In-tube DNA methylation profiling by fluorescence melting curve analysis.
BACKGROUND
Most PCR assays for detection of 5-methylcytosine in genomic DNA entail a two-step procedure, comprising initial PCR amplification and subsequent product analysis in separate operations that usually require manual transfer. These methods generally provide information about methylation of only a few CpG dinucleotides within the target sequence.


METHODS
An in-tube methylation assay is described that integrates amplification of bisulfite-treated DNA and melting analysis by using a thermal cycler coupled to a fluorometer (LightCycler). DNA melting curves were acquired by measuring the fluorescence of a double-stranded DNA-binding dye (SYBR Green I) during a linear temperature transition.


RESULTS
Analysis of a region comprising 11 CpG sites at the SNRPN promoter CpG island showed that the melting temperature (T(m)) differed by approximately 3 degrees C between unmethylated and fully methylated alleles. This assay could easily distinguish patients with Prader-Willi syndrome or Angelman syndrome from individuals without these conditions. Melting curve analysis also allowed resolution of methylation "mosaicism" at the p15(Ink4b) promoter in bone marrow samples from patients with acute myeloid leukemia (AML). AML samples representing pools of heterogeneously methylated p15(Ink4b) alleles showed broadened melting peaks with overall T(m)s between those of the unmethylated and fully methylated alleles.


CONCLUSIONS
Integration of PCR and fluorescence melting analysis may be useful for simple and cost-effective detection of aberrant methylation patterns.
Worm
J
J
Department of Tumor Cell Biology, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark.
Aggerholm
A
A
Guldberg
P
P
D000208:Acute Disease; D017204:Angelman Syndrome / Q000235:genetics; D001324:Autoantigens / Q000235:genetics; D001853:Bone Marrow / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016172:DNA Fingerprinting / Q000379:methods*; D019175:DNA Methylation; D005470:Fluorometry; D006801:Humans; D007202:Indicators and Reagents; D007951:Leukemia, Myeloid / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011218:Prader-Willi Syndrome / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics; D013447:Sulfites; D025521:Tumor Suppressor Proteins; D055517:snRNP Core Proteins
D001324:Autoantigens; C493966:CDKN2B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D007202:Indicators and Reagents; D017411:Ribonucleoproteins, Small Nuclear; C526417:SNRPN protein, human; D013447:Sulfites; D025521:Tumor Suppressor Proteins; D055517:snRNP Core Proteins; C042345:hydrogen sulfite