Flashcards in LMP 301 Lecture 20: Molecular Diagnostics Deck (55)
molecular diagnosis includes...
- identity testing (forensics)
- molecular genetics
- molecular oncology
- infectious diseases
what is pharmacogenetics?
- personalized medicine
- prescribe drugs to person based on their genome
what type of disorder is sickle cell anemia (genetics)?
what part of the genome has a problem in those with sickle cell anemia?
single point mutation in B-globulin gene
- E -> V
- restriction enzyme (MstII) is no longer able to recognize the sequence that it's supposed to digest
MstII cuts between...
C and T
restriction fragment length polymorphism
RFLP is used to...
1. digest the genome with restriction enzymes
2. use southern blot to detect length of digested pieces
3. compare to normal to see if there is mutation / what the mutation affects
what differs in a mutant phenotype when looking at RFLP results?
usually, some sites are not cut (no longer recognized by the restriction enzyme). This will lead to larger segments when compared with the normal phenotype
How long does southern blot take?
about 1 week
process of southern blot (RFLP method)
1. restriction enzyme digest sample
2. electrophoresis on sample
limitations of southern blot based on RFLP method
- labour intensive
- time consuming / slow turn around time
- require operator skills / not automated
- use radioactive isotopes
steps of PCR
1. denature DNA (95*C)
2. anneal primers (55*C)
3. extension - Taq adds nucleotides (72*C)
30 cycles of PCR can produce...
1 billion PCR products (new strands)
PCR allows us to look at target gene without...
highly sensitive probes (because there's so many!)
turn-around time of PCR-RFLP
process of PCR-RFLP
1. PCR reaction
2. Restriction enzyme digestion
3. Gel electrophoresis
limitations with PCR-RFLP
- based on known sequence (get right primer & restriction enzymes)
- risk of contamination (will amplify mistakes)
- miss heterozygous large insertion/deletion
what technique is used to examine multiple mutations associated with a disease?
- use multiple primers to amplify several DNA fragments in 1 run
- genotype based on electrophoresis pattern
what disease is genotyped using multiplexed PCR-RFLP?
Hereditary hemocromatosis (HH)
which gene is affected in those with HH?
mutations of CF
many mutations in the disease-causing gene
effect of CF / why is it named this way?
scarring (fibrosis) and cyst formation in the pancreas
what is the most common life-limiting autosomal recessive disease among Caucasians?
what type of disease is CF (genetics)
incidence of disease
1 in 2500
incidence of carrier for CF
1 in 25
what is the affected gene in CF?
cystic fibrosis transmembrane conductance regulator (CFTR)
protein which is responsible for transport of Cl- ions across membrane (lungs, pancreas, liver, digestive tract, reproductive tract, skin)