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All Journal ANNALES BOGORIENSES
BUGI RATNO BUDIARTO
Research Center for Biotechnology, Indonesian Institute of Sciences Jalan Raya Bogor km 46, Cibinong 16911, Bogor, Indonesia
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Immunology & microbiology Medicine & Pharmacology

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Improvement of HER2I655V TARMS-PCR Performance by DNA Quality Analysis Budiarto, Bugi Ratno; Azamris, Azamris; Desriani, Desriani
ANNALES BOGORIENSES Vol 21, No 2 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ab.v21i2.308

Abstract

Reliable TARMS-PCR is a prerequisite in constructing a solid conclusion in genetic diagnostics. The validity of data generated by this molecular technique is hampered by a false positive result. In attempt to develop a  TARMS-PCR for HER2I655V genotyping with no interfering of bias we used DNase I to eliminate DNA contaminant resided in PCR reagent. TARMS-PCR without enzyme treatment using recombinant plasmids that contained HER2I655V gene with represented its alleles was used to evaluate the presence of false positive  result while DNase I treated-PCR reagent was used in TARMS-PCR to evaluate the effective dose of the enzyme and further to adjust the TARMS-PCR conditions.  PCR master mix kit used in this study produced a false positive result on HER2I655V TARMS-PCR as proven by the presence of multiple PCR products in Non-Template Control (NTC) and 0.1 U of the enzyme could eliminate this DNA contaminant effectively, although this pretreatment altered the specificity of HER2I655V TARMS-PCR genotyping on certain genotype. Combination of touchdown TARMS-PCR with another allele-specific primer recovered specificity of detection on this model system. Interestingly, this optimized HER2I655V TARMS-PCR can only be used for genotyping the clinical samples if only further optimization was done using genomic DNA as template
ILE655VAL Genotyping Study of HER2 - Positive Breast Cancer of Patients from Padang - Indonesia With High Resolution Melting Technique Wulandari, Dwi; Azamris, Azamris; Nurhayati, Isna; Warisman, M Ali; Budiarto, Bugi Ratno; Desriani, Desriani
ANNALES BOGORIENSES Vol 21, No 2 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (282.104 KB) | DOI: 10.14203/ab.v21i2.310

Abstract

Transtuzumab has proven to be a great improvement in the treatment of HER2 + of breast cancer patients, but it is associated with relevant adverse cardiac events and with significantly elevated cost of treatment.  One of the risk factors for cardiotoxicity due to transtuzumab is the I655V HER2 polymorphism (GTC> ATC mutation) in which the allele mutant (Ile val or val / val) has a higher risk than the wild type (Ile/Ile). The detection of specific alleles is very important for therapeutic decision-making. In this study, our group have developed a HRM with EvaGreen dye method to discriminate specific allele of I655V HER2 (wild type, heterozygote mutant or homozygote mutant) in 66 frozen section samples of HER2+ of breast cancer patients. Our group reported the wild type is the most prevalent allele (77,27%), whereas heterozygous mutation is significantly present in this research (21,21%) and only 1,52% of sample detected as minor allele.  It showed that only one sample detected as a minor allele (val/val) and may have relatively low abundance in the population. This method has been compared with sanger sequencing and giving 100% of validity.
Co-Authors Arika Wahyuningsih, Arika Azamris, Azamris Desriani Desriani Dwi Wulandari Isna Nurhayati, Isna Warisman, M Ali