Introduction

Epigenetics refers to the heritable, but reversible, regulation of various genomic functions mediated through partially stable modifications of DNA and chromatin histones (1). DNA methylation [i.e., cytosine methylation (^metC) at CpG and occasionally non-CpG sites] is one of the best-understood epigenetic mechanisms and has been investigated using a myriad of laboratory techniques (2). Over the last 15 years, the gold standard technique for fine mapping of ^metC has been based on the treatment of genomic DNA with sodium bisulfite, which converts unmethylated cytosines to uracils (and subsequently, via PCR, to thymidines), while methylated cytosines are resistant to bisulfite and remain unchanged (3). After sodium bisulfite treatment, DNA regions of interest are amplified and sequenced to identify C → T transitions or stable C positions, respectively corresponding to unmethylated and methylated cytosines in the native DNA. Typically, PCR products are either sequenced directly to provide a strand-specific average sequence for the population of DNA molecules or cloned and sequenced to provide methylation maps of single DNA molecules (3,4). An alternative approach to quantitatively assess the methylation level at specific cytosine sites within a product is methylation-sensitive single nucleotide primer extension (Ms-SNuPE) (5,6).

Conventional sodium bisulfite treatment is beset by a number of problems that result from the fact that to ensure the full conversion of unmethylated cytosines, the reaction is necessarily harsh and thus causes the large-scale degradation of genomic DNA (7). Alternative bisulfite-conversion protocols have been developed in which DNA is embedded in agarose during treatment to reduce DNA loss and ensure efficient conversion (8), but these procedures only partially reduce the degradation and are not suited to automated analysis. In many epigenetic studies, the amount of genomic DNA starting material is limited, especially in experiments utilizing valuable clinical samples, for example oocytes, laser capture micro-dissected cells, and microscope slides. Following bisulfite treatment, converted DNA is single-stranded and prone to further denaturation unless stored at −80°C. The degradation of DNA during and after sodium bisulfite treatment is a major hurdle to successful studies of DNA methylation.

In this report, we examine the applicability of whole genome amplification (WGA) to bisulfite-treated DNA. WGA methods are routinely used on normal genomic DNA for genotyping and sequence analysis when the amount of starting template is extremely low. One common application of WGA, for example, is in forensic analyses where it is used to improve both the quality and quantity of DNA and allows accurate genetic profiling from single cells (9). Two commonly used WGA strategies are (i) primer extension preamplification (PEP), a Taq DNA polymerase PCR-based reaction (10) and (ii) multiple displacement amplification (MDA), an isothermal genome amplification using φ29 DNA polymerase (11). Both methods are utilized widely for genotyping, with several studies demonstrating the reliability of data produced from WGA templates (12,13). In this study, we have primarily used an optimized version of the original PEP protocol to amplify sodium bisulfite-treated DNA. In addition, we tested a commercially available multiple displacement amplification (MDA) protocol (REPLI-g®) on a number of samples. Following gene-specific PCR, we assessed methylation profiles using three approaches: (i) direct sequencing of the entire product; (ii) the sequencing of cloned PCR products; and (iii) single-nucleotide primer-extension, and compared data obtained from usual bisulfite DNA (non-WGA DNA) to that obtained from WGA bisulfite-treated DNA (WGA DNA).

Materials and Methods

Bisulfite Treatment of Genomic DNA

Bisulfite treatment was performed using a standard protocol as described by Clark et al. (14). Briefly, approximately 500 ng genomic DNA was denatured in 0.3 M NaOH for 15 min at 37°C. After adding freshly prepared 3.5 M sodium metabisulfite and 1 mM hydroquinone solution (both from Sigma, St. Louis, MO, USA), samples were subjected to a 5-h incubation at 55°C under exclusion of light. The samples were then purified using MinElute® PCR purification columns (Qiagen, Valencia, CA, USA). Recovered samples were desulfonated in 0.3 M NaOH for 15 min at 37°C and neutralized. DNA was precipitated overnight in ethanol at −20°C and resuspended in 50 µL elution buffer (Buffer EB; Qiagen).