| Summary: | 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.
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