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DHPLC screening of cystic fibrosis gene mutationsRavnik-Glavač, Metka ...Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the ... potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFžTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) (Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations (Ravnik-Glavač et al., 1994) we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations.Source: Human mutation. - ISSN 1059-7794 (Letn. 19, št. 4, 2002, str. 374-383)Type of material - article, component partPublish date - 2002Language - englishCOBISS.SI-ID - 15728089
Author
Ravnik-Glavač, Metka |
Atkinson, Andrew |
Glavač, Damjan |
Dean, Michael
Topics
Cystic Fibrosis |
Genetics |
Cystic Fibrosis Transmembrane Conductance Regulator |
Genetics |
Dna Mutational Analysis |
Genetic Screening |
Mutation |
Time Factors |
Temperature |
Software |
Sensitivity And Specificity |
Polymorphism, Single-Stranded Conformational |
Nucleic Acid Denaturation |
Algorithms |
Chromatography, High Pressure Liquid |
Cistična fibroza |
Cistična fibroza, regulator transmembranske prevodnosti |
DNA mutacijska analiza |
Genetsko presejanje |
Mutacija |
Algoritmi |
Kromatografija visokotlačna, tekočinska |
Nukleinska kislina, denaturacija |
Občutljivost in specifičnost |
Polimorfizem, enojne-vijačnice konformacijske |
Programska oprema |
Temperatura |
Časovni faktorji
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Database name | Field | Year |
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Ravnik-Glavač, Metka | 01502 |
Glavač, Damjan | 09275 |
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