Thymine.

Figure 1.23: Sodium bisulfite conversion of Cytosine to Uracil

Treatment of Cytosine (1) with sodium bisulfite under acidic conditions (pH 5) yields 5,6-dihydroCytosine-6-sulfonate (12). This product is susceptible to hydrolytic deamination at C4 and is easily converted to 5,6-dihydroUracil-6- sulfonate (13). After desulfonation by treatment with NaOH, the final product is Uracil (14). (Taken from Shiraishi et al, 2002).

Bisulfite conversion can be combined with a number of techniques in order to effectively assess DNA methylation. Techniques include bisulfite sequencing, combined bisulfite restriction analysis (COBRA) and melt curve analysis. COBRA relies on the detection of a fluorescent tag on one of the primers after bisulfite- specific PCR (BS-PCR); restriction of the BS-PCR product allows the detection of different fragments from that product which indicate its methylation profile. One of the most comprehensive studies on DNA methylation profiling was undertaken at the end of 2006. Using bisulfite sequencing of genomic DNA, high resolution methylation profiles were obtained for 12 different tissue types including: leukocyte subsets CD4+ and CD8+ and placenta on chromosomes 6, 20 and 22. This was one of the first major attempts to assess DNA methylation at the genomic level. In total 2524 amplicons on 873 genes were analysed on chromosomes 6, 20, and 22; including 5’ untranslated regions, intronic regions, exonic regions and transcription factor binding sites (Eckhardt et al, 2006).

Approximately 27% of loci were hypomethylated (<20% methylation), and 42% of loci were hypermethylated (>80% methylation), with 30% of CpGs methylated in a heterogeneous pattern (20-80% methylation). This study is an important landmark, showing that bisulfite sequencing can be used to produce high throughput methylation profiles of genomic regions at the resolution of single nucleotides. The data generated from this study is an important resource, which in the context of

NIPD could be used to provide targets for candidate biomarkers (Eckhardt et al, 2006).

Restriction enzyme digestion can be used to reveal DNA methylation dependent sequence differences in bisulfite-specific PCR products. This procedure is termed COBRA and can be used to analyse the fractions of methylated and unmethylated DNA from a BS-PCR product (Xiong and Laird, 1997). The sequence conversion of a C → T can lead to creation of a new restriction site, as well as the retention of the Cytosine can lead to the retention of a restriction site. In a mixed sample of methylated and unmethylated DNA molecules, the methylated and unmethylated fraction can be determined by COBRA, to give a direct measurement of the percentage of each fraction, by the detection of a fluorescent tag in the primer of each product by a genetic analyser (Xiong and Laird, 1997).

Another technique termed melt curve analysis (MCA) can be performed after BS- PCR. The PCR product can be melted over a temperature gradient in order to assess the level of DNA methylation. Because G:C base pairs between complementary DNA strands contain 3 hydrogen bonds and A:T base pairs have 2 hydrogen bonds, DNA sequences with a higher G:C content have a higher melting temperature (denaturing temperature) Tm than sequences with a higher A:T ratio. Thus BS-PCR is followed by temperature gradient melting of DNA. Methylated sequences have a higher Tm than unmethylated sequences as they retain their G:C content (Guldberg et al, 2002). Most real-time PCR machines such as the BIORAD I-cycler carry out post-amplification melt profile analysis as standard.

In review MeDIP is a useful technique when combined with CGH or microarray analysis, it can provide methylation patterns for entire chromosomes in a single experiment, which can be used to target regions of interest. MSRE analysis is a simple technique, which is amenable to high throughput of techniques such as 96 well PCR and can also be used to provide information on methylation at the level of a single CpG. Bisulfite conversion is a laborious technique, as is not easily amenable to high through put analysis; however, its use in high throughput methods can yield high resolution information on selected regions of the genome.