title: Denaturing HPLC analysis of DNA deletions and insertions.
abstract: Denaturing HPLC (DHPLC) is a useful technique for the fast screening of known and unknown heterozygous gene mutations. Most DNA mutations causing genetic disorders consist of nucleotide substitutions, but insertions and deletions occur, albeit less frequently. The heteroduplexes with insertions/deletions have gaps that may affect molecular stability differently from the mismatches caused by substitutions. Therefore, gaps and mismatches may be distinguished by DHPLC analysis, which is based on the differential thermal stability of amplicons with different characteristics. To verify this hypothesis, we examined 12 DNA samples containing insertions and deletions of different sizes (one to 29 residues) from four different genes (ABCA4, CFTR, FTL, and SLC11A3). We found that all of them were detected by DHPLC runs at 50 degrees C, which is considered a non-denaturing temperature, as well as by runs at the temperature optimized for mismatch recognition. The finding confirms that gaps reduce heteroduplex stability more than mismatches, and indicates that DHPLC analysis at low temperature may be applied to distinguish DNA deletions/insertions from substitutions.
authors.affiliation: IRCCS H. San Raffaele, Unit of Genomics for Diagnosis of Human Pathologies, Milan, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Cremonesi
authors.forename: Stefania
authors.initials: S
authors.lastname: Stenirri
authors.forename: Isabella
authors.initials: I
authors.lastname: Fermo
authors.forename: Rita
authors.initials: R
authors.lastname: Paroni
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
authors.forename: Mario
authors.initials: M
authors.lastname: Cazzola
authors.forename: Paolo
authors.initials: P
authors.lastname: Arosio
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D027682:Cation Transport Proteins / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002872:Chromosome Deletion; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D005293:Ferritins / Q000235:genetics; D006801:Humans; D016254:Mutagenesis, Insertional / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009691:Nucleic Acid Denaturation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012374:Rod Cell Outer Segment; D000098742:Ferroportin
chemical_list: D020121:5' Untranslated Regions; C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters; C505032:CFTR protein, human; D027682:Cation Transport Proteins; D000098742:Ferroportin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D005293:Ferritins