US 9,850,528 B2 · Determination of the normalized degree of DNA methylation

Abstract

The present invention provides oligonucleotides and processes for determining the normalized methylation level of DNA, and for determining the relative methylation level of DNA between at least two samples. The invention makes use of the random distribution of transposons in the genome. The disclosed oligonucleotides and processes are of importance, in particular, for clinical diagnostics.

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Claims15

Dependent claims

A process for determining the normalized DNA methylation level, comprising the steps:

a) quantitatively determining the presence of a transposon or fragment thereof in a DNA wherein this quantitative determination comprises amplifying a non-bisulfited DNA with at least one primer pair that is specific for the transposon or fragment thereof, or amplifying of a bisulfited DNA with at least one primer pair that is specific for a bisulfited transposon or fragment thereof, wherein the primers do not include a differentially methylated position of the transposon;
b) quantitatively determining the presence of at least one differentially methylated C of a CpG dinucleotide within the same transposon or fragment thereof wherein this determination comprises amplifying the bisulfited DNA with at least one primer pair that is specific for the transposon or fragment thereof, and that includes at least one primer comprising at least one differentially methylated position of the transposon;
and
c) determining the normalized DNA methylation level via the values determined in steps a) and b) wherein this determination comprises determining the normalized DNA methylation level via the ratio of the amplificates formed in steps a) and b), wherein the same amounts of DNA are employed in steps a) and b), having been isolated from one sample, wherein the primer pair in step a) and the primer pair in step b) are selected so that a first amplifying region in step a) and a second amplifying region in step b) have a distance of ≦6000 bp apart on the transposon.

1 more element

Dependencies are inferred from the claim text.

Description14,686 words

Summary13,649 words

#This application is a national stage filing of PCT application number PCT/EP2010/050687 filed on Jan. 21, 2010, which claims priority to European patent application serial number 09151141.0 filed on Jan. 22, 2009, both of which are incorporated herein by reference.

#The invention belongs to the field of epigenetics, especially DNA methylation. It provides an amplification process for the detection of epigenetic changes that are relevant, in particular, to clinical diagnostics. Further, specific primers for this amplification process are provided.

#Epigenetic mechanisms cause changes in gene expression that are not accompanied by a change of the coding sequence of the genes, but can be inherited, for example, mitotically. The DNA methylation patterns are transferred from the parent cell to the daughter cells in a manner that is coupled to replication. Thus, the inheritance of epigenetic information is ensured. In higher eukaryotes, DNA methylation is the best studied epigenetic mechanism in addition to RNA-associated silencing and histone modification (Serman et al., Coll Anthropol. 2006; 30(3):665-71).

Brief description of the drawings2 figures

#FIG. 1 shows the result of the determination of the relative DNA methylation of tumor DNA in different dilution stages (with healthy urothelium) according to a preferred embodiment of the invention.

#FIG. 2 shows the result of the determination of the relative DNA demethylation in 4 patient samples as compared to healthy subjects according to a preferred embodiment of the invention.

Detailed description976 words

#DNA from the urothelial carcinoma cell line HT1376, DNA from a urothelial carcinoma and from healthy bladder epithelium were isolated by means of a commercially available kit (Qiagen; QIAamp DNA blood kit). The DNA of the urothelial carcinoma cell line was diluted with DNA from the healthy bladder epithelium in different dilution stages. This was followed by a bisulfitation of the DNA in the different compositions by means of the EpiTect Bisulfite Kit (Qiagen). Subsequently, the determination according to the invention of the relative methylation level of the individual samples as compared to the DNA from the healthy bladder epithelium is performed.

#For each sample, each value was determined in triplicate, and mean values were calculated.

#The amplification was performed by means of real time PCR. Primers were used that were specific for the following LINE-1 promoter sequences:

Facts

Application

13/145,164Filed 21 Jan 2010

Priority

EP 0915114122 Jan 2009

Published

US 2011/0318739 A129 Dec 2011

Granted

26 Dec 2017

Term ends

21 Jan 2030

Estimated · how

20 years from earliest known nonprovisional domestic filing plus recorded patent term adjustment. Maintenance-fee lapses, terminal disclaimers and statutory extensions have not been checked. The continuity chain may be incomplete; this is not an enforceability determination.

Classification4 codes

C12Q 1/6858
Nucleic acid amplification reactions: allele-specific amplification
C12Q 1/6886
For diseases caused by alterations of genetic material: for cancer
C12Q 2600/154
Oligonucleotides characterized by their use: methylation markers
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms

Timeline

22 Jan 2009Priority claimedEP 09151141
21 Jan 2010Application filedUS 13/145,164
27 Jul 2011Assigned to Heinrich-Heine-Universitat Dusseldorfassignment of assignor's interest
29 Dec 2011PublishedUS 2011/0318739 A1
26 Dec 2017GrantedUS 9,850,528 B2
12 Oct 2026TodayWithin its term
21 Jan 2030Term endsEstimated

Determination of the normalized degree of DNA methylation

Life of this patent

Determination of the normalized degree of DNA methylation

Life of this patent

Life of this patent

Abstract

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Drawings

Claims15

Description14,686 words

Classification4 codes

Life of this patent

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Description14,686 words