Download Mutants: Their Uses

Mutants: Their Uses
Pick Kuen Chan
What is a Mutation?
• permanent change in the DNA sequence
• Range from single DNA base to a large
segment of a chromosome
What Causes DNA Mutations?
• DNA damage from environmental agents
- ultraviolet light (sunshine)
- nuclear radiation
- certain chemicals
• Mistakes that occur when a cell copies its
DNA in preparation for cell division
Types of Mutations
• Point mutations
single nucleotide base changes in a gene's DNA
sequence
- Missense mutations
- Nonsense mutations
- Silent mutations
• Insertion and deletion mutations
add or remove one or more DNA bases. Insertion and
deletion mutations cause frameshift mutations
The Central Dogma of Biology
DNA
Transcription
RNA
Translation
Protein
Genome to Gene Function
• Genome sequence
• Gene discovery
• Gene functional annotation
– In silico prediction
– Gene expression
– Protein-protein interaction
– Mutant analysis
Mutant Analysis
• Known as forward genetics
• Phenotype-centric process
• Reliable means to assign gene function
• Enormous effort to identify each gene
responsible for a particular phenotype
Mutant Analysis
• Reverse genetics
• Prediction of function based on DNA
sequence
• Required large collections gene-indexed
mutation
Mutant Analysis
Forward Genetics
Reverse Genetics
Select a biological process
Select a gene or genes of
interest
Generate highly redundant
mutant populations
Screen for mutants with a
desired phenotype
Map and clone the gene
responsible for the phenotype
Generate highly redundant
mutant populations
Develop and conduct
sequence-based mutant
screens
Analyse the phenotype of
the mutants
Alfonso and Ecker, 2006
Generation of Mutant Population
• Chemical agents
– Ethyl methanesulphonate (EMS) and
nitrosomethylurea (NMU)
• Physical agents
– Fast neutrons, X-ray and carbon ions
• Biological agents
– Transposons and transferred-DNA (T-DNA)
EMS Mutagenesis
• Efficient mutagens in plant
- Soybean, pea, Lotus japonicus,
Medicago truncatula
• Generate a broad range of DNA alteration
- Point mutation
• Unbiased distribution of mutation
Supernodulation mutant
EMS
Wild type
nts1007
Ethylene insensitive mutants
Wild type
Skl
Penmetsa et al., 2003
Fast Neutron Mutagenesis
• Intermediate efficiency
• Produce double stranded DNA breaks that
are then repaired
• DNA alteration
- Big chromosomal insertions, deletions
and rearrangement
Examples
ga1-3 transgenic
mutant
ga1-3
wild
type
Sun et al., 1992
FN37
wild type
Men et al., 2002
Biological agents
• DNA or RNA molecules as mutagen
• Introduction of engineered DNA/RNA
molecule into living cell.
- Agrobacterium tumefaciens as shuttle
- Plant-associated bacteria
- DNA/RNA viruses
- Particle bombardment
Application in Plant Transformatiom
• Insertional mutagenesis
• Ectopic expression
• Gene silencing
• Homologous recombination
Criteria for T-DNA
• Selectable marker
- Herbicide resistance gene, antibiotic
resistance gene
• Reporter genes
- GFP, β-glucuronidase (GUS), luciferase
• Gene/s of interest
Schematic Diagram on
Transformation
Examples
Ottenschläger et al., 2003
Workshop on Mycorhizal