Development of multiplex methylation-specific PCR for detecting methylated/un-methylated PTEN promoter in breast cancer patients using a single closed tube

Abstract

PTEN is a suppressor gene that methylation in its promoter is potentially used for diagnosis and prognosis for breast cancer and other cancer. We aim to develop a simple, fast, and economical Multiple Methylation-Specific PCR (MMSP) PTEN, recognizing a PTEN promoter gene for methylation and un-methylation developed in one single closed tube. This trueexperimental study uses a recombinant plasmid prepared with a TA cloning system. For the development of PTEN with Multiple MSPs, two formulations for MMSP PTEN were designed and compared, in which the best was to study specificity, limit of detection, reproducibility, and repeatability. The methods were validated with a clinical sample that identified its methylation status using Methylation-Specific PCR (MSP). As a result, the best formulation for MMSP PTEN was formulation 2, which comprises of 1:3 methylation and un-methylation primer, with only high specificity for detecting its target and high sensitivity of 1.5625%, while its limit detection was 0.00032 ng/µl. It has high reproducibility and repeatability, which showed consistent data in the experiments with clinical samples in this activity. This simple molecular evidence aids in detecting biomarkers that are potentially used for the diagnosis and prognosis of cancer and the epidemiological study of methylated PTEN promoters.

Keywords

• pten
• methylation
• recombinant plasmid
• breast cancer
• detection
• diagnosis
• multiple