Improvement of the methylation specific PCR technical conditions for the detection of p16 promoter hypermethylation in small amounts of tumor DNA.
Methylation specific PCR (MSP) is a technique that enables the detection of hypermethylation at a CpG island. The objective of this study is the introduction of small modifications to the MSP technique to make it more suitable for the study of promoter hypermethylation at tumor suppressor genes whenever there is a shortage of material available for study. This commonly happens in the case of using archival material from the Pathology departments. Tumor DNA was extracted from a collection of 40 fresh-frozen soft tissue sarcomas and 19 paraffin-embedded PNETs (primitive neuroectodermal tumors). The MSP technique was performed to detect hypermethylation at the p16 promoter. Also, blood genomic DNA was mixed with herry sperm genomic DNA as a carrier, in nine different combinations, in order to test for the best conditions that could produce MSP bands even when low amounts of genomic tumor DNA is available for study. We demonstrate the benefit of using herry sperm carrier DNA up to 10 microg together with small quantities of tumor DNA. This result will facilitate the incorporation of paraffin-embedded samples for study of promoter hypermethylation at tumor suppressor genes. Other technical conditions for the MSP technique are also studied.
Fan
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X
Molecular Neuro-Oncology Laboratory, University of Navarre Medical School, E-31008 Pamplona, Spain.
Inda
M M
MM
Tunon
T
T
Castresana
J S
JS
D001932:Brain Neoplasms / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics*; D016147:Genes, Tumor Suppressor; D006801:Humans; D008297:Male; D018242:Neuroectodermal Tumors, Primitive / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012509:Sarcoma / Q000235:genetics*; D012983:Soft Tissue Neoplasms / Q000235:genetics*; D013094:Spermatozoa / Q000737:chemistry; D013447:Sulfites / Q000494:pharmacology
D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D013447:Sulfites; C009279:sodium bisulfite