Technology

What are CpG sites? Where are they located?

CpG sites are dinucleotides CG or cytosine-guanosine designated CpG. In animals these sites can be epigenetically changed by addition of a methyl group to cytosine, which is then called “a methylated cytosine”. CpG sites exist throughout the genome, every 500-700 nucleotides on average, but in some regions the frequency of CpG sites can be very high. These regions are called “CpG islands”, often abbreviated as CGI. CpG sites – and CpG Islands – are frequent at the beginning of the gene (CpG island of the promoter/first exon), where they participate in gene regulation.

US Biomarkers identifies CpG that are different in disease and health, e.g. methylated in health and UNmethylated in disease. Such disease-specific CpG will be BIOMARKERS for the disease.

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Can biomarkers be made for any region of DNA?

Biomarkers can be designed for any section of DNA that contains GCGC (site for the Hin6I enzyme that discriminates between methylated and unmethylated cytosines).

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How can you target and analyze specific regions with CpG?

We analyze specific CpG dinucleotides that are parts of the enzyme recognition site GCGC. If a DNA fragment contains UNmethylated site, it will be attacked, and "digested" (cut at the site), so its integrity will be destroyed, and PCR primers located across this GCGC site will produce not product. If the site is methylated (meC), the enzyme will not cut, and PCR primers will generate product. In short, PCR primers allow precise targeting, the enzyme differentiates methylated vs unmethylated, and PCR generates the result.

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Do all genes contain these GC-rich regions?

Some of them do, others don't. Those that do not have the site for the enzyme will not be informative and will be excluded from analysis.

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How do you select GC-rich regions for analysis?

For proof-of-principle studies we used the MethDet-56, which contained 56 promoters selected from those that some years ago were known to be changed in cancer. Now we use genome-wide analysis to analyze EVERYTHING. Then the most informative sites (20-30) are tested individually by PCR in individual samples and their informative value confirmed.

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What exactly does the MethDet array detect?

The MethDet array detects DNA methylation in a specific section of the gene (almost always - in the CpG island, which is located in promoter and/or first exon of the gene). The MethDet technology tests methylation in all fragments on the array, collecting all available information for each sample.

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Are the MethDet arrays proprietary?

The MethDet-56 array was proprietary and was custom-produced for proof-of-principle runs. The MethDet-244k array is produced by Agilent Technologies, Inc. For whole genome screening we use Affymetrix Human Tiling arrays 2.0, which tests the whole genome at high resolution (35 bp). Importantly, these devices are used ONLY for selection of candidate DNA fragments for the biomarker. Our customers will test the biomarker in a simple qPCR-based assay.

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What are the benefits of the MethDet technology for blood-based analysis?

The MethDet technology was developed for comprehensive analysis of DNA methylation in ultra-small samples. This is important because clinical samples contain little DNA and are very precious. For example, healthy people have only 5-6 ng of cfDNA per milliliter of plasma, so comprehensive analysis is difficult without the MethDet. When the biomarker has been developed only a few fragments are included, so a couple drops of blood will provide all DNA for analysis.

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Why can’t you amplify DNA first and analyze its methylation after amplification?

PCR will make more DNA, that is correct, but this DNA will not be methylated, so all disease-specific methylation marks will be lost. To get information about disease-specific methylation the test has to use the original DNA from the patient and cannot use PCR product.

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