Ligase/polymerase method for detecting cytosine methylation in dna samples
AssigneeEpigenomics AG
InventorsAlexander Olek, Kurt Berlin
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Abstract
Described is a method for detecting 5-methylcytosine in genomic DNA samples. First, a genomic DNA from a DNA sample is chemically converted with a reagent, 5-methylcytosine and cytosine reacting differently, and the pretreated DNA is subsequently amplified using a polymerase and at least one primer. In the next step, the amplified genomic DNA is hybridized to at least two oligonucleotides, the latter being joined by inserting at least one oligonucleotide. In the ligation product, one nucleotide carries a detectable label, and the elongation depends on the methylation status of the specific cytosine in the genomic DNA sample. In the next step, the elongated oligonucleotides are analyzed for the presence of the label.
Claims36
A method for detecting 5-methylcytosine in genomic DNA samples, characterized in that the following steps are carried out:
- (a) a genomic DNA from a DNA sample is chemically converted with a reagent, 5-methylcytosine and cytosine reacting differently, thus exhibiting different base pairing behaviors in the DNA duplex subsequent to the reaction;
- (b) the pretreated DNA is amplified using a polymerase and at least one oligonucleotide (type A) as a primer;
- (c) a set of oligonucleotides is hybridized to the amplified genomic DNA, forming a duplex;
- this set of oligonucleotides comprising different species of type B and of type C;
- and said hybridized oligonucleotides of type B, with their 3′-ends, immediately or at a distance of up to 10 bases, adjoining the positions to be analyzed with regard to their methylation in the genomic DNA sample;
- and said second oligonucleotide (type C) hybridizing to a second region of the target molecule so that the 5′-end of the second oligonucleotide (type C) is separated from the 3′-end of the first oligonucleotide (type B) at the location of said selected position by a gap of the size of a single nucleotide or of up to 10 nucleotides;
- (d) the oligonucleotide (type B) having a known sequence of n nucleotides is elongated by means of a polymerase by not more than the number of nucleotides lying between the 3′-end of the oligonucleotide of type B and the 5′-end of the oligonucleotide of type C, the elongation depending on the methylation status of the specific cytosine in the genomic DNA sample;
- (e) the oligonucleotides are incubated in the presence of a ligase, the adjoining first oligonucleotide of type B elongated by the polymerase reaction and the second oligonucleotide of type C being linked, resulting in a ligation product provided that the oligonucleotide of type B has been elongated in the preceding step in such a manner that now the 3′-end with existing 3′-hydroxy function immediately adjoins the 5′-end of the oligonucleotide of type C;
- (f) it is detected whether a ligation product has formed.
The method as recited in one of the preceding claims, characterized in that, prior to the amplification, the DNA is treated with a bisulfite solution (=disulfite, hydrogen sulfite).
The method as recited in one of the preceding claims, characterized in that the amplification is carried out by means of the polymerase chain reaction (PCR).
The method as recited in one of the preceding claims, characterized in that the oligonucleotides of type B and/or C used either contain only the bases T, A and C or else the bases T, A und G.
The method as recited in one of the preceding claims, characterized in that the ligation products and/or the elongation products are provided with a detectable label for detection.
The method as recited in one of the preceding claims, characterized in that the labels are fluorescence labels.
The method as recited in one of the preceding claims, characterized in that the detection of the elongated oligonucleotides and/or of the ligation products is carried out and visualized by means of matrix assisted laser desorption/ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
The method as recited in one of the preceding claims, wherein the polymerases are heat-resistant DNA-polymerases and/or the ligases are thermostable ligases.
The method as recited in one of the preceding claims, wherein SNPs are also detected and visualized in addition to the DNA methylation.
The method as recited in one of the preceding claims, wherein the used nucleotides are terminating (type D 2) and/or chain-elongating nucleotides (type D 1).
The method as recited in one of the preceding claims, characterized in that the amplification of several DNA segments is carried out in one reaction vessel.
The method as recited in one of the preceding claims, characterized in that the 3′-end of the oligonucleotide of type B elongated in step d is formed by the nucleotide 2′-desoxyadenosine which, through its 3′-hydroxy function, enables the ligation in step e, whereas a 2′,3′-didesoxyguanosine at the 3′-end of the oligonucleotide does not give rise to a ligation product.
The method as recited in one of the preceding claims, characterized in that that the 3′-end of the oligonucleotide of type B elongated in step d is formed by the nucleotide 2′-desoxyguanosine which, through its 3′-hydroxy function, enables the ligation in step e, whereas a 2′,3′-didesoxyadenosine at the 3′-end of the oligonucleotide does not give rise to a ligation product.
The method as recited in one of the preceding claims, characterized in that the 3′-end of the oligonucleotide of type B elongated in step d is formed by the nucleotide thymidine which, through its 3′-hydroxy function, enables the ligation in step e, whereas a 2′,3′-didesoxycytidine at the 3′-end of the oligonucleotide does not give rise to a ligation product.
The method as recited in one of the preceding claims, characterized in that the 3′-end of the oligonucleotide of type B elongated in step d is formed by the nucleotide 2′-desoxycytidine which, through its 3′-hydroxy function, enables the ligation in step e, whereas a 2′,3′-didesoxythymidine at the 3′-end of the oligonucleotide does not give rise to a ligation product.
The method as recited in one of the preceding claims, characterized in that the methylation analyses of the upper and lower DNA strands are carried out concurrently.
Dependencies are inferred from the claim text.