Quantitative analysis of DNA methylation in the promoter region of the methylguanine‐O⁶‐DNA‐methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis

Abstract

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples.

Capillary Electrophoresis: Trends and Recent Advances

“…Although often the identification of nucleic acids is done by polyacrylamide gel electrophoresis, or PAGE, and polymerase chain reaction, or PCR, analysis, the capability of attaining insights in post-transcriptional modifications is limited. 100 Recently, CE-MS was used to identify and quantify these modifications. 101 The authors could detect two endogenous human circulating miRNAs isolated from B-cell chronic lymphocytic leukemia serum.…”

Application of Capillary Electrophoresis with Laser-Induced Fluorescence to Immunoassays and Enzyme Assays

“…Consequently, the use of CE-LIF in bioanalytical assays has drawn significant attention with the publication of numerous papers dealing with the analysis of biomolecules based on the two significant bio-reaction classes: (1) immune reaction and (2) enzyme reaction. The application of CE based on these two reactions has been extensively reviewed, summarized in [28,29,30,31,32,33,34,35,36].…”

Διερεύνηση Και Κλινική Αξιολόγηση Νέων Δεικτών Μεθυλίωσης Του DNA Στον Καρκίνο, Στα Πλαίσια Της Υγρής Βιοψίας

“…Τα προϊόντα της πέψης μπορούν να αναλυθούν με ηλεκτροφόρηση σε πηκτή αγαρόζης, ενώ για ημιποσοτική ανάλυση μπορούν να αποδιαταχθούν, να διαχωριστούν με ηλεκτροφόρηση σε πηκτή πολυακρυλαμιδίου με αποδιατακτικό και στη συνέχεια, μεταφερόμενα σε μεμβράνη με ηλεκτροστύπωμα να υβριδοποιηθούν με ραδιοσημασμένα ολιγονουκλεοτίδια. Σχετική ποσοτικοποίηση μπορεί να πραγματοποιηθεί χρησιμοποιώντας ένα phosphorimager και το ποσοστό των πλήρως μεθυλιωμένων θέσεων στο γενωμικό δείγμα να υπολογιστεί ως ο λόγος του PCR προϊόντος που έχει υποστεί πέψη προς τη συνολική ποσότητα του PCR προϊόντος (161).…”