Quantitative 1-step DNA methylation analysis with native genomic DNA as template.
BACKGROUND
DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis.


METHODS
The assay is based on combining methylation-sensitive FastDigest(R) endonuclease digestion and quantitative real-time PCR (qPCR) in a single reaction. The first step consists of DNA digestion, followed by endonuclease inactivation and qPCR. The degree of DNA methylation is evaluated by comparing the quantification cycles of a reaction containing a methylation-sensitive endonuclease with the reaction of a sham mixture containing no endonuclease. Control reactions interrogating an unmethylated locus allow the detection and correction of artifacts caused by endonuclease inhibitors, while simultaneously permitting copy number assessment of the locus of interest.


RESULTS
With our novel approach, we correctly diagnosed the imprinting disorders Prader-Willi syndrome and Angelman syndrome in 35 individuals by measuring methylation levels and copy numbers for the SNRPN (small nuclear ribonucleoprotein polypeptide N) promoter. We also demonstrated that the proposed correction model significantly (P < 0.05) increases the assay's accuracy with low-quality DNA, allowing analysis of DNA samples with decreased digestibility, as is often the case in retrospective studies.


CONCLUSIONS
Our novel DNA methylation assay reduces both the hands-on time and errors caused by handling and pipetting and allows methylation analyses to be completed within 90 min after DNA extraction. Combined with its precision and reliability, these features make the assay well suited for diagnostic procedures as well as high-throughput analyses.
von Kanel
Thomas
T
Division of Human Genetics, Department of Pediatrics, Inselspital, University of Bern, Bern, Switzerland. thomas.von_kaenel@alumni.unibe.ch
Gerber
Dominik
D
Schaller
André
A
Baumer
Alessandra
A
Wey
Eva
E
Jackson
Christopher B
CB
Gisler
Franziska M
FM
Heinimann
Karl
K
Gallati
Sabina
S
D017204:Angelman Syndrome / Q000175:diagnosis* / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000032:analysis*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000737:chemistry; D005240:Feasibility Studies; D018628:Gene Dosage; D056426:Genetic Loci; D015894:Genome, Human; D018392:Genomic Imprinting; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011218:Prader-Willi Syndrome / Q000175:diagnosis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D055517:snRNP Core Proteins / Q000235:genetics*
C526417:SNRPN protein, human; D055517:snRNP Core Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D004262:DNA Restriction Enzymes