Phase Change Studies • Genetic and molecular genetic approaches, – isolate mutants that fail in some way to change phase properly, – study genes, gene products and associated molecules, and resulting structures.
Forward vs. Reverse Genetics • Treat thousands of organisms with a mutagen, – random mutagenesis, • Identify an individual with a phenotype of interest, • Identify the gene. Forward • Treat thousands of organisms with a mutagen (usually), – random mutagenesis, • Identify an individual with a genotype of interest, • Identify the phenotype. Reverse
T-DNA (Transfer DNA) Laboratory Construct T-DNA selection genes virulence genes …can put other genes. transform, select for agro with T-DNA Agrobacterium infect plant, select for plants with T-DNA …if the T-DNA lands in a gene, the gene is disrupted.
Set-Up DNA Pooling Maintain lines as pools of seed. Seeds (9) Germinate and grow seeds in liquid culture. Seedlings (225) Extract DNA, DNA (225) Super Pool DNA, 1 2 3 4 5 PCR Screen 6 …30 Super Pools (2025)
PCR Strategy • Polymerase Chain Reaction (PCR), – with oligonucleotide primers with homology to the 5’ and 3’ ends of your gene, amplify the DNA sequence between the primers. Reaction: Product: 5’ Your gene Your gene amplified 3’