DEVELOPMENT AND VALIDATION OF A SCREENING-TEST FOR 12 COMMON MUTATIONS OF THE CYSTIC-FIBROSIS CFTR GENE

The results obtained using deoxyribonucleic acid (DNA) amplification-b ased tests must be accurate and reproducible, One such test that simul taneously detects any of 12 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene is presented in thi s report. An investigation was conducted into how changes of primer, D NA template and Taq DNA polymerase concentrations and of polymerase ch ain reaction annealing temperatures affect the test, A total of 383 DN A samples obtained from different laboratories was then examined. The preliminary studies defined the conditions under which accurate result s are obtained even if the test is performed under suboptimal conditio ns, Subsequently, 377 (98.4%) of the DNA samples analysed were in full agreement with DNA typing results derived by other methods. The remai ning 1.6% of samples were not mistyped, rather they were not scored ow ing to failure to detect control DNA sequences. These were also archiv al DNA preparations rather than freshly prepared samples from venous b lood. Careful primer design and optimization of reaction conditions ar e important in the development of multiplex deoxyribonucleic acid ampl ification-based diagnostic tests, Providing the recommended protocols are followed, the test described here is simple to carry out, gives ac curate results and works well if performed within defined operational windows for each reaction variable.