title: DNA from buccal swab is suitable for rapid genotyping of angiotensin-converting enzyme insertion/deletion (I/D) polymorphism.
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.
authors.affiliation: Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy. Electronic address: angelo.minucci@virgilio.it.
authors.forename: Angelo
authors.initials: A
authors.lastname: Minucci
authors.affiliation: Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Canu
authors.affiliation: Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Concolino
authors.affiliation: Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy.
authors.forename: Donatella
authors.initials: D
authors.lastname: Guarino
authors.affiliation: Institute of Public Health, Section of Hygiene, Department of Public Health, Catholic University, Rome, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Boccia
authors.affiliation: Department of Chemical Sciences, University of Messina, Italy.
authors.forename: Silvana
authors.initials: S
authors.lastname: Ficarra
authors.affiliation: Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Zuppi
authors.affiliation: Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Giardina
authors.affiliation: Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy. Electronic address: ecapoluongo@rm.unicatt.it.
authors.forename: Ettore
authors.initials: E
authors.lastname: Capoluongo
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004847:Epithelial Cells / Q000737:chemistry; D017353:Gene Deletion; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009061:Mouth Mucosa / Q000737:chemistry*; D016254:Mutagenesis, Insertional; D009691:Nucleic Acid Denaturation; D007703:Peptidyl-Dipeptidase A / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results
chemical_list: D004247:DNA; D007703:Peptidyl-Dipeptidase A
keywords: Angiotensin converting enzyme gene; Buccal genomic DNA; High resolution melting analysis; I/D polymorphism