- 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
- use always freshly prepared bisulphite solution:
- 5.41 g Na-bisulfit (ACS reagent of Sigma) (I store it under
- 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
- cover with 200 µl mineral oil and incubate in the dark
for 4 hours at 55°C.
- use the Promega Wizard DNA purification kit to separate DNA
- elute the DNA in 105 µl 1 mM TRIS pH 8.
- 100 µl of the DNA + 11 µl 3 M NaOH
- incubate at 37°C for 20 min (up to a maximum of 50 min).
- 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)
Nested PCR in 2 subsequent reactions.
- 30% mismatch C, replaced by T (or complementary A)
- 30% G
- 20% A
- 20% T
- no CpGs
- 25...30 nucleotides long
Use in a 50 µl reaction 2.5 µl of the bisulphite
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
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
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)
- no PCR-product
- test your DNA with published primer pairs and PCR
- 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
Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW,
Molloy PL, Paul CL.
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.
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.
and resolution of artifacts in bisulfite sequencing." Methods.