Molecular Basis of Mutation (3 possible mutations can occur at the…
Molecular Basis of Mutation
Site Directed Mutagenesis
Technique that introduces mutations at specific locations in vitro by deleting or inserting 1/more nucleotides.
5 Step Procedure: 1. Determine the sequenced gene being studied 2. Synthesize a short synthetic ssDNA primer containing the desired mutation (must be complementary to the DNA strand) 3. DNA polymerase extends the single-strand primer by copying all of its genes 4. Copied gene is placed in a vectore and introduced into a host cell and cloned. 5. Mutants are selected by DNA Sequencing to verify that they have the mutations.
3 possible mutations can occur at the molecular basis:
Missense Mutation- changing one nucleotide of the triplet, therefore, changing the codon and the amino acid produced.
Nonsense Mutations -Triplet changed to a stop codon so transcription of proteins is stopped.
Silent Mutation - alters the codon but no change n the amino acid produced
H J Muler 1927
X-ray caused mutations in the egg and sperm X150 more than spontaneous mutations
Studied CIB genes in Drosophila
Ame's Test (mutagens , carcinogens)
Studied bacterium strain of Salmonella Typhimurium
his D gene unable to synthesize amino acid histidine from ingredients in its culture medium.
Mutagenic effect because some of the bacteris will regain their ability to make histidine forming a cluster colony around the disc on plates that lack histidine. Scattered colonies around the edges of the plate = spontaneous revertants
Gene Knockout-Knockout Mice
introduction of null mutation into a gene that is then introduced into an organism (usually mice) using transgenic techniques. the organism eventually loses its functions of the gene.
Steps in the procedure:
Create a targeting vector (must have Marker gene that is antibiotic resistant eg : Neomycin Neoand Negative selection Dna marker gene eg Thyamidine Kinasww TK
Insert the targeting vector/sequence into the Mouses Embryonic Stem Cells via electroporation allowing the gene to be incoorporated into the embryo
Identify the cells with the correct gene knocked out with standard molecular biology techniques
Some cells will contain the Targeting Vector which combines with targeting gene and knocks out 1 target gene) = Heterozygous for Knockout Mutation
Injecting cells into a developing embryo and transfer into a chimeric mouse (mixed cell types)
Success determined by the coat colour
Knockout gene of 2 parents = Black dominant coat then transplanted into white recessive coated parents
Success=patches of heterozygous= black and white fur