Identifying and Analysis of genes and Genetic Analysis of Gene Function
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How much does it cost to sequence your genome?
£7,800
Give two ways we can find genes in the nucleotide sequence
Use gene prediction software Use computer to translate the DNA in all 6 reading frames and then search for similarity proteins called blasts
What is gene prediction software?
Where a sequence is scanned for promoters, start and stop code sequences and intron splicing sites One cant be sure if this prediction is true
Similarity between two proteins suggests what?
That proteins have evolved from the same common ancestor and they have similar molecular functions
What are mircroarrays?
Allow us to compare the transcriptomes of different tissues to each other High throughput, small scale, fast and automated
What manufactures an array?
A very precise robot Each position on the grid contains one cDNA and there is a spot for every gene in the genoeme
What are the three different ways to identify genes?
1) By making a library of cDNA clones from mRNA 2) By making a library of genomic clones then make predictons based upon genomic sequence 3) Identify sets of interesting genes using microarray
Give a way to analyse a gene
Reverse genetics eg in mice
Describe gene replacement in a mouse
Usually makes small changes to the endogenous gene eg mutations in humans could be tested for causing disease symptoms by making the same corresponding mutation in mice genes
What is a knock out?
Where the gene is completetly removed to determine its function To begin a knock out project you must first have a genomic clone of your gene
How can we knockout a single gene in mice?
First acquire a genomic clone of the gene and insert NEO directly on to the econ (destroying the activity of the gene) TK is placed off to one side
Why are the sequences from the target genes called homologous arms?
Because they are the only sequence with homology to the mouse genome
The DNA contrsuct is inserted in to what?
Embryonic stem cells
Is the process of recombining the construct in to the mouse genome efficient?
No
When homologous recombination does occur, which gene is lost?
TK
How are knock outs identified?
Double selection
How are cells that are a result of homologous recombination selected for?
Cells that have integrated NEO are now able to grow on a neomycin media Cells that have integrated TK and Neo wil die when grown on GANC media
How is a single gene targeted in mice?
1) The selected cell line is reintroduced in to the mice embryos 2) The first generation is mosaic and so are their gonads 3) These mosaic animals are then bred to generate non-mosaic carriers of the transgene 4) The carriers are then interbred to create homozygous mutant mice
What is one in forward genetics?
1) Randomly mutate the genome 2) Look for interesting phenotypes in the offspring 3) Identify the gene that causes the defect
Which are the two main model organisms used in forward genetic screens?
Drosophila and zebrafish
What is reverse genetics?
Where we already know the gene and we want to find its function
Describe the steps in a genetic fly screen
1) Mutagenise male: each serm has a different set of mutations by EMS - this male is heterozygous for the mutation 2) Outcross male to wildtype female 3) Incross the family to see what homozygour embryos look like 4) 1/4 of the offspring will show the phenotype if there is one
How many generations does it take to make a mutation homozygous?
3
How do you test if mutations are on different alleles if two mutations show the same phenotype?
Complementation testing