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Analysis of methylation using bisulphite sequencing
Contributed by Dr. A. Gratchev

This method allows precise analysis of methylation in a certain region by converting all nonmethylated cytosines into tymines, while methylated cytosines remain unchanged. This method requires small amount of genomic DNA and therefore seems to be very useful for the analysis of clinical samples, where the material amount is limited. However I suggest optimising the method using genomic DNA from a cell line and then apply it to valuable samples.

Before starting the experiment you have to develop primers for bisulphite converted DNA. You can generate a model of bisulphite treated DNA by substituting all cytosines which are not in CG context into tymines. And then design your primers in the way that they don not contain any CG. If this is impossible, you have to use C/T at the place of C in CG context. Usually primer selection is the most critical in bisulphite based methylation analysis, since the complexity of DNA is reduced. Therefore I would suggest to select 2-3 pairs of primers, check them on bisulphite modified DNA, and use the most specific ones.

Protocol

  1. Isolate genomic DNA with the quality sufficient for restriction enzyme digestion.
  2. Digest DNA (50 to 200 ng) with any enzyme which does not cut the region of interest, but resulting in as short fragments as possible in smallest possible volume. Note: increasing the amount of DNA will make denaturing not efficient enough and therefore make bisulphite reaction incomplete!
  3. Stop the reaction by boiling DNA for 5 min.
  4. Add 10N NaOH to 0.3N final concentration and denature DNA 37° C for 15 min.
  5. Prepare 4-5 eppendorf tubes with cold mineral oil.
  6. Add 2x volume of 2% low melting agarose to the DNA solution, mix by pipetting up and down.
  7. Form agarose beads by pipetting 10 µl aliquots of DNA/agarose mixture into cold mineral oil. Note: Don't pipett the second aliquot in the tube where you already have one bead!
  8. Transfer beads in the tube containing 1 ml of modifying solution (5M sodium bisulphite (2.5M sodium metabisulphite), 100mM Hydroquinon).
  9. Incubate the tubes 4 h at 50° C in the dark.
  10. Wash the beads 6 times for 15 min in TE pH 8.0.
  11. Complete the modification by incubating the beads 2 times for 15 min in 0.2 N NaOH.
  12. Wash the beads 3 times for 15 min in double distilled H2O.
  13. Use one ul of the obtained DNA for PCR with selected primers.

Obtained PCR product can be sequenced directly, in this case you will obtain more or less reliable results about the percentage of methylated cytosine in every position. Another possibility is to clone the product and then sequence 10 or more individual clones. This method is much more time consuming. Third method which can be used for the analysis of bisulphite treated DNA is Single Nucleotide Primer Extension (SNuPE).