Validation of high-resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
High-resolution melting analysis of polymerase chain reaction products for mutation scanning, which began in the early 2000s, is based on monitoring of the fluorescence released during the melting of double-stranded DNA labeled with specifically developed saturation dye, such as LC-Green. We report here the validation of this method to scan 98% of the coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We designed 32 pairs of primers to amplify and analyze the 27 exons of the gene. Thanks to the addition of a small GC-clamp at the 5' ends of the primers, one single melting domain and one identical annealing temperature were obtained to co-amplify all of the fragments. A total of 307 DNA samples, extracted by the salt precipitation method, carrying 221 mutations and 21 polymorphisms, plus 20 control samples free from variations (confirmed by denaturing high-performance liquid chromatography analysis), was used. With the conditions described in this study, 100% of samples that carry heterozygous mutations and 60% of those with homozygous mutations were identified. The study of a cohort of 136 idiopathic chronic pancreatitis patients enabled us to prospectively evaluate this technique. Thus, high-resolution melting analysis is a robust and sensitive single-tube technique for screening mutations in a gene and promises to become the gold standard over denaturing high-performance liquid chromatography, particularly for highly mutated genes such as CFTR, and appears suitable for use in reference diagnostic laboratories.
Audrezet
Marie-Pierre
MP
INSERM, U613, Brest F-29200, France. marie-pierre.audrezet@univ-brest.fr
Dabricot
Aurélia
A
Le Marechal
Cédric
C
Ferec
Claude
C
D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid; D015331:Cohort Studies; D004396:Coloring Agents / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D005091:Exons; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008086:Lissamine Green Dyes / Q000378:metabolism; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D009691:Nucleic Acid Denaturation; D050500:Pancreatitis, Chronic / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011446:Prospective Studies; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D004396:Coloring Agents; D017931:DNA Primers; D008086:Lissamine Green Dyes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA