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 24 or absence of a 288 bp Alu sequence DNA fragment. Methods: The present paper regards the development of a single-tube High ResolutionMelting 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: HRMAwas 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.

DNA from buccal swab is suitable for rapid genotyping of 2 angiotensin-converting enzyme insertion/deletion (I/D) polymorphism

FICARRA, Silvana;
2014

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 24 or absence of a 288 bp Alu sequence DNA fragment. Methods: The present paper regards the development of a single-tube High ResolutionMelting 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: HRMAwas 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.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11570/2671201
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