Optimization of DCR1 and DCR2 epigenetic annealing temperatures for breast cancer biomarker using in-silico and in-vitro study

Abstract

The epigenetics of methylated and unmethylated DCR1 and DCR2 (decoy receptors 1 and 2) are genes encoding membrane receptors that can bind to TRAIL causing TRAIL inhibition in the apoptotic pathway. Epigenetic detection of DCR1 and DCR2 was developed as a biomarker of breast cancer. One of the detection methods is using PCR. The most important step in the PCR process is the determination of the annealing temperature. This research performs Tm analysis using the insilico program from Neb, insilico, Thermofisher, and Promega and in vitro optimization. Methylated DCR1 can be amplified at annealing temperatures of 51.4°C, 52.4°C, 53.6°C, 54.7°C measuring about 600bp according to Tm analysis of insilico and promega. DCR1 could also be amplified at annealing temperatures of 50,1, 49, and 48.8 but the primers were also amplified at non-specific sites. Methylated DCR2 could be amplified at annealing temperatures of 48.8°C, 49°C and 50.1°C and a specific size of about 500 bp according to the Tm analysis of promega. Unmethylated DCR1 and DCR2 genes could not be amplified at the annealing temperature which were analyzed using Neb, insilico, promega, and thermofisher.

Keywords

• melting temperature
• annealing temperature
• pcr