Lecture 11 - Mutant genes

Question 1

What are genetic markers?

Answer 1

gene loci conferring an observable trait

Question 2

What are physical markers and two examples

Answer 2

Variation in sequence

• sequence length (repeat regions, insertions/deletions)
• base changes (SNPs)

Question 3

What are 5 detecting markers?

Answer 3

RFLPs: restriction fragment length polymophisms
CAPS: cleaved amplified polymorphoc sequences
dCAPS: derived CAPS
ASP: allele-specific PCR
STR: short tandem repeats

Question 4

How are RFLP used?

Answer 4

1) cut genomic DNA with restriction enzyme
2) separate by gel electrophoresis
3) blot on membrane
4) use labelled probe complementary to specific chromosomal region to detect fragments

Question 5

How are CAPS used?

Answer 5

1) PCR amplifies chromsomal locus
2) Cut PCR product with restriction enzyme
3) separate products by gel electrophoresis

Question 6

How are dCAPS used?

Answer 6

1) When SNPs not within a restriction site, primers designed so that a restriction site is created within one of the alleles
2) mismatch of primer does not prevent amplification
3) restriction site is created in PCR products of certain allele
4) restriction sites cleaved by restriction endonuclease (e.g. MboI)
5) separate products by gel electrphoresis

Question 7

How are ASPs used?

Answer 7

1)use allele-specific primers in two parallel reactions
2)
a)allele 1 with primer 1 and common reverse primer - amplified product
b)allele 1 with primer 2 and common reverse primer - no product
c)allele 2 with primer 1 and common reverse primer - no product
d)allele 2 with primer 2 and common reverse primer - amplified product

Question 8

How are STRs used?

Answer 8

1) PCR amplifies STR region, different alleles have different number of repeats
2) separate products by gel electrophoresis (or higher resolution by capillary electrophoresis)

Question 9

Why do STR loci change frequently in a population?

Answer 9

base slippage during replication

Question 10

How can a microsatellite locus show linkage to a disease gene?

Answer 10

1) identify parental alleles when one parent shows disease. Parental alleles who will have different STR
2) compare the banding pattern of the parents with the banding pattern of the offspring and identify common alleles between those who have the disease to show disease gene

Question 11

How can physical and recombination maps be aligned?

Answer 11

20kb on physical map

corresponds to 1 map unit on recombination map

Question 12

What is a forward genetic screen?

Answer 12

When you know mutant phenotype but not the gene affected

Question 13

What is required for forward genetic screen?

Answer 13

Double heterozygotes for GOI and for markers

look for recombinants between the mutant locus and the other two alleles, where there are less recombinants, there is less genetic space between the alleles

Question 14

How do you begin a genetic screen?

Answer 14

1) test markers on all chromosomes for linkage with mutant locus to find out on which chromosome the mutant is

Question 15

How do you do a forward genetic screen?

Answer 15

1) obtain double heterozygotes by crossing of mutant with wild type in a different genetic background
2) Self F1 progeny
3) test individuals with mutant phenotype in F2 for given marker
4) 50% recombinants shows independent assortment, mutant not linked to this locus, may reside on another chromosome or far away on the same
5) test individuals for a different marker
6) if recombination frequency is low i.e. 10% then mutant is linked to that marker and suggest mutant is on that chromosome

Question 16

Why in actual genetic screens do you not need to test many individual samples for different markers?

Answer 16

Can use bulked segregant analysis

• assume that if no linkage, two alleles of a marker should be present in 50/50 ratio
• isolate DNA from individuals that show mutant phenotype
• mix aliquots of all samples to obtain DNA pool
• test for the present of marker allelles against control DNA from F1 (verify that equal band intensities are acheived)
• when in F2 one allele is underrepresented when compared to F1, the non-unrepressented allele must be linked to the mutant allele

Question 17

What occurs after identifying on what chromosome the mutant allele is?

Answer 17

1) select a number of different markers on the arm of the chromsome the marker loci was on
2) test DNA from individuals with mutant phenotype
3) identify chromosomal segments in recombinant chromsomes that correspond to parental mutant genotype
4) use new markers within interval found

Question 18

How can we do a genetic screen when we do not know the genome sequence?

Answer 18

Positional cloning/map-based cloning

• must clone the chromosomal locus
• polymorphisms must be found
• chromosome walking may be needed

Question 19

What is a chromosome walk?

Answer 19

create a collection of overlapping clones to cover the entire refion between markers

Question 20

What do genome wide association studies allow?

Answer 20

large scale analysis of polymorphic markers thoughout a population to associate chromosomal loci with traits and diseases

Question 21

How can you use association mapping to identify genes for disease susceptibilty?

Answer 21

find common genes (high frequency in population) between those who have certain disease

Question 22

Once mapping and association ahve provided candidate genes how can their function be demonstrated?

Answer 22

by complementation of mutants with wild type gene