Mapping mutations Flashcards
How do we choose a mapping allele when mapping with visible markers?
Randomly
What is the genotype of the tester in a testcross?
Homozygous recessive for all loci under consideration
Which generation do we look at to determine if the gametes produced by a double heterozygote are parental or recombinant
Look at the parental generation, not F1. A gamete with the same genotype of the original parent is parental
What is the ratio of the progeny phenotypes if the new mutation and the mapping allele are unlinked?
1:1:1:1
What is the ratio of the progeny phenotypes if the new mutation and the mapping allele are linked?
Not 1:1:1:1, will have more parentals than recombinants
Can we tell which side of a mapping allele the new mutation is once we get the distance?
No, need to crosses with other mutations to triangulate the position
Why can’t we always get a precise location of the new mutation when using visible alleles?
There isn’t too many of them. Also depends on the organism, i.e. far fewer visible phenotypes in C. elegans but more in Drosophila
How could we increase the resolution of our mapping and get a more precise location of our new mutation?
Molecular markers
What is a SNP?
Particular nucleotides in the genome where variation among individuals from the same species exists
How many alleles do SNPs tend to have?
Usually 2, but sometimes more. One tends to be more common than the others
How many SNPs are there in the genome? Where are they in the genome?
Millions in a genome, very abundant. Found along the entire length of the chromosome in every eukaryote
What is a Snip SNP?
A very special SNP that is inside a restriction enzyme cut site
How do we detect Snip SNPs?
Isolate genomic DNA and amplify the area surrounding the SNP with PCR, then subject the product to a restriction digest and run it on a gel. The enzyme will cut in one SNP allele but not the other and we have differentiate them
Where do the primers bind when amplifying SNP alleles?
The regions flanking the SNP, which will be the same regardless of what SNP is there
What are 3 ways to detect a SNP?
- Snip SNPs
- ASO hybridization
- Primer extension
What is ASO hybridization?
Use small, labelled oligonucleotide probes that will hybridize to either 1 SNP allele or the other
How do we do ASO hybridization?
Isolate genomic DNA, then spot in duplicate onto a solid support. Add the probes and see which ones hybridize by the colours
What do ASO hybridization results look like if an individual is homozygous?
Only one probe hybridizes, so we only get one signal or the other
What do ASO hybridization results look like if an individual is heterozygous?
Both probes hybridize, so the signal is coming from both membranes/both probes are fluorescing
What do Snip SNP results look like if an individual is homozygous?
The site either gets cut and we see two bands, or it doesn’t get cut and we see one larger band
What do Snip SNP results look like if an individual is heterozygous?
One allele gets cleaved and shows up as two bands, the other doesn’t and it shows as one band, so it would have 3 bands total
What is primer extension?
Using labelled ddNTPs to extend a primer by one nucleotide and determine which SNP is present
What is in the reaction mix for primer extension?
Taq polymerase, buffer, differentially labelled ddNTPs
Where does the primer bind when doing primer extension?
The 3’ end stops one base before the SNP
Why does the primer stop one base before the SNP in primer extension?
The next nucleotide to get incorporated will be the ddNTP that is complementary to the SNP and will terminate the chain, and since they all have different labels we can tell which ddNTP will get incorporated
How do we determine which SNP is present after extending the primer by one nucleotide?
Separate the extension products by capillary electrophoresis and detect the fluorescent product
Why is primer extension difficult for the average lab to do?
It requires a DNA sequencing machine, which is expensive
What are microsatellites?
Polymorphic loci based on a 2-5 bp repeating unit
What is the difference between different alleles of a microsatellite or minisatellite?
The number of repeats
What causes the different number of repeats in microsatellite and minisatellite alleles?
Slippage during replication. Repeating regions are mutation hotspots
How do you determine which microsatellite allele is present?
Isolate genomic DNA, then do PCR and run the products on a polyacrylamide gel
What do microsatellite results look like if an individual is homozygous?
One band
What do microsatellite results look like if an individual is heterozygous?
Two bands
What are minisatellites?
Same idea as microsatellites, but the repeating units are much larger (can be thousands of nucleotides)
How do we genotype using minisatellites?
Use restriction enzymes that cleave outside the minisatellite, then run that on a gel and do a southern blot
