Forward Genetic Techniques

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Mutagenesis

If there is not a natural change in the phenotype of interest, the first stage in forward genetic screens is to cause a mutation. This can be done using chemical agents such as ethylmethanesulfonate (EMS), irradiation (X-ray, UV-rays, ionising particles) or by introducing a retroviruses into the genome of the animal model.

Identifying mutations

Firstly it needs to be determined if a mutation is dominant or recessive. This can be achieved using a genetic crosses. When a Wild Type/ Mutant cross produces F1 progeny displaying the mutant phenotype then the mutation is dominant. If this observation is not made then backcrossing the F2 progeny with F1 will produce a homozygous mutant which should then show the abnormal phenotype and confirm the recessive mutation.

To define the location of the mutation, linkage analysis is used to determine if the position of the mutation is close to a known gene marker. Recombination between genes occurs more frequently the further two genes are apart, therefore finding a gene with a low recombination between the parent and disease gene allele suggests that the disease gene is close to this marker.

This shows a general location for the mutation however to narrow the position down further positional cloning is used. Chromosome fragments from the closest known genetic marker are used as a probe to find and sequence nucleotides. A section of the end of this fragment is then used as a probe to search the genome for further fragements. This process is repeated until there is a series of overlapping DNA fragments which can be compared to a DNA library in order to identify mutations.