Objective.- By the end of this session you will have simulated the direct diagnosis of genetic disease using (virtual) DNA methods, relating the information obtained from this lab to the previous section on pedigrees. You will select a family from the 'virtual freezer' (see below), and apply a range of tests to identify the mutation(s) segregating in that family. In your report, you should state the family you have chosen and the mutation(s) found, using the standard rules of nomenclature that apply to mutations (see below). You should describe the methods used.

The 'DNA samples' for this lab are under the Tools button on the left, where you will also request a variety of on-line simulated tests. The nature of the tests, (such as DGGE, etc) is discussed under their specific name in the buttons on the left.

In the laboratory, you contemplate two situations;

1) Having to identify an unknown mutation from a patient that has been clinically diagnosed as having certain disease, (here Cystic Fibrosis) and

2) Having to search for the presence of a known mutation in relatives of the patient.

To identify an unknown mutation, particularly if the gene is large, you need first to identify the exon that harbors the mutation. For this you use Denaturing Gradient Gel Electrophoresis (DGGE). If you knew that a mutation is particularly prevalent in a population, you may search the corresponding exon first. If, as in CF, the most prevalent mutation is a small deletion, instead of DGGE you may start by doing Heteroduplex Analysis of the relevant exon.

In particular you should be able to understand the thinking behind the experiments and use the tools mentioned on the left (green) column of this page:

• To understand the routine used in detection of sequences bearing unknown mutations, using a simulation of Denaturing Gradient Gel Electrophoresis.
• Ditto sequences bearing small deletions, using Heteroduplex Analysis.
• To characterize mutations by Sequencing, for instance using Sanger’s Dideoxy method.
• To automatically locate mutations on a sequence, by sequence alignment of wild type and mutant sequences.
• To use some rules for mutation nomenclature.
• To translate DNA and obtain the complementary strand from the coding strand. This is useful to establish whether a mutation is expected to alter the normal protein sequence, for instance by replacing one aminoacid by another.

Once the mutation(s) segregating in a family have been determined, it is easier to screen other members of the family for the presence of those mutations. For instance, the mutation may create or destroy a restriction site, in which case restriction enzyme analysis may be used to screen specifically for such mutation. For these studies, the Polymerase Chain Reaction (PCR) is often used, and the digestion is performed on PCR products. The first step should be, therefore, to select a set of primers to amplify the relevant exon(s) in such a way that the product spans the region with the mutation. If the mutation(s) do not alter restriction sites, you may use 'taylor-made' PCR oligo primers specific for the mutation, in a test called Amplification-refractory mutation system (A.R.M.S.). In brief, to screen members of a family for known mutations, you may need;

• To search for Restriction Enzyme cutting sites in a DNA sequence.
• To obtain optimized oligo primers from your DNA sequence for PCR reactions.
• To prepare a kit for the simultaneous detection of several known mutations.
• To speculate on the role of chromosomal background in the phenotypic variability of cystic fibrosis.
• Current examples refer to simple family structures where cystic fibrosis is segregating. Once again, our emphasis here has been on practical application, and we assume that the theory behind the subjects has been covered in the lecture courses. What has been said about families could be applied to populations.