Mapping mutations

Question 1

How do we choose a mapping allele when mapping with visible markers?

Answer 1

Randomly

Question 2

What is the genotype of the tester in a testcross?

Answer 2

Homozygous recessive for all loci under consideration

Question 3

Which generation do we look at to determine if the gametes produced by a double heterozygote are parental or recombinant

Answer 3

Look at the parental generation, not F1. A gamete with the same genotype of the original parent is parental

Question 4

What is the ratio of the progeny phenotypes if the new mutation and the mapping allele are unlinked?

Answer 4

1:1:1:1

Question 5

What is the ratio of the progeny phenotypes if the new mutation and the mapping allele are linked?

Answer 5

Not 1:1:1:1, will have more parentals than recombinants

Question 6

Can we tell which side of a mapping allele the new mutation is once we get the distance?

Answer 6

No, need to crosses with other mutations to triangulate the position

Question 7

Why can’t we always get a precise location of the new mutation when using visible alleles?

Answer 7

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

Question 8

How could we increase the resolution of our mapping and get a more precise location of our new mutation?

Answer 8

Molecular markers

Question 9

What is a SNP?

Answer 9

Particular nucleotides in the genome where variation among individuals from the same species exists

Question 10

How many alleles do SNPs tend to have?

Answer 10

Usually 2, but sometimes more. One tends to be more common than the others

Question 11

How many SNPs are there in the genome? Where are they in the genome?

Answer 11

Millions in a genome, very abundant. Found along the entire length of the chromosome in every eukaryote

Question 12

What is a Snip SNP?

Answer 12

A very special SNP that is inside a restriction enzyme cut site

Question 13

How do we detect Snip SNPs?

Answer 13

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

Question 14

Where do the primers bind when amplifying SNP alleles?

Answer 14

The regions flanking the SNP, which will be the same regardless of what SNP is there

Question 15

What are 3 ways to detect a SNP?

Answer 15

1. Snip SNPs
2. ASO hybridization
3. Primer extension

Question 16

What is ASO hybridization?

Answer 16

Use small, labelled oligonucleotide probes that will hybridize to either 1 SNP allele or the other

Question 17

How do we do ASO hybridization?

Answer 17

Isolate genomic DNA, then spot in duplicate onto a solid support. Add the probes and see which ones hybridize by the colours

Question 18

What do ASO hybridization results look like if an individual is homozygous?

Answer 18

Only one probe hybridizes, so we only get one signal or the other

Question 19

What do ASO hybridization results look like if an individual is heterozygous?

Answer 19

Both probes hybridize, so the signal is coming from both membranes/both probes are fluorescing

Question 20

What do Snip SNP results look like if an individual is homozygous?

Answer 20

The site either gets cut and we see two bands, or it doesn’t get cut and we see one larger band

Question 21

What do Snip SNP results look like if an individual is heterozygous?

Answer 21

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

Question 22

What is primer extension?

Answer 22

Using labelled ddNTPs to extend a primer by one nucleotide and determine which SNP is present

Question 23

What is in the reaction mix for primer extension?

Answer 23

Taq polymerase, buffer, differentially labelled ddNTPs

Question 24

Where does the primer bind when doing primer extension?

Answer 24

The 3’ end stops one base before the SNP

Question 25

Why does the primer stop one base before the SNP in primer extension?

Answer 25

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

Question 26

How do we determine which SNP is present after extending the primer by one nucleotide?

Answer 26

Separate the extension products by capillary electrophoresis and detect the fluorescent product

Question 27

Why is primer extension difficult for the average lab to do?

Answer 27

It requires a DNA sequencing machine, which is expensive

Question 28

What are microsatellites?

Answer 28

Polymorphic loci based on a 2-5 bp repeating unit

Question 29

What is the difference between different alleles of a microsatellite or minisatellite?

Answer 29

The number of repeats

Question 30

What causes the different number of repeats in microsatellite and minisatellite alleles?

Answer 30

Slippage during replication. Repeating regions are mutation hotspots

Question 31

How do you determine which microsatellite allele is present?

Answer 31

Isolate genomic DNA, then do PCR and run the products on a polyacrylamide gel

Question 32

What do microsatellite results look like if an individual is homozygous?

Answer 32

One band

Question 33

What do microsatellite results look like if an individual is heterozygous?

Answer 33

Two bands

Question 34

What are minisatellites?

Answer 34

Same idea as microsatellites, but the repeating units are much larger (can be thousands of nucleotides)

Question 35

How do we genotype using minisatellites?

Answer 35

Use restriction enzymes that cleave outside the minisatellite, then run that on a gel and do a southern blot