DNA Methylation Protocols (Methods in Molecular Biology Vol by by Ken I. Mills (Editor), Bernie H. Ramsahoye (Editor)

By by Ken I. Mills (Editor), Bernie H. Ramsahoye (Editor)

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Extra resources for DNA Methylation Protocols (Methods in Molecular Biology Vol 200)

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13. , Martsen, E. , and Lapeyre, J. N. (1998) Re-amplification of short primer-generated bands from RAPD and methylation-sensitive restriction fingerprinting by discrimination primers. Biotechniques 24, 402–403. 52 Davies Two-Dimensional DNA Profiling 53 6 Restriction Landmark Genome Scanning Joseph F. Costello, Christoph Plass, and Webster K. Cavenee 1. Introduction Restriction landmark genomic scanning (RLGS) is a method that provides both a quantitative genetic and epigenetic (cytosine methylation) assessment of thousands of CpG islands in a single gel without prior knowledge of gene sequence (1).

Phenol. Chloroform. Isoamyl alcohol. Dialysis clips/closures (Spectra/Por). Proteinase K. RNAse A (Boehringer Mannheim). Sarkosyl (Fluka). 8-hydroxyquinoline (Sigma). 0, and 1% sarkosyl. PCI: phenolϺ chloroformϺ isoamylalcohol in the ratio 50Ϻ49Ϻ1. 56 Costello et al. 2. Enzymatic Processing of Genomic DNA 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Wide-bore pipet tips. Dithiothreitol (DTT). Triton X-100. Bovine serum albumin (BSA). dGTPαS, dCTPαS, ddATP, ddTTP, ddGTP, ddCTP (Pharmacia).

Using a methylation-sensitive enzyme, the technique has been termed RLGS-M (3). The resulting RLGS profile displays both the copy number and methylation status of the CpG islands. These profiles are highly reproducible and are therefore amenable to inter- and intra-individual DNA sample comparisons. To increase the number of fragments analyzed by RLGS, the DNA samples can be processed with a different series of enzymes. The choice of a “landmark” enzyme is critical since this site determines the bias of the displayed fragments.

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