Detection of methylated DNA
The present invention is based on the discovery of sensitive and specific methylation detection by a) controlling excessive DNA degradation prior to conversion by incubating DNA conversion reagent (e.g. bisulfite reagent) directly with a nucleic acid containing sample without requiring prior nucleic acid purification from the sample and without requiring prior nucleic acid denaturation at elevated temperatures of 98° C. and, b) optimizing bisulfite removal by controlling the pumping rate flow of the bisulfite treated sample over an extraction membrane inside an automated system.
1. A method for detecting nucleic acid methylation status in an automated system comprising an extraction chamber comprising an extraction membrane, said method comprising the steps of:
a) introducing a source of the nucleic acid into the automated system, wherein the source of the nucleic acid is a biological sample comprising nucleic acids or cells comprising DNA, adding bisulfite to the source of the nucleic acid and immediately followed by heating the source of the nucleic acid to produce a lysate comprising the nucleic acid and to deaminate an unmethylated cytosine base present in said nucleic acid;
b) adding binding buffer to the lysate to produce a binding buffer-and-lysate-solution;
c) binding the nucleic acid to the extraction membrane and removing the bisulfite by transporting the binding buffer-and-lysate-solution over the extraction membrane by controlled pumping;
d) adding desulphonation buffer to the extraction chamber and transporting the desulphonation buffer over the extraction membrane by controlled pumping to desulphonate the base deaminated in a previous step in the nucleic acid and to convert said base to an uracil base; and
e) eluting the converted nucleic acid from the extraction membrane; and
f) analyzing the converted nucleic acid wherein the transporting by controlled pumping in step d) is slower than the transporting by controlled pumping in step c).
2. The method according to claim 1 , wherein the heating of the source of the nucleic acid is performed at a temperature between about 40° C. and about 80° C.
3. The method according to claim 1 , wherein the analysis of the converted nucleic acid is performed in said automated system.
4. The method according to claim 1 , wherein the heating of the source of the nucleic acid is performed at a temperature between 40° C. and 60° C.
5. The method according to claim 1 , wherein the transporting by controlled pumping in step c) and the transporting by controlled pumping in step d) are at a flow rate between 0.001 ml/s and 0.1 ml/s.
6. The method according to claim 1 , wherein the extraction membrane is a silica membrane, or a composite membrane comprising 2 or 3 silica membrane layers.
7. The method according to claim 1 , wherein the controlled pumping in step c) and step d) is performed at a flow rate slower than or equal to 0.1 ml/s.