Tumor suppressor genes are tasked with preventing excessive growth of cells. 5-methylcytosine is formed following the addition of a methyl group to cytosine in the cytosine-guanine dinucleotide, CpG, which is found in the promoter region of tumor suppressor genes. This mutation occurs at early stages of cancer even before the symptoms are noted. Solid tumors such as melanoma shed the methylated genes, circulating tumor DNA, into the blood circulation following the programmed cell death and necrosis of tumor cells.
Recently, Stanford scientists: Dr. Jared Nesvet, Dr. Giovanni Rizzi, and Professor Shan X. Wang developed a new analysis method (integration of melt curve analysis) on giant magnetoresistive biosensors with methylation specific polymerase chain reaction. The method is highly sensitive and resulted in an increased ability to detect methylated genes by increasing the DNA hybridization sensitivity. This is very important in the clinic because it can detect low concentration of methylated genes at early stages of oncogenesis. They used specific methylation primers which have the advantage of giving a high ratio of methylated and unmethylated genes which means detecting much lower concentrations of the methylated genes than any other method before. Their research work is published in Biosensors and Bioelectronics.
The research team used synthetic probes that could detect unmethylated and methylated DNA by binding at the cytosine guanine dinucleotide in the amplicon of the methylation specific PCR. The difference in the melting temperature between the two probes were measured which showed that the analysis could be achieved at the lowest limit of 0.1 percent of methylated DNA. The ratio of methylated to umethylated genes was assessed using linear regression of a graph of melting temperatures in different mixtures of the genes.They postulated that it can be used in determining the prognosis of the cancer and even diagnosis. The researchers also proved that giant magnetoresisitive assay could be used to achieve multiplexity. It could resolve a mixture of two genes even when their amplicons melting temperature overlapped. In addition, it had increased sensitivity.
The researchers also analyzed a curve of methylation-sensitive melting with the aim of finding the concentration of the methylated and unmethylated retinoic acid receptor beta, RARB, which is expressed in melanoma. They showed the presence of a mixture of unmethylated and methylated RARB genes when two peaks were observed. They also found that selective amplification of methylated retinoic receptor beta genes was higher as compared with unmethylated genes under high annealing temperature.
They also showed that increased sensitivity of the detection of the methylated genes was achieved using the analysis of the giant magnetoresistive melting. It could be integrated with methylation specific genes so that a sample with 0.1% methylated genes, lowest concentration, could be detected with high specificity. In addition, the sensors of GMR could be functionalized with KIT and RARB, which are methylation genes in melanoma. It thus gave the ability of the methylation-specific genes to detect multiple methylated genes in a single malignancy.
In a nutshell, the method reported by Stanford scientists where methylation specific PCR integrated with GMR is associated with highest sensitivity, multiplexity, and specificity in detecting low concentrations of circulating methylated genes.
Nesvet, J., Rizzi, G., & Wang, S. X. (2019). Highly sensitive detection of DNA hypermethylation in melanoma cancer cells. Biosensors and Bioelectronics, 124, 136-142.Go To Biosensors and Bioelectronics