This activity focused on the CFTR protein and its gene that harbors mutations that cause cystic fibrosis. We used bioinformatics tools to assess two main perspectives of disease-causing mutations: characterizing the resulting defect in the protein and diagnosing of mutation carriers.
Using Prosite we searched for structural and functional motifs in CFTR protein. We found that CFTR belongs to a family of proteins called ABC (ATP-Binding Cassette). The proteins in this family share two types of conserved motifs: one that anchors the protein to the cell membrane (ABC_TM1F) and forms a channel, and another that binds the ATP required for ion transport against the concentration gradient through the channel (ABC_TRANSPORTER2). The motifs we identified in the protein, their function and location, helped us learn about the relationship between the structure of a protein and its function. Analyzing the sequence of the F508del mutant protein using Prosite, we found the motif that is affected by the mutation.
Then, we used the tool Primer3Plus to design primers for a PCR designed to differentiate between DNA from a healthy subject and DNA from a subject that carries the F508del mutation in the CFTR gene. We designed the primers for different experimental approaches: A. choosing primers from both side of the mutation site, for amplification and sequencing, and identifying the origin of the DNA by comparing the resulting sequence to the known sequences of normal and mutant alleles. B. Mutation-dependent amplification – choosing one primer to match the mutation site itself, that is specific to the mutant allele, and then separating the PCR products on a gel, expecting to obtain a product only if the tested subject has the gene mutation, in at least one allele. A rational and thoughtful design of primers is important for a successful PCR.