Limits...
Design, measurement and processing of region-specific DNA methylation assays: the mass spectrometry-based method EpiTYPER.

Suchiman HE, Slieker RC, Kremer D, Slagboom PE, Heijmans BT, Tobi EW - Front Genet (2015)

Bottom Line: The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution.It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies.Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

View Article: PubMed Central - PubMed

Affiliation: Molecular Epidemiology, Department of Medical Statistics and Bioinformatics, Leiden University Medical Center Leiden, Netherlands.

ABSTRACT
EpiTYPER® is a mass spectrometry-based bisulfite sequencing method that enables region-specific DNA methylation analysis in a quantitative and high-throughput fashion. The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution. It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies. Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

No MeSH data available.


Biochemical steps and methylation quantification with EpiTYPER®. EpiTYPER® resolves on re-sequencing DNA on mass. After bisulfite conversion DNA is amplified by PCR and the resulting PCR product is transcribed from the reverse strand of the PCR product. This single stranded product is cleaved after each T resulting in fragments of known mass. Fragments with a methylated cytosine are 16 Dalton heavier than fragments with a non-methylated cytosine, the size of the two peaks can be used to calculate the methylation ratio of the CpG dinucleotide. It is easy to envision that fragments can arise with the same mass or with a mass extremely close to that of another fragment. Such circumstances limit the detection of certain CpG dinucleotides.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585020&req=5

Figure 1: Biochemical steps and methylation quantification with EpiTYPER®. EpiTYPER® resolves on re-sequencing DNA on mass. After bisulfite conversion DNA is amplified by PCR and the resulting PCR product is transcribed from the reverse strand of the PCR product. This single stranded product is cleaved after each T resulting in fragments of known mass. Fragments with a methylated cytosine are 16 Dalton heavier than fragments with a non-methylated cytosine, the size of the two peaks can be used to calculate the methylation ratio of the CpG dinucleotide. It is easy to envision that fragments can arise with the same mass or with a mass extremely close to that of another fragment. Such circumstances limit the detection of certain CpG dinucleotides.

Mentions: The principle of EpiTYPER is mass based re-sequencing of PCR amplified bisulfite converted DNA with a mass spectrometer and involves several biochemical steps (Figure 1) (Ehrich et al., 2005, 2007). In short, genomic DNA (gDNA) is treated with bisulfite which leads to a conversion of all unmethylated cytosines, while methylated cytosines remain unaffected. A PCR is performed on the bisulfite converted DNA with primers tagged with a T7 promoter. After shrimp alkaline phosphatase treatment, to discard unincorporated DNA nucleotides, the T7 promoter added during PCR is used to transcribe the PCR product from the reverse strand, yielding a single stranded RNA product. This RNA product is cleaved with RNase A resulting in a specific fragmentation of the RNA product. After a final cleaning step with a resin the samples are loaded on a SpectroCHIP® II Array, which prepares the fragments for separation on mass with the mass spectrometer. The mass spectrometer is a Matrix Assisted Laser Desorption-Ionization Time of Flight (MALDI-TOF) device. The matrix on the SpectroCHIP II absorbs the energy of the laser and transfers it to the RNA fragments which subsequently become ionized. The ionized fragments are separated by the time it takes to arrive at the detector in at the end of the mass spectrometer's flight tube under the influence of an electric field. The time of flight increases with higher mass. A fragment containing one or more CpG dinucleotides is called a CpG unit. If a CpG dinucleotide was methylated and protected from bisulfite conversion, the corresponding RNA fragment, the CpG unit, will be 16 Da heavier in mass when the CpG dinucleotide was methylated, resulting in a 16 Da shift in the mass spectrum. The signal detected by the mass spectrometer for either fragments is proportional to the number of fragments. The number of fragments is quantified by the surface area of the corresponding peaks in the mass spectrum. The DNA methylation percentage of a given CpG is calculated by dividing the surface area of the peak representing the methylated fragment by the total surface area of the peaks of both the methylated and unmethylated fragment.


Design, measurement and processing of region-specific DNA methylation assays: the mass spectrometry-based method EpiTYPER.

Suchiman HE, Slieker RC, Kremer D, Slagboom PE, Heijmans BT, Tobi EW - Front Genet (2015)

Biochemical steps and methylation quantification with EpiTYPER®. EpiTYPER® resolves on re-sequencing DNA on mass. After bisulfite conversion DNA is amplified by PCR and the resulting PCR product is transcribed from the reverse strand of the PCR product. This single stranded product is cleaved after each T resulting in fragments of known mass. Fragments with a methylated cytosine are 16 Dalton heavier than fragments with a non-methylated cytosine, the size of the two peaks can be used to calculate the methylation ratio of the CpG dinucleotide. It is easy to envision that fragments can arise with the same mass or with a mass extremely close to that of another fragment. Such circumstances limit the detection of certain CpG dinucleotides.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585020&req=5

Figure 1: Biochemical steps and methylation quantification with EpiTYPER®. EpiTYPER® resolves on re-sequencing DNA on mass. After bisulfite conversion DNA is amplified by PCR and the resulting PCR product is transcribed from the reverse strand of the PCR product. This single stranded product is cleaved after each T resulting in fragments of known mass. Fragments with a methylated cytosine are 16 Dalton heavier than fragments with a non-methylated cytosine, the size of the two peaks can be used to calculate the methylation ratio of the CpG dinucleotide. It is easy to envision that fragments can arise with the same mass or with a mass extremely close to that of another fragment. Such circumstances limit the detection of certain CpG dinucleotides.
Mentions: The principle of EpiTYPER is mass based re-sequencing of PCR amplified bisulfite converted DNA with a mass spectrometer and involves several biochemical steps (Figure 1) (Ehrich et al., 2005, 2007). In short, genomic DNA (gDNA) is treated with bisulfite which leads to a conversion of all unmethylated cytosines, while methylated cytosines remain unaffected. A PCR is performed on the bisulfite converted DNA with primers tagged with a T7 promoter. After shrimp alkaline phosphatase treatment, to discard unincorporated DNA nucleotides, the T7 promoter added during PCR is used to transcribe the PCR product from the reverse strand, yielding a single stranded RNA product. This RNA product is cleaved with RNase A resulting in a specific fragmentation of the RNA product. After a final cleaning step with a resin the samples are loaded on a SpectroCHIP® II Array, which prepares the fragments for separation on mass with the mass spectrometer. The mass spectrometer is a Matrix Assisted Laser Desorption-Ionization Time of Flight (MALDI-TOF) device. The matrix on the SpectroCHIP II absorbs the energy of the laser and transfers it to the RNA fragments which subsequently become ionized. The ionized fragments are separated by the time it takes to arrive at the detector in at the end of the mass spectrometer's flight tube under the influence of an electric field. The time of flight increases with higher mass. A fragment containing one or more CpG dinucleotides is called a CpG unit. If a CpG dinucleotide was methylated and protected from bisulfite conversion, the corresponding RNA fragment, the CpG unit, will be 16 Da heavier in mass when the CpG dinucleotide was methylated, resulting in a 16 Da shift in the mass spectrum. The signal detected by the mass spectrometer for either fragments is proportional to the number of fragments. The number of fragments is quantified by the surface area of the corresponding peaks in the mass spectrum. The DNA methylation percentage of a given CpG is calculated by dividing the surface area of the peak representing the methylated fragment by the total surface area of the peaks of both the methylated and unmethylated fragment.

Bottom Line: The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution.It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies.Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

View Article: PubMed Central - PubMed

Affiliation: Molecular Epidemiology, Department of Medical Statistics and Bioinformatics, Leiden University Medical Center Leiden, Netherlands.

ABSTRACT
EpiTYPER® is a mass spectrometry-based bisulfite sequencing method that enables region-specific DNA methylation analysis in a quantitative and high-throughput fashion. The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution. It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies. Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

No MeSH data available.