Forward and Reverse Genetics

If we know what goes wrong within an organism when a gene is mutated, we infer the genes function in its Wild-Type state.

• mutant phenotypes are known before their corresponding genes have been identified.

• much work went into mapping and cloning the mutant genes using pedigree or association studies and “clone walking”

• Many genes were named after their mutant phenotype before they were cloned.

Treating an organism with a mutagen, then screening the offspring for particular phenotypes of interest.

X-rays:

Cause breaks in double-stranded DNA, resulting in large deletion or re-arrangements of chromosome pieces.

pro: strong mutations

cons: 1. may disrupt multiple genes 2. labarious cloning

Chemical:

Use of a chemical (e.g. EMS) causes point mutations (single nucleotide).

May be non-sense (introduce stop codon)

Or miss-sense (cause amino acid replacement)

Or in a non-coding-sequence affecting splycing signals or gene regulation (expression)

pro: 1.full spectrum of mutations 2. random distribution

cons: muation detection difficult

Insertional:

Transposable Elements containing marker genes are inserted into either amino acid sequence (aa replacement) or non coding DNA (regulate expression)

pro: 1. mutation is tagged 2. reversible

con: nonrandom distribution

We now know all of the genes in an organism, but we do not know the function of many of them

Instead of screenig for a phenotype, we look at nucleotide changes.

This is done by performing PCR on the gene of interest and looking for differences of the product in a gel or column.

1. use forward mutagenesis (for example EMS), except instead of screening for a particular phenotype, screen your gene of interest for nucleotide changes.

2. This typically requires that you screen 1000’s or 10,000’s of individuals.

3. This is done by performing PCR on your gene of interest and looking for slight differences in the migration of the PCR product on a gel or column.

4. In theory, you could sequence the DNA of each individual and look for changes, but there are more efficient methods of detection.

   Some examples:

   DHPLC, DGGE, SSCP

1. Denaturing PCR product (heat)

2. Renaturation

   Here you can get a homoduplex: e.g. A-T

   Or a heteroduplex: e.g. A-C

3. Heteroduplexes migrate slower through a DHPLC column.

1. Introducing a plasmid with a marker gene, that has matching flanking regions to the gene of intrest.

2. Natural recombination inserts the marker gene and knocks out the target gene

A form of Transposable element excision.

It stands for white-Hobo-yellow.

1. insert contains 2 nested TEs with 2 reporter genes (white and yellow)

2. knock out Hobo

   target gene right of hobo:

target left of hobo:

RNA interferenz is a way to regulate gene expression without altering the genome. (knock down)

1. introduce double stranded RNA

   1. via injection

   2. or feeding/soaking

   3. or with transgenic methods (permanently inserted into the genome)

2. binds to protein complex

3. binds target mRNA

4. gets degraded before translation

1. Introduce guide RNA with a matching target sequence to the gene of interest as well as the Cas9 endonuclease

2. Cas9 cuts both strands

3. During cell repair some DNA is lost resulting in a knockout of the target gene

4. In step 4 foreign DNA can be knocked in aswell

Can make exact nucleotide changes in vitro on a gene cloned into a plasmid (via QuickChange mutagenisis method) which is inserted back into the genome.

Applications:

1. Use an easily detectable gene from a different organism to test for regulatory sequences.

2. Drosophila flies with point mutations in highly conserved region of Adh gene had 2-fold increse in alcohol dehydrogenase -> increased expression