[Epigenetics 2:2, 86-95; April/May/June 2007]; ©2007 Landes Bioscience
Research Paper
Improved Quantification of DNA Methylation Using Methylation-Sensitive Restriction Enzymes and Real-Time PCR Ko Hashimoto1 Shoichi Kokubun1 Eiji Itoi1 Helmtrud I. Roach2,* 1Department
of Orthopaedics; Tohoku University Graduate School of Medicine; Sendai, Japan 2Bone
& Joint Research Group; Institure of Developmental Sciences; General Hospital; Southampton UK *Correspondence to: Helmtrud I. Roach; Bone & Joint Research Group; Institure of Developmental Sciences, MP887, General Hospital; Southampton SO16 6YD UK; Tel.: +44.2380.794.316; Fax: +44.2380.796.141; Email:
[email protected] Original manuscript submitted: 12/14/06 Manuscript accepted: 03/27/07 Previously published online as an Epigenetics E-publication: http://www.landesbioscience.com/journals/epigenetics/abstract.php?id=4203.
Key words DNA methylation, real-time PCR, quantitative method, MMP-13, methylation sensitive restriction enzyme
Abstract Heterogeneity of cells with respect to the DNA methylation status at a specific CpG site is a problem when assessing methylation status. We have developed a simple two‑step method for the quantification of the percent of cells that display methylation at a specific CpG site in the promoter of a specific gene. The first step is overnight diges‑ tion of genomic DNA (optimal conc. 20ng/5ml) with a relevant methylation‑sensitive restriction enzyme (optimal 2 units). This is followed by real time PCR, using the SYBR® Green method, with primers that bracket the site cleaved by the enzyme. By including fully methylated and fully non-methylated DNA in each PCR plate, the errors caused by non-specific digestion or incomplete digestion can be measured and used to adjust the raw results and thus increase specificity. The method can detect differences in methyla‑ tion status if these are more than 10%. No specialized equipment is required beyond the real‑time PCR system and the method can be adapted for any of the 53 commercially available methylation‑sensitive restriction enzymes.
DNA methylation, together with histone post‑translational modifications, are epigenetic mechanisms by which cells maintain a stable chromatin configuration that usually represses transcription. Epigenetics is defined as heritable changes in DNA structure without changes in the sequence. In the last 20 years, the study of epigenetics in general and DNA methylation in particular has expanded enormously as evidence has accrued about their crucial roles in embryogenesis,1 development2,3,4 as well as in disease processes, such as cancer5,6,7 or osteoarthritis.8,9 Definitive knowledge regarding the methylation status of specific CpG sites in specific promoters of particular somatic cells is still scarce, yet such knowledge would be crucial for a full understanding of how changes in DNA methylation influence gene transcription in normal development or disease. This difficulty is partly due to the cell‑selective nature of DNA methylation changes, partly to methodological problems of assessing methylation status. Unlike the genetic code, which is identical in every somatic cell in the body, the DNA methylation status varies between different cells/tissues or even between cells within a tissue. Hence there is a need for quantitative methods to assess the percentage of cells methylated at the CpG site of interest within a population with heterogeneous CpG methylation status.
Methods for Detection of DNA Methylation Two common methods for analysis of DNA methylation are methods based on bisulfite modification and those based on methylation‑sensitive restriction enzymes (MSREs). In the first, sodium bisulfite is used to deaminate cytosine to uracil while leaving methylated cytosines unchanged.10 Using primers designed to be complementary to the bisulfite‑converted sequences, but positioned so that the primers themselves do not contain CpG sites,11 the PCR‑amplified DNA is either purified and sequenced directly, or cloned into a vector before sequencing. In the sequence, cytosines of the CpG sites will be replaced by thymine when they are un‑methylated, but remain cytosines when methylated. This method provides information about the methylation status of every individual CpG site within the region bracketed by the primers. However, the method is cumbersome, bisulfite modified DNA is unstable and bisulfite may induce strand breaks, hence the product lengths for sequencing should not exceed 300 bp.11,12 Variations of the bisulfite method include methylation‑specific PCR (MSP)13 and Combined Bisulfite Restriction Analysis (COBRA).14 A major alternative method is the Methylation Sensitive Restriction Enzyme (MSRE) method.8,15,16 This utilizes the methylation sensitivity of the 53 currently commercially available restriction enzymes: When the CpG site is not methylated, the enzymes cleave the DNA and subsequent PCR amplification is abrogated. However, if the CpG site 86
Epigenetics
2007; Vol. 2 Issue 2
Quantification of DNA Methylation by Real-Time PCR
distinguish between methylated and non-methylated CpG sites.17,18,19 These techniques are sensitive and specific, providing the methylation states of all CpG sites within the primer or probe sequence are identical, but the methods depend on cumbersome bisulfite modification. Using the simpler MSRE method, there are several possibilities to quantify methylation. To quantify methylation of the gluthionine S‑transferase gene (GSTP1) in prostate cancer, digestion with HpaII was carried out and primers designed to bracket a promoter region containing six HpaII cleavage sites.20,21 The methylation‑insensitive isoschizomer MspI was used to determine whether complete digestion has taken place. A very complex method, termed COMPARE-MS,22 digests the genomic DNA with AluI together with HpaII. All methylated DNA fragments are captured using recombinant methyl‑binding domain proteins MBD2‑His6 magnetic bead construct prior to real‑time PCR with primers bracketing the HpaII site. These methods work well for the genes examined, but do not have wide applicability, because the CpG site of interest may be cleaved by MSREs other than HpaII and most MSREs do not have Figure 1. Optimization of amounts of genomic DNA and enzyme. Fully methylated or fully non-methylated DNA (2.5 to 250 ng/8.3µl reaction mixture) were digested with the methyla‑ methylation‑insensitive isoschizomers. tion-sensitive enzyme HpyCH4IV, with 0.5 to 4units/ reaction. The CpG site investigated was Here we report a simple real‑time PCR method, at -110bp in the MMP-13 promoter region. From these data, the optimal amounts were >10ng, which follows a one‑step digest with a MSRE and but
