Angelo Minucci1, Giulia Canu2, Paola Concolino2, Donatella Guarino2, Stefania Boccia3, Silvana Ficarra4, Cecilia Zuppi2, Bruno Giardina2, Ettore Capoluongo5. 1. Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy. Electronic address: angelo.minucci@virgilio.it. 2. Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy. 3. Institute of Public Health, Section of Hygiene, Department of Public Health, Catholic University, Rome, Italy. 4. Department of Chemical Sciences, University of Messina, Italy. 5. Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy. Electronic address: ecapoluongo@rm.unicatt.it.
Abstract
BACKGROUND: Plasma angiotensin-converting enzyme (ACE) variability between individuals is the results of an insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene. The I and D alleles differ for the presence or absence of a 288 bp Alu sequence DNA fragment. METHODS: The present paper regards the development of a single-tube High Resolution Melting Analysis (HRMA), applied to DNA extracted by buccal swabs, for determining three ACE I/I, I/D, D/D genotypes, in order to obtain a rapid and high throughput method. This method takes advantage of the presence of the 288 bp DNA fragment. Primer design was performed taking into account the possible different efficiency of allele I amplification compared to allele D, avoiding the misclassification of I/D with D/D genotypes. RESULTS: 50 samples previously genotyped by "conventional" PCR protocol already published in literature were 100% concordant with the HRMA results, showing high reproducibility, sensitivity and specificity. ACE genotypes were distinguished by normalized temperature melting curves and by derivate fluorescence plots. CONCLUSIONS: HRMA was confirmed as particularly suitable for the identification of ACE I/D polymorphism. Simple setup and rapidity of the analysis (about 1.5 h for 96 samples, including data interpretation) are other important advantages along with low-costs, making this technique useful in clinical research and diagnostics.
BACKGROUND: Plasma angiotensin-converting enzyme (ACE) variability between individuals is the results of an insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene. The I and D alleles differ for the presence or absence of a 288 bp Alu sequence DNA fragment. METHODS: The present paper regards the development of a single-tube High Resolution Melting Analysis (HRMA), applied to DNA extracted by buccal swabs, for determining three ACE I/I, I/D, D/D genotypes, in order to obtain a rapid and high throughput method. This method takes advantage of the presence of the 288 bp DNA fragment. Primer design was performed taking into account the possible different efficiency of allele I amplification compared to allele D, avoiding the misclassification of I/D with D/D genotypes. RESULTS: 50 samples previously genotyped by "conventional" PCR protocol already published in literature were 100% concordant with the HRMA results, showing high reproducibility, sensitivity and specificity. ACE genotypes were distinguished by normalized temperature melting curves and by derivate fluorescence plots. CONCLUSIONS: HRMA was confirmed as particularly suitable for the identification of ACE I/D polymorphism. Simple setup and rapidity of the analysis (about 1.5 h for 96 samples, including data interpretation) are other important advantages along with low-costs, making this technique useful in clinical research and diagnostics.
Authors: Dona Jeanne Alladagbin; Carlos Gustavo Regis da Silva; Luciano Kalabric Silva; Washington Lc Dos-Santos; Geraldo Gileno de Sá Oliveira Journal: BMC Nephrol Date: 2022-10-10 Impact factor: 2.585