Bisulfite Genomic Sequencing (Microcon version)

© 2004 Christoph Grunau,

Denaturation:

  • approximately 300 ng 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)
    • 18 µl DNA (16 ng/µl in sterile destilled water)
    • + 2 µl 1 g/l tRNA or mRNA as carrier
    • + 2.2 µl 3 M NaOH
  • incubate at 42°C 20 min

Sulfonation/Deamination:

  • use always freshly prepared bisulfite solution:
    • 5.41 g Na-bisulfit (ACS reagent of Sigma)
    • add approximately 7 ml distilled water
    • + 0.5 ml Hydroquinone in distilled water (0.022g/10 ml)
    • + 400 µl 10 M NaOH - dissolve without vigorous shaking (takes a while)
    • adjust to 10 ml with distilled water
    • the pH should now be 5
    • filter through a 0.45 µm filter
  • give 240 µl of this solution directly into the denatured DNA
  • incubate in the dark for 4 hours at 55°C (mineral oil cover not necessary).

Desalting:

  • use Amico Ultra (UFC510024 Millipore) (formerly Microcon YM-100) filter device to separate DNA und Bisulfite.
  • add 200 µl distilled sterile water to the DNA+bisulfite solution
  • apply on the filter column, centrifuge at 12000 g 10°C 5 min
  • discard flow-through
  • wash 3 times with 350 µl distilled sterile water, centrifuge each time 12000 g 10°C 5 min
  • optional: continue washing if necessary until no more schlieren are visible when you add the water, it is necessary to remove all the remaining bisulfite

Desulfonation:

  • add 350 µl 0.1 M NaOH to the DNA in the Microcon column
  • centrifuge at 12000 g 10°C 10 min, discard flow-through
  • add 350 µl distilled sterile water, centrifuge 12000 g 10°C 5 min

Neutralization/Recovery:

  • add 50 µl 10 mM TRIS/Cl pH 7.5-8.0 to the DNA in the Microcon column
  • incubate at RT 5 min
  • invert the tube and collect the DNA by centrifugation (1000 g, 3 min)
  • the DNA is ready to use and can be stored at least 3 month at -80°C

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 (if you can't avoid CpG replace C with a non-T mismatch = A or G and complements on the reverse strand)
  6. 25...30 nucleotides long

Use in a 50 µl reaction 2.5 µl of the bisulfite 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 (consult MethDB or the methPrimerDB )
    • 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.:

Boyd VL, Zon G. "Bisulfite conversion of genomic DNA for methylation analysis: protocol simplification with higher recovery applicable to limited samples and increased throughput." Anal Biochem. 2004 Mar 15;326(2):278-80.

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.

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