Methods for analyzing chromosomal DNA, including chromatin, are provided.

Methods for analyzing chromosomal DNA, including chromatin, are provided.

The present invention relates to a method of screening for the presence of methylated DNA in a biological sample by using multiple methylation-sensitive restriction endonucleases. More particularly, the present invention relates to a method of quantitatively screening for the level of one or more methylated genes of interest without the requirement that an undigested internal reference sample is used as a point of reference against which relative quantification is calculated. The present invention is useful in a range of applications including, but not limited to, providing a simpler and more accurate means to determine DNA methylation status, such as in the context of diagnosing or monitoring conditions characterised by changes to DNA methylation.

The present invention relates to a method of screening for the presence of methylated DNA in a biological sample by using multiple methylation-sensitive restriction endonucleases. More particularly, the present invention relates to a method of quantitatively screening for the level of one or more methylated genes of interest without the requirement that an undigested internal reference sample is used as a point of reference against which relative quantification is calculated. The present invention is useful in a range of applications including, but not limited to, providing a simpler and more accurate means to determine DNA methylation status, such as in the context of diagnosing or monitoring conditions characterised by changes to DNA methylation.

The present disclosure concerns embodiments related to methods of enriching particular DNA for analysis of methylation status and/or profiles, for example in the process of diagnosis of cancer. In particular embodiments, the methods utilize cell-free DNA as a source of DNA instead of genomic DNA and allow for focused enrichment of fragments having two or more enzyme digestion sites and containing at least one CpG site.

The present disclosure concerns embodiments related to methods of enriching particular DNA for analysis of methylation status and/or profiles, for example in the process of diagnosis of cancer. In particular embodiments, the methods utilize cell-free DNA as a source of DNA instead of genomic DNA and allow for focused enrichment of fragments having two or more enzyme digestion sites and containing at least one CpG site.

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.

Reagents and methods for analysis of DNA hydroxymethylation are provided. Methods comprise modification of hydroxymethylated cytosine residues with a bulky moiety to protect hydroxymethylated positions from cleavage with a DNA endonuclease. For example, methods may comprise contacting DNA with a glucosyltransferase to glucosylate hydroxymethylated DNA positions and digesting the DNA with a DNA endonuclease to cleave DNA in positions lacking hydroxymethylation. Reagents and kits for hydroxymethylated DNA analysis are also provided.

Reagents and methods for analysis of DNA hydroxymethylation are provided. Methods comprise modification of hydroxymethylated cytosine residues with a bulky moiety to protect hydroxymethylated positions from cleavage with a DNA endonuclease. For example, methods may comprise contacting DNA with a glucosyltransferase to glucosylate hydroxymethylated DNA positions and digesting the DNA with a DNA endonuclease to cleave DNA in positions lacking hydroxymethylation. Reagents and kits for hydroxymethylated DNA analysis are also provided.