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New Solutions for Epigenetics Research
Dr. Gerald Schock, Global Product Manager, QIAGEN GmbH, QIAGEN Strasse 1,40724 Hilden, Germany
gerald.schock@qiagen.com
www.qiagen.com
QIAGEN offers complete solutions for epigenetics analysis.
Summary
Epigenetics is an emerging field of science that is being studied with increasing intensity. The term “epigenetics” describes the study of heritable changes in gene function that occur without a change in the structural DNA sequence. In eukaryotes, a major epigenetic mechanism is DNA methylation. This article outlines the physiological significance of DNA methylation and describes new solutions for analyzing the methylation status of DNA.
Physiological Significance of DNA Methylation
Methylation of CpG islands by DNA methylases represses transcription and occurs both physiologically and in diseases, notably cancer. Physiologically, the processes of apoptosis and DNA repair are known to be influenced by DNA methylation. Methylation also regulates the expression of many genes, and the promoters of genes with tissue-specific expression patterns are methylated in non-expressing tissues, repressing their transcription (1). Methylation silences methylated imprinted alleles and brings about inactivation of one X chromosome. For example, the promoter region of the imprinted SNRPN gene contains a CpG island that is methylated in the maternally derived allele and unmethylated in the paternally derived allele. Aberrant methylation patterns of this gene cause Prader-Willi syndrome (only the methylated allele is present) or Angelman syndrome (only the unmethylated allele is present; 2).
The most widespread disease caused by aberrant methylation patterns is cancer. The abnormal patterns of gene expression found in cancers are frequently brought about by changes in methylation: for example, tumor suppressor genes in tumors are frequently inactivated by methylation of CpG islands in their promoters (3). Researchers are focusing on characterizing aberrant DNA methylation patterns in different tumor types, aiming to reveal patterns that might have diagnostic or clinical uses. Reversal of DNA methylation at hypermethylated sites is a potential therapeutic strategy, as this may restore expression of transcriptionally silenced genes.
Complete Solutions for DNA Methylation Research
The widespread occurrence of DNA methylation means that robust techniques to analyze the methylation status of DNA are of critical importance in epigenetics research. Both the location and the frequency of methylated sites appear to be significant, which presents a challenge to researchers, as they may need to focus on subtle changes in DNA methylation patterns.
Methylation analysis of DNA requires the flowing steps: isolation of high-quality DNA, conversion of non-methylated cytosines to uracil, and methylation analysis to distinguish methylated cytosines from uracils through PCR or sequencing. QIAGEN provides standardized solutions for each of these steps (Figure 1).
Isolation of high-quality genomic DNA is critical, as the DNA must be sufficiently pure to ensure complete conversion by sodium bisulfite (4). QIAGEN DNeasy Kits provide high-quality DNA from animal and plant samples, while manual QIAamp and PAXgene Kits and automated EZ1 Kits provide maximum recovery of genomic DNA from various clinical sample types.
Following DNA isolation, non-methylated cytosines must be converted to uracil so that the methylation status of the DNA can be subsequently determined using PCR or sequencing. QIAGEN now offers the EpiTect Bisulfite Kit to meet this need. This kit provides rapid and efficient bisulfite conversion and is described in more detail in subsequent sections of this article.
Typically, methylation-specific PCR is used to determine the methylation status of target DNA after sodium bisulfite treatment. The technique uses two sets of primers (one set annealing to unchanged, methylated DNA and the second set annealing to uracil). Stringent PCR conditions are needed to avoid nonspecific primer binding and amplification of DNA artifacts. QIAGEN HotStarTaq Plus DNA Polymerase, HotStarTaq DNA Polymerase, and the QIAGEN Multiplex PCR Kit provide highly specific and sensitive analysis of a range of targets in methylation analysis. Where full sequence analysis is required, this can be facilitated by use of QIAGEN PCR cleanup kits, PCR cloning kits, and a comprehensive range of plasmid prep kits.
Fast and Easy Bisulfite Conversion Using the EpiTect Bisulfite Kit
The EpiTect Bisulfite Kit is a new addition to QIAGEN’s product portfolio that provides all reagents needed for conversion of non-methylated cytosines to uracil. Standard sodium bisulfite protocols for this procedure are labor intensive and time consuming, requiring up to 19 hours and resulting in a low yields of highly degraded and only partially converted DNA (Table 1). In contrast, the EpiTect protocol requires less than six hours, converts more than 99 percent of unmethylated cytosines, and provides an innovative buffer that significantly reduces DNA degradation.
Unique DNA Protection
The EpiTect Bisulfite Kit includes DNA Protect Buffer, which is uniquely formulated to prevent the degradation of DNA that usually occurs during bisulfite treatment (Figure 2A). The inclusion of an indicator dye in the buffer provides confirmation of the correct pH for cytosine conversion (Figure 2B). The prevention of DNA degradation leads to accurate results, even when amplifying over 700 bp from very low amounts of template (Figure 3). The column-based purification ensures removal of all bisulfite treatment reagents, allowing storage of the converted DNA for at least six months without reduction in DNA quality. The EpiTect Kit also provides optimized protocols for challenging sample types, such as microdissected and formalin-fixed, paraffin-embedded tissues, which can be extremely valuable for identifying disease markers. Even very small or archived samples can be used.
The Future of Epigenetics Analysis at QIAGEN
Epigenetic analysis is a valuable tool with potential research and therapeutic implications for a range of conditions, including cancer. QIAGEN will extend its product portfolio for methylation analysis with the development of methylation detecting assays. In addition, through a strategic collaboration between QIAGEN and the molecular diagnostics company Epigenomics, solutions for methylation analysis will be developed that are optimized for use in molecular diagnostics protocols. These developments reflect the commitment by QIAGEN to developing streamlined protocols for epigenetics analysis.
Figure 1. Complete Solutions for Methylation Research
Table 1. Fast and Easy Bisulfite Conversion
Figure 2.
A. DNA Protect Buffer: A unique solution preventing DNA fragmentation during bisulfite treatment. The innovative formulation protects the DNA from the harsh conditions required for conversion (i.e., low pH, high temperature, and high bisulfite salt concentrations) and facilitates the formation of single-stranded DNA, enabling complete bisulfite conversion. B. The pH-indicator dye in the DNA Protect Buffer enables visualization of complete reaction mixing, ensuring the correct pH is achieved for complete cytosine conversion.
Figure 3.
Human genomic DNA was purified from blood using the QIAamp DNA Blood Mini Kit, and various amounts (1 ng to 1 µg) were converted using the EpiTect Bisulfite Kit. PCR was performed using the HotStarTaq Master Mix Kit and two sets of primers designed to amplify converted DNA. Five microliters of each PCR were loaded onto a 1.3 percent agarose gel. As little as 1 ng DNA is sufficient for conversion using the EpiTect Bisulfite Kit. C: Untreated genomic DNA (negative control). M: Marker.\
References
1. Russo, V.E.A. et al. Epigenetic Mechanism of Gene Regulation, Cold Spring Harbor Laboratory Press, 1996, p. 173-194.
2. Glenn, C.C. et al. “Gene structure, DNA methylation, and imprinted expression of the human SNRPN gene.” Am J Hum Genet 58, 335-46; 1996.
3. Jones, P.A., and S.B. Baylin. “The fundamental role of epigenetic events in cancer.” Nat Rev Genet 3, 415-28; 2002.
4. Warnecke, P.M. et al. “Identification and resolution of artifacts in bisulfite sequencing.” Methods 27, 101-7; 2002.
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