Sensitive measurement of unmethylated repeat DNA sequences by end-specific PCR

• Published in: BioTechniques Vol. 49; no. 4; pp. xiii - xvii
• Main Authors: Rand, Keith N, Molloy, Peter L
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.10.2010; Eaton
• Subjects: Alu Elements; assay; Biological and medical sciences; Cell Line, Tumor; Diverse techniques; DNA - analysis; DNA - genetics; DNA - metabolism; DNA Methylation; DNA Restriction Enzymes - metabolism; elements; Fundamental and applied biological sciences. Psychology; Humans; hypomethylation; Male; Molecular and cellular biology; PCR; Polymerase Chain Reaction - methods; Sequence Analysis, DNA - methods; single-tube; Polymerase chain reaction; End; Nucleotide sequence; Repeated sequence
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/000113494

We describe a new method that is well-suited for the determination of the methylation level of repetitive sequences such as human elements. We have applied the method to the analysis of cell and tissue DNAs and expect it to have wide utility in studies of DNA methylation in cancer and other disease states, in monitoring response to epigenetic cancer therapies and in epidemiological studies. Only 1 ng DNA is needed for a duplex, one-tube real-time PCR in which methylation level and the amount of input DNA are concurrently measured. The relative cutting by the methylation-sensitive enzyme UI is compared with that of the methylation-insensitive enzyme I to give a measure of DNA methylation. The method depends upon the use of 5′-tailed, 3′-blocked oligonucleotides called facilitator oligonucleotides (Foligos). Only cut DNAs with specific matching sequences at their 3′ ends can copy the tails of the Foligos and thus become tagged and available for subsequent PCR. Both the tagging and PCR are carried out by the same enzyme, DNA polymerase. Because amplification only occurs if suitable ends have been generated in the target DNA, we have called this method end-specific PCR (ESPCR). ESPCR avoids the bisulfite treatment step that is usually required to measure methylation.