Sodium bisulfite treatment followed by PCR and DNA sequencing is widely considered the gold standard for the analysis of DNA methylation patterns. However, this technique generally requires substantial quantities of genomic DNA as starting material and is often associated with degradation of DNA. Here, we assess the feasibility of performing bisulfite sequencing on DNA isolated from 3-mm diameter punches of dried blood Guthrie spots. We demonstrate that it is possible to perform bisulfite sequencing from both freshly prepared and archived dried blood spots, using a combination of high purity DNA extraction and efficient bisulfite conversion. With the number of new technologies available for DNA methylation studies, we have extended this analysis and have successfully used a high-throughput mass spectrometry method for DNA methylation analysis on these samples. This provides a new source of material for epigenetic analysis of birth samples and provides an invaluable reference point to track temporal change in epigenetic profiles possibly linked with health and disease.
Dried blood or “Guthrie” spots are taken from children born in developed countries for newborn screening for a number of metabolic disorders including phenylketonuria (PKU) (1) and genetic testing using PCR-based assays (2). However, the utility of such samples for DNA methylation analysis has remained unclear due to the general requirement of microgram quantities of genomic DNA as starting material (3). More recently, bisulfite sequencing on limited amounts of DNA from microdissected samples has been demonstrated (4). Bisulfite sequencing is capable of discriminating methylated cytosine from unmethylated cytosine in genomic DNA (3). However, traditional bisulfite-based DNA modification has the inherent problem of DNA degradation due to the destructive nature of the modification process (5). Often, this has deleterious effects on the ability to successfully generate test PCR products for analysis. Furthermore, there is evidence for DNA fragmentation over time with storage of dried blood spots (6), and this can also influence the efficiency of any downstream, PCR-based applications. More recently, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry has been used as a means of high-throughput DNA methylation analysis (7) and could potentially be used to analyze such samples en masse. The degree to which these factors will influence the utility of bisulfite-based methylation analysis on Guthrie spots has not previously been tested.
Here, we aim to investigate the utility of DNA methylation analysis by bisulfite PCR-based DNA sequencing and SEQUENOM analysis on dried blood spot Guthrie samples. We analyzed the methylation status of two gene promoters, predicted to be either methylated [DNA methyltransferase 3-like (DNMT3L) gene promoter] or unmethylated [vitamin D receptor (VDR) gene promoter], by bisulfite PCR and sequencing of freshly prepared and aged, dried blood Guthrie spots. We further demonstrated the utility of this approach by examining the methylation levels at the H19 imprinted region using the SEQUENOM MassArray EpiTYPER analysis. The demonstration of archived DNA blood spots as a suitable template for DNA methylation analysis represents a unique resource for the retrospective analysis of epigenetic status of gene regulatory region methylation in whole blood specimens at birth.Materials and Methods
Seven genomic DNA extraction kits using a range of extraction methods including resin-based [Instagene Matrix and Chelex 100 (both from Bio-Rad Laboratories, Hercules, CA, USA)], lysis-based [Instagene Dx (Bio-Rad Laboratories), Extract-N-Amp (Sigma-Aldrich, St. Louis, MO, USA), Blood DNA Mini kit (Qiagen, Hilden, Germany), and ReadyAmp DNA Purification System (Promega, Madison, WI, USA)], or magnetic bead-based (ChargeSwitch Forensic DNA Purification kit; Invitrogen, Carlsbad, CA, USA) were used on 3-mm punches of freshly prepared dried blood Guthrie spots (Table 1). Where indicated, modification of the recommended protocol for dried blood spots was also carried out according to manufacturer's instructions.
Sequence tagged site (STS) marker PCR amplification of D6S510 and D6S265 (primers listed in Table 2) was performed on 1 µL extracted genomic DNA. PCR Mastermix (Promega) was used to amplify the STS markers, and the conditions used for amplification are as follows: denaturation at 95°C for 2 min, followed by 25 cycles of 95°C for 30 s, 53°C for 30 s, and 72°C for 30 s.
The yield of genomic DNA from each extraction method was determined by spectrophotometry (Nanodrop, Wilmington, DE, USA) and was further characterized by using a serial 2-fold standard curve and absolute quantitative PCR using primers to D6S510 and SYBR GreenER qPCR Supermix (Invitrogen, Carlsbad, CA, USA) using the following amplification conditions: polymerase activation at 95°C for 30 s, 53°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s. Quantitative PCR was analyzed using an ABI 7300 (Applied Biosystems, Foster City, CA, USA).
Four bisulfite modification methods were then evaluated on the extracted DNA in addition to a commonly used in-house method (based on Reference 3): EZ DNA Methylation kit (Zymo Research, Orange, CA, USA), CpGenome DNA Modification kit (Millipore, Billerica, MA, USA), and MethylEasy kit (Human Genetic Signatures, Sydney, Australia). Protocols were performed according to the manufacturer's instructions for the minimum amount of genomic DNA as starting material recommended for each kit.