Back to : Contents . BioLab Help . Sample Experiments
Formerly known as cystic fibrosis of the pancreas, this disease has increasingly been labelled simply 'cystic fibrosis.' Symptoms relate to malfunctions of the pancreas, intestinal glands, biliary tree, bronchial glands, and sweat glands. Infertility occurs sometimes in males and females. People with cystic fibrosis make too much mucus in their lungs. The mucus is thicker than normal and collects in the lungs, making gaseous exchange difficult and providing a breeding ground for bacteria. The thick mucus is also made in the pancreas. It blocks the pancreatic duct, stopping digestive enzymes from flowing into the duodenum.
Cystic fibrosis is a genetic disease. A genetic disease is caused by a mutation in one or more genes. In the case of cystic fibrosis, the gene is for an ion channel (called Cystic fibrosis transmembrane conductance regulator (CFTR)), which controls chloride transport as well as regulates many other transport pathways. A mutation in CFTR will cause the production of a malfunctioning channel protein.
We have two alleles of each gene. Cystic fibrosis will only develop when both alleles are defective. Indeed, if one allele is normal, the correct protein will be made in sufficient amounts to balance the effect of the incorrect protein synthesised from the other allele. The dominant allele (F) makes a normal protein; the recessive allele (f) makes the incorrect protein. These are three genotypes.
Finding a diagnostic
The CFTR gene has been completely sequenced as well as some parts of the chromosome surrounding it. The coding sequence can be found in the EMBL database at accession number M28668. Many mutations in this gene have been characterised, and more than 120 are listed which are linked to cystic fibrosis symptoms. One of them is a deletion of three nucleotides (ttt) leading to the omission of one amino acid, Phe at position 508 of the protein. Unlike the other two examples we have studied so far, this change does not affect any restriction sites. We can still design a diagnostic test using PCR . It is possible to design sets of PCR primers that will only amplify a specific fragment in the mutated or the normal gene.
To do so, we need to use four primers. Two of these primers, ("F" for forward and "R" for reverse) will amplify a fragment in the cftr gene, in both the normal and mutated gene. It is our control set. We have two additional primers."W", for wild, coupled with R will amplify a fragment only in the normal gene. W will only hybridize with the DNA sequence if the three Ts are present. "M" for mutant, coupled with F, will amplify a fragment only in the mutated gene. It recognises the a 26 bp nucleotide sequence in which the three Ts are absent. This is illustrated in figure 1
The amplified fragments have different sizes so it is easy to differentiate them on a gel.
This diagnostic test therefore involves:
Load experiment 13 into BioLab. It contains 13 samples.
Samples 1-3 contain DNA extracted from different individuals
Sample 4 - 8 contain respectively primers F, R, W and M
Samples 9-13 will be used later
PCR the DNA samples
You should see that the normal DNA (F/F: homozygous for normal CFTR gene) and the mutant DNA (f/f: homozygous for the mutated CFTR) both give two PCR fragments, but they differ by their sizes. The sample containing both normal and mutant DNA (F/f: heterozygous), simulating an heterozygote, gives four fragments, two of them being so close in size that the two bands could be mistaken as one.
Use this procedure to determine whether samples 8-13 are from:
Click here to check your results!