Primariadewi Rustamadji, Azamris, Nunung Ainur Rahmah, Elsa Septiani Putri, Rosita Wati, Nadya Shifani, Yeremiah Rubin Camin, Endry Nugroho Prasetyo, Kartini Eriani, Elvan Wiyarta, and Desriani

DOI: 10.14456/sjst-psu.2022.116

pp. 869 - 875

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.