By Levi Clancy for Student Reader on
- 5'-Deletion Mutants
- cDNA Microarray
- Cloning Vectors
- DNA Fingerprinting
- DNA Miniprep
- Gel Shift Assay
- Gene Control in Development: Laboratory Techniques
- Gene Targeting
- Genetic Engineering
- Genetic screen
- In Vitro Nuclear Run-on Experiment
- Interrupted Mating Experiment
- Knockout mutation
- Linkage analysis
- Polymerase Chain Reaction
- Promoter (Transcriptional) (RNA) Fusion
- Reporter Gene
- Restriction Enzymes (Endonucleases )
- Sequence Alignment
- Shotgun sequencing
- Temperature Sensitive Mutant Experiment
- Translational (Protein) Fusion
- Transposon Tagging
The Ames Test is used to determine the strength of a mutagen.
Auxotrophic strains of Salmonella Typhimurium (his-) are mutagenized, then assayed for revertants. In other words, you mutagenize strains with a single mutation in a single gene. You then assay for how many cells were reverted to WT. There are 3 commonly used Ames strains:
Contains a GC bp inserted into hisC gene.
Contains a TA for AT transversion in hisC gene.
Contains a CG for AT transversion in hisC gene.
The Ames strains contain 3 mutations. The first mutation was described above (hisC). The other two mutations (a) increase permeability of the cell so that it can uptake more mutagen (called the rfa mutation) and (b) make it so that the cell is unable to repair mutations. When the cells were engineered to contain these mutations, though, the biotin gene was accidentally deactivated. As a result, Ames strains are unable to synthesize their own biotin. This means that in order for an Ames strain to grow, biotin must be supplemented.
Pretend that you want to find out if Chemical A is a mutagen. You expose the three different strains to dH2O (negative control) and the mutagen. After incubation, you plate the cells on GMA + biotin. The number of colonies which develop is an indication of how powerful the mutagen is. By comparing the mutagenized cells to the negative control, you can find out not only the relative strength of the mutagen but also what kind of mutations it induces.
Incubate bacteria with mutagen for ~45 minutes (in TYE + Mg2+)
plate onto GMA + biotin plates
score for revertants, above the number on the control plate
spontaneous mutations will occur; how many occur on the control plate will reflect the rate of spontaneous reversion
can determine what type of mutations a mutagen causes by observing which strain can be reverted
observe the effects of the rfa mutation
crystal violet, when it penetrates the cell sufficiently, is lethal
half of a plate is â€œpaintedâ€ with a lawn of an rfa strain, and a wildtype on the other half
filter disks dipped in crystal violet are placed on each strain
how far the crystal violet diffuses on the plate will be visible
observe how close each strain can grow to the crystal violet disk
expect the wildtype strain to tolerate higher of crystal violet