Bisulphite Genomic Sequencing

Denaturation:

  • approximately 2 µg genomic DNA (don't have to be ultra-pure, common methods for preparation are sufficient)
  • if you use DNA of CsCl gradient quality -> cut with suitable restriction enzymes to facilitate denaturation (of course not within the region of interest)
  • DNA in a total volume of 90 µl (if necessary adjust with sterile destilled water)
    • + 10 µl 1 g/l tRNA or mRNA as carrier
    • + 11 µl 3 M NaOH
  • incubate at 37°C 20 min or (in case of high G+C content) at 42°C 20 min

Sulfonation/Deamination:

  • use always freshly prepared bisulphite solution:
    • 5.41 g Na-bisulfit (ACS reagent of Sigma) (I store it under vacuum)
    • add approximately 7 ml destilled water
    • + 0.5 ml Hydroquinone in destilled water (0.022g/10 ml)
    • + 400 µl 10 M NaOH - disolve without vigorous shaking (takes a while)
    • adjust to 10 ml with destilled water
    • the pH should now be 5
    • filter through a 0.45 µm filter
  • give 1200 µl of this solution directly into the denatured DNA
  • cover with 200 µl mineral oil and incubate in the dark for 4 hours at 55°C.

Desalting:

  • use the Promega Wizard DNA purification kit to separate DNA und Bisulphite.
  • elute the DNA in 105 µl 1 mM TRIS pH 8.

Desulfonation:

  • 100 µl of the DNA + 11 µl 3 M NaOH
  • incubate at 37°C for 20 min (up to a maximum of 50 min).

Neutralization:

  • the 111 µl DNA-NaOH
    • + 47 µl 10 M filtered ammonium acetate
    • + 1 µl 1 g/l tRNA or mRNA as carrier
    • + 500 µl 96% Ethanol
  • precipitate at -20°C over night
  • centrifuge, wash with 500 µl 70% Ethanol, dry
  • disolve in 100 µl 1 mM TRIS pH 8 (not water - it might be acid, depends on your local conditions!).
  • Store at -20°C (up to 10 month without loss of PCR yield)

PCR:

Nested PCR in 2 subsequent reactions.

    ideal primer:
  1. 30% mismatch C, replaced by T (or complementary A)
  2. 30% G
  3. 20% A
  4. 20% T
  5. no CpGs
  6. 25...30 nucleotides long

Use in a 50 µl reaction 2.5 µl of the bisulphite converted DNA.

     

      

    5 x

    25 x

    94°C 2 min

    94°C 1 min

    94°C 0:30 min

    72°C 10 min 

    50°C 2 min 

    50°C 2:00 min 

    72°C 3 min

    72°C 1:30 min

     

Use in the second PCR again 2.5 µl of the first PCR, same programm.
Try several primer combinations. (For the beginning order at least 6 to 8 primers.) Use as a control primers that have been published. As soon as you get a PCR product, optimize the programm. (It might be necessary to initially decrease the annealing temperature to 45°C.)

Sequencing:

Direct DyeTerminator sequencing of the PCR products does not deliver nice results. Better:
a) gel purify PCR products and subcloning or
b) make primer with M13 "tail" and do DyePrimer sequenzing (further information in the related literature)

Troubleshooting:

  • no PCR-product
    • test your DNA with published primer pairs and PCR conditions
    • decrease annealing temperature
    • change primer site and sequence
  • no or rare conversion
    • remove proteins from DNA (proteinase treatment)
    • digest DNA before denaturation
    • increase denaturation temperature
    • add urea to bisulfite solution

Ref.:

Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL. "A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands."Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827-31.

Grunau C, Clark SJ, Rosenthal A. "Bisulfite genomic sequencing: systematic investigation of critical experimental parameters." Nucleic Acids Res. 2001 Jul 1;29(13):E65-5.

Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clark SJ. "Identification and resolution of artifacts in bisulfite sequencing." Methods. 2002 Jun;27(2):101-7.