DETEKSI SINGLE NUCLEOTIDE POLYMORPHISM NQO1 P187S, p53 P72R, dan MDM2 T309G DENGAN TEKNIK PCR-RFLP DAN PCR-HRM PADA SAMPEL FORMALIN FIXED PARAFFIN-EMBEDDED (FFPE) PASIEN KANKER PAYUDARA

Abstract

Detection test for Single Nucleotide Polymorphism (SNP) recently carried out by PCR-RFLP. This method needs post-PCR procedure to determine the result. Discovery of real time PCR lead to the new method for SNP detection, called PCR-HRM. This alternative method was being analyzed to find better detection method. Detection of SNP NQO1 P187S, p53 P72R, and MDM2 T309G was carried out because they are having a relation on the metabolism response on anthracycline chemotherapy. Formalin Fixed Paraffin-Embedded (FFPE) samples are routine samples that collected in cancer hospital. The riches of this samples can be used as useable diagnostic samples. Nevertheless, quality of DNA which is isolated from FFPE�s sample is poor because of formalin. were There two aims of this research. First was to know the benefit of FFPE sample�s in SNP detection. Second was to know the benefot of PCR-HRM and HRM�s profile in SNP NQO1 P187S, p53 P72R, and MDM2 T309G�s detection. FFPE�s samples were collected from operation. The samples were DNA�s isolated, and amplified using PCR (Polymerase Chain Reaction) for NQO1, p53, dan MDM2. Restriction Fragment Lenght Polymorphism (RFLP) was conducted using restriction enzymes HinfI, BstUI, dan MspA1I, respectively for NQO1, p53, dan MDM2. The second method was PCR-High Resolution Melting (PCR-HRM). This method was done using real time PCR Corbett Rotor Gene. The results was annalyzed using melting curve, normalization curve, and differentiation plot curve.The comparison between PCR-RFLP and PCR-HRM has been done to detect three SNP. Sequencing technique has been used as standart technique if there are any differences found between PCR-RFLP and PCR-HRM. There are 6 differences found in comparison between PCR-RFLP and PCR-HRM. The PCRRFLP �results showed 66,67% similarity with sequencing�s results. FFPE�s sample can be used for SNP NQO1 P187S, p53 P72R, and MDM2 T309G� detection. HRM�s profile can be analyzed using three curves. There are melting curve, normalization curve, and differentiation plot curve. The comparison between PCR-RFLP and PCR-HRM has done to detect three SNP. PCR-RFLP more reliable method than PCR-HRM if primer which is used not specially design for PCR-HRM. PCR-HRM is a faster method than PCR-RFLP for SNP NQO1 P187S, p53 P72R, and MDM2 T309G�s detection