PCR with Bisulfite for Detecting DNA Methylation Biomarkers in Cancer

Several traditional and new PCR-based methods have been developed for detecting DNA methylation at single loci. All have characteristic advantages and disadvantages, particularly with regard to use in clinical settings. In order to detect methyla- tion patterns on DNA, one needs greater amounts of it than can be extracted from a small patient sample. In order to achieve this, one can increase the amount of DNA extracted from minute samples rather than use larger samples. Use of PCR for amplifying DNA results in the loss all information on the positions of methylcytosine.

To overcome the limitations of conventional PCR, Epigenomics Inc. uses a procedure that is based on modification of all non-methylated cytosines to a different base, uracil, by the chemical bisulphite. Uracil’s hybridization behavior is identical to that of thymine. Thus, in DNA treated with bisulphite, methylcytosine can easily be detected by hybridizing to guanine. This enables the use of variations of established methods of molecular biology, most notably PCR, hybridization, oligonucleotide-arrays, and mass-spectrometry. The remaining cytosines are present in the sequence context 5'-cytosine-guanine-3' (CpG). The total number of CpG positions in the human genome is ~40 million, up to 10% of which are located in non-repetitive, relevant sections of DNA.

MDxHealth Inc’s methylation-specific PCR (MSP) can rapidly assess the meth- ylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. An MSP assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches, which relies on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. Patent-protected MSP platform is the only scalable technology that enables a sensitive and specific detection of methylated genes in a background of normal cells, critical for early diagnosis or detection of micrometastases in serum, saliva or sputum samples. The process employs an initial bisulfite reaction to modify the DNA, followed by PCR amplification with specific primers designed to distinguish methylated from unmethylated DNA. This specific alteration enables the detection of a few cancer cells embedded in otherwise normal tissue. This process is universal and can be applied to the detection of promoter hypermethylation of relevant tumor suppressor genes or any other cellular genes related to cancer. A practical aspect of this diagnostic marker strategy is the concentration on specific genes known to play an important role in tumorigenesis. This approach is far less labor intensive and more amenable to high throughput screening than microarray assays.

Orion’s MethylScreen® technology leverages biomarkers discovered using its MethylScope technology to develop of a new class of oncology diagnostic kits. MethylScreen is a reliable enzyme-based real-time PCR technology that is compatible with testing platforms widely used in clinical laboratories. The sensitivity of MethylScreen assays enables Orion’s scientists to measure unique qualities of epigenetic DNA that are indicative of disease progression. MethylScreen assays provide critical clinical information about disease progression using blood serum and other easily collected patient samples. MethylScreen is unique in that it is the only platform that does not rely on bisulfite conversion, a harsh chemical process that has been shown to destroy more than 94% of the tumor DNA in patient samples.

MethySYBR is a SYBR green-based PCR assay for the dual analysis of DNA methylation and CpG methylation density (Lo et al. 2009). MethySYBR begins with multiplex PCR to enable the simultaneous amplification of many discrete target alleles in a single reaction using bisulfite-converted DNA. In the second round of PCR, the specific methylated target is quantified from multiplex products using both nested methylation-independent and methylation-specific primer sets. Moreover, the use of SYBR green dye during quantitative PCR enables melting curve analysis of target amplicons to determine the methylation density of CpG sites on target alleles. To establish proof of principle, two cancer-specific methylated genes, RASSF1A and OGDHL, were assessed by MethySYBR. MethySYBR sensitively detected methylated alleles in the presence of a 100,000-fold excess of unmethylated allele. Furthermore, MethySYBR was shown to be capable of analyzing minute amounts of DNA from paraffin-embedded tissue. Therefore, the MethySYBR assay is a simple, highly specific, highly sensitive, high-throughput, and cost-effective method that is widely applicable to basic and clinical studies of DNA methylation.

PCR-based methods that use sodium bisulfite-treated DNA as a template are generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at single loci (Kristensen and Hansen 2009). A number of new methods, such as methylation-specific fluorescent amplicon generation (MS-FLAG), methylation-sensitive high-resolution melting (MS-HRM), and sensitive melting analysis after real-time methylation-specific PCR (SMART-MSP), now complement the traditional PCR-based methods and promise to be valuable diagnostic tools. In particular, the HRM technique shows great potential as a diagnostic tool because of its closed-tube format and cost effectiveness.

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