Lecture 11 - Mutant genes Flashcards
What are genetic markers?
gene loci conferring an observable trait
What are physical markers and two examples
Variation in sequence
- sequence length (repeat regions, insertions/deletions)
- base changes (SNPs)
What are 5 detecting markers?
RFLPs: restriction fragment length polymophisms
CAPS: cleaved amplified polymorphoc sequences
dCAPS: derived CAPS
ASP: allele-specific PCR
STR: short tandem repeats
How are RFLP used?
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
How are CAPS used?
1) PCR amplifies chromsomal locus
2) Cut PCR product with restriction enzyme
3) separate products by gel electrophoresis
How are dCAPS used?
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
How are ASPs used?
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
How are STRs used?
1) PCR amplifies STR region, different alleles have different number of repeats
2) separate products by gel electrophoresis (or higher resolution by capillary electrophoresis)
Why do STR loci change frequently in a population?
base slippage during replication
How can a microsatellite locus show linkage to a disease gene?
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
How can physical and recombination maps be aligned?
20kb on physical map
corresponds to 1 map unit on recombination map
What is a forward genetic screen?
When you know mutant phenotype but not the gene affected
What is required for forward genetic screen?
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
How do you begin a genetic screen?
1) test markers on all chromosomes for linkage with mutant locus to find out on which chromosome the mutant is
How do you do a forward genetic screen?
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
Why in actual genetic screens do you not need to test many individual samples for different markers?
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
What occurs after identifying on what chromosome the mutant allele is?
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
How can we do a genetic screen when we do not know the genome sequence?
Positional cloning/map-based cloning
- must clone the chromosomal locus
- polymorphisms must be found
- chromosome walking may be needed
What is a chromosome walk?
create a collection of overlapping clones to cover the entire refion between markers
What do genome wide association studies allow?
large scale analysis of polymorphic markers thoughout a population to associate chromosomal loci with traits and diseases
How can you use association mapping to identify genes for disease susceptibilty?
find common genes (high frequency in population) between those who have certain disease
Once mapping and association ahve provided candidate genes how can their function be demonstrated?
by complementation of mutants with wild type gene
