LMP 301 Lecture 20: Molecular Diagnostics Flashcards Preview

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Flashcards in LMP 301 Lecture 20: Molecular Diagnostics Deck (55)
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1

molecular diagnosis includes...

- pharmacogenetics
- identity testing (forensics)
- molecular genetics
- hematology
- molecular oncology
- infectious diseases

2

what is pharmacogenetics?

- personalized medicine
- prescribe drugs to person based on their genome

3

what type of disorder is sickle cell anemia (genetics)?

Autosomal recessive

4

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

5

MstII cuts between...

C and T

6

RFLP

restriction fragment length polymorphism

7

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

8

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

9

How long does southern blot take?

about 1 week

10

process of southern blot (RFLP method)

1. restriction enzyme digest sample
2. electrophoresis on sample
3. immobilization
4. hybridization
5. detection

11

limitations of southern blot based on RFLP method

- labour intensive
- time consuming / slow turn around time
- require operator skills / not automated
- use radioactive isotopes

12

steps of PCR

1. denature DNA (95*C)
2. anneal primers (55*C)
3. extension - Taq adds nucleotides (72*C)

13

30 cycles of PCR can produce...

1 billion PCR products (new strands)

14

PCR allows us to look at target gene without...

highly sensitive probes (because there's so many!)

15

turn-around time of PCR-RFLP

1-2 days

16

process of PCR-RFLP

1. PCR reaction
2. Restriction enzyme digestion
3. Gel electrophoresis

17

limitations with PCR-RFLP

- based on known sequence (get right primer & restriction enzymes)
- risk of contamination (will amplify mistakes)
- miss heterozygous large insertion/deletion

18

what technique is used to examine multiple mutations associated with a disease?

multiplexed PCR-RFLP

19

multiplexed PCR-RFLP

- use multiple primers to amplify several DNA fragments in 1 run
- genotype based on electrophoresis pattern

20

what disease is genotyped using multiplexed PCR-RFLP?

Hereditary hemocromatosis (HH)

21

which gene is affected in those with HH?

HFE

22

mutations of CF

many mutations in the disease-causing gene

23

effect of CF / why is it named this way?

scarring (fibrosis) and cyst formation in the pancreas

24

what is the most common life-limiting autosomal recessive disease among Caucasians?

CF

25

what type of disease is CF (genetics)

autosomal recessive

26

incidence of disease

1 in 2500

27

incidence of carrier for CF

1 in 25

28

what is the affected gene in CF?

cystic fibrosis transmembrane conductance regulator (CFTR)

29

CFTR codes...

protein which is responsible for transport of Cl- ions across membrane (lungs, pancreas, liver, digestive tract, reproductive tract, skin)

30

CFTR is a ___ transporter

ABC (ATP-binding cassette)