Forward Genetics And Development Flashcards
Outline forward genetics
Identification of a gene responsible for a phenotype.
Give examples of physical mutagens
UV
Ionising radiation (X-rays, Gamma rays)
give examples of chemical mutagens
Base analogues
Alkylating agents
Give examples of biological membranes
Transposable elements (move from one part of the genome to another- disrupt function of a gene)
Give examples of biological mutagens
Transposable elements (move from one part of the genome to another- disrupt function of a gene)
Outline the steps of forward genetics
1- create mutants in a model organism
2- identify mutants with a phenotype of interest
3- define genes responsible for these phenotypes (complementation)
4- clone/identify the genes
5- analyse the genes to predict encoded proteins
Outline step one of forward genetics
Mutagenesis
-physical mutagens
-chemical mutagens
-biological mutagens
Outline step two of forward genetics
Identify mutants with a phenotype of interest
Genetic selection- involves killing individuals which don’t have the phenotype of interest
Genetic screening- examine phenotypes of interest
Use of replica plating for identification of auxotrophic mutants
Mutants have lost their ability to make something themselves
Outline step three of forward genetics
Genetic complementation tests
Outline step 4 of forward genetics
Cloning genes by mutant rescue (Complementation)
Mutant is transformed using a library from a wild type strain
What library is used for eukaryotic
cDNA library
What library is used for prokaryotes
Genomic library
Outline step five in forward genetics
Isolate the plasmid that rescued the mutant, use PCR to amplify the cDNA insert, DNA sequencing, conduct BLAST, use expression vector to purify protein and make antibodies
