Optimization of PCR Condition: The First Study of High Resolution Melting Technique for Screening ofAPOA1Variance.
High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on the different melting points of DNA fragments. The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly for screening of a large number of samples.APOA1encodes apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C). This study aimed to obtain an optimal quantitative polymerase chain reaction (qPCR)-HRM condition for screening ofAPOA1variance.



Genomic DNA was isolated from a peripheral blood sample using the salting out method.APOA1was amplified using the RotorGeneQ 5Plex HRM. The PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The melting profile was confirmed by looking at the melting curve.



Five sets of primers covering the translated region ofAPOA1exons were designed with expected PCR product size of 100-400 bps. The amplified segments of DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons 2, 3 and 4B were optimized at an annealing temperature of 60 °C at 40 PCR cycles. Amplicon 4A was optimized at an annealing temperature of 62 °C at 45 PCR cycles. Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles.



In addition to the suitable procedures of DNA isolation and quantification, primer design and an estimated PCR product size, the data of this study showed that appropriate annealing temperature and PCR cycles were important factors in optimization of HRM technique for variant screening inAPOA1.
Center for Biomedical Research, Diponegoro University Faculty of Medicine, Semarang, Indonesia.
Hesty
H
Wahyuningsih
Center for Biomedical Research, Diponegoro University Faculty of Medicine, Semarang, Indonesia.
Ferdy
F
K Cayami
Center for Biomedical Research, Diponegoro University Faculty of Medicine, Semarang, Indonesia; †Department of Cardiology and Vascular Medicine, Diponegoro University Faculty of Medicine, Semarang, Indonesia.
Udin
U
Bahrudin
†Department of Cardiology and Vascular Medicine, Diponegoro University Faculty of Medicine, Semarang, Indonesia; ‡Department of Pharmacology and Therapeutic, Diponegoro University Faculty of Medicine, Semarang, Indonesia.
Mochamad
M
A Sobirin
Center for Biomedical Research, Diponegoro University Faculty of Medicine, Semarang, Indonesia.
Farmaditya
F
Ep Mundhofir
Center for Biomedical Research, Diponegoro University Faculty of Medicine, Semarang, Indonesia.
Sultana
S
Mh Faradz
§Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago 683-8503, Japan.
Ichiro
I
Hisatome
APOA1; HRM; optimization