Biology 3594A Lecture Notes - Lecture 22: Histidine, Mutagen, Wild Type
13 May 2018
School
Department
Course
Professor
Ames Test
Reverse mutations –have a mutant and see if a compound will change the DNA sequence from mutant state back to the wild type
Use a specific strain of salmonella
Auxotrophic –mutants can’t produce the nutrient but the wild type can
Provide histidine for them on a plate or they won’t grow
Assume compound X would be mutagenic
Prototrophic –back to original state before mutation, can make their own histidine
Different strains of salmonella bacteria
Don’t need to memorize these
You know what mutation each strain has
From the different strains used in the test (use a lot to cover a lot of mutational types)
Whichever strain is being fixed, that’s the type of mutation the compound is causing
Specificity –specific mutations
Sensitivity
How much the colonies grow
Membrane around the cell –wreck it and more likely the mutagen will enter the cell
Salmonella strain
Control: plate with a bit of histidine
(want the histidine to have time to be used up, then colonies will die unless they reverted back to the wild type)
Add rat liver extract –create microsomal environment that mimics metabolism in the human body
Called the S9 mix, it’s called that in papers
When you add to the plate it’ll help metabolize compound down and see if the metabolites are carcinogenic and mutagenic
Mystery compound X is indirectly mutagenic
His- media: limited histidine on it
His- bacteria: can’t synthesize histidine
Results suggest that the metabolites of compound X are mutagenic or carcinogenic
Not directly: because compound X alone doesn’t do anything
After metabolism has taken place, no carcinogenic or mutagenic effects
ddPCR
PCR reaction happening in each tiny droplet
Fluorescent probes binding to target DNA
Certain droplets that don’t have the target won’t fluoresce
Droplets are fed through a reader one at a time
We’ll know which droplets had the target DNA in it
Very specific and very sensitive technique
Top: target DNA and how primers would anneal
castPCR
Good for detecting rare mutations and variants of small quantity (same as ddPCR)
First Collect DNA samples and purify them
An allele specific primer –need to know what the mutation is that you’re looking for (specific base pair change and where it is in the genome)
Since T matches A in the spot on the left you’ll get preferential amplification for the mutant not the wild type
Amplify in other direction for other probe so you get more copies faster
Tutorial 7 Mutation Detection Assays II
May 13, 2018
3:08 PM
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