Towards a low-cost clinical multiple mutation diagnostic: cystic fibrosis as a model

Cystic fibrosis is used in this work as an example of a genetic disease where early diagnosis and medical intervention can improve quality of life. Current methods of cystic fibrosis diagnosis rely heavily on the sweat test, a biochemical method of measuring the concentration of sodium and chloride in sweat. A repeat test is required for sodium levels between 40 and 70 mmol per litre, which may occur in babies with either mild or no cystic fibrosis. The advantage of a DNA test is that a cut off value is not necessary, this reduces false positive and negative results. A mutation is either present in a person’s DNA or it is absent. Cystic fibrosis has been associated with over 500 mutations, therefore a type of mutation detection was investigated which could potentially examine a large proportion of these in one test. A simple, low cost method of mutation detection is required for use within the National Health Service. The reverse dot blot hybridisation allows a known sequence of DNA to be placed on a membrane (mutant and wild type) and hybridised with an unknown sequence (patients’ DNA). Covalent coupling of oligonucleotides to membrane produced increased attachment over physical attachment as examined via radioactive labels. Polystyrene slides were chemically modified (using 5% potassium permanganate in 1.2M H2SO4) to provide carboxyl groups for the covalent attachment of amino terminated oligonucleotides. Human DNA was amplified by PCR, labelled with biotin and detected via chemiluminescence. Genomic DNA was extracted from blood samples with the Qiagen QIAamp system and both wild type and mutant sequences were amplified. Hybridisations were performed on nylon membranes and modified polystyrene slides. Hybridisation was specific at high stringency (0.1%SDS/0.1xSSC). Further work is required to produce a prototype diagnostic device.