Highly sensitive method for the detection of cytosine methylation patterns

Abstract

The present invention concerns a method for the detection of cytosine methylation in DNA samples, wherein the following steps are conducted: (a) a genomic DNA sample, which comprises the DNA to be investigated and background DNA, is chemically treated in such a way that all of the unmethylated cytosine bases are converted to uracil, whereas the 5-methylcytosine bases remain unchanged; (b) the chemically treated DNA sample is amplified with the use of at least 1 primer oligonucleotide as well as a polymerase, whereby the DNA to be investigated is preferred as the template over the background DNA, and (c) the amplified products are analyzed and the methylation status in the DNA to be investigated is concluded from the presence of an amplified product and / or from the analysis of additional positions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application is a continuation of U.S. patent application Ser. No. 10 / 229,370, which in turn is a continuation-in-part of International Application No. PCT / EP02 / 02572, which has an international filing date of Mar. 8, 2002, now abandoned, both of which are incorporated herein by reference. Said international application was published under PCT Article 21(2) in a language other than English.BACKGROUND OF THE INVENTION[0002]The present invention concerns a method for the detection of cytosine methylation in DNA samples.[0003]The levels of observation that have been well studied due to method developments in recent years in molecular biology include the genes themselves, as well as transcription and translation of these genes into RNA and the proteins arising there from. During the course of development of an individual, when a gene is turned on and how the activation and inhibition of certain genes in certain cells and tissues ar...

AI Technical Summary

Benefits of technology

[0033]It is also particularly preferred that one of the additional oligonucleotides and / or PNA oligomers prevents the binding of the forward primer, while the other prevents the binding of the reverse primer.

[0172]In order to determine the relative sensitivity, PCR of GSTp1 was conducted with 10 ng of template DNA mixtures (see Example 8) with and without blocker oligonucleotides (FIG. 16). The calculation of the “threshold cycle” was made by the TaqMan software and gives the number of PCR cycles at which the GSTP1 PCR product can be detected for the first time with a higher signal than the negative control, comparable to the “crossing point” values of the LightCycler. This means that the lower the “threshold cycle” value is, the more efficient the amplification of the GSTp1 fragment was.

Problems solved by technology

In addition, in the case of a PCR amplification, the epigenetic information, which is borne by 5-methylcytosines, is completely lost.

Of course, previously, only individual regions of up to approximately 3000 base pairs in length have been investigated; a global investigation of cells for thousands of possible methylation analyses is not possible.

Of course, this method also cannot reliably analyze very small fragments of small sample quantities.

These are lost despite the protection from diffusion through the matrix.

For nucleic acids, which have a backbone with multiple negative charges, the ionization process through the matrix is essentially less efficient.

For example, it is not possible to conclude the quantity present in serum from the detectability of an amplified fragment of the DNA to be investigated.

Even minimal quantities of such DNA are successful for obtaining a positive result, which is an advantage, on the one hand, but can also act in a very unfavorable manner, if one wishes to evaluate, for example, the effect of a tumor resection on serum DNA.

The greatest disadvantage, however, is that many methylation positions are present, in which the DNA to be investigated and the background DNA differ only in degree.

In contrast, it is typical in a tumor tissue that a specific position is present in the methylated state, e.g., in 95% of the tumor cells, in which the otherwise present background DNA, however, is present in the methylated state only to a maximum of 5%, so that it is not possible with the MSP method to produce informative results, since a quantification of the template DNA by means of PCR in principle is not possible or can be accomplished only with increased expenditure.

The disadvantage of this method, however, is that it cannot supply accurate information in those cases in which the background DNA greatly predominates, since this information is amplified accurately along with the DNA to be investigated and both are analyzed in the mixture.

This problem does not exist in the analysis of solid tumors, in which the material to be investigated can be selected in a targeted manner, but the analysis, for example, of serum DNA is made difficult.

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