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
Q
molecular diagnosis includes...
A
- pharmacogenetics
- identity testing (forensics)
- molecular genetics
- hematology
- molecular oncology
- infectious diseases
2
Q
what is pharmacogenetics?
A
- personalized medicine
- prescribe drugs to person based on their genome
3
Q
what type of disorder is sickle cell anemia (genetics)?
A
Autosomal recessive
4
Q
what part of the genome has a problem in those with sickle cell anemia?
A
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
Q
MstII cuts between...
A
C and T
6
Q
RFLP
A
restriction fragment length polymorphism
7
Q
RFLP is used to...
A
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
Q
what differs in a mutant phenotype when looking at RFLP results?
A
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
Q
How long does southern blot take?
A
about 1 week
10
Q
process of southern blot (RFLP method)
A
1. restriction enzyme digest sample
2. electrophoresis on sample
3. immobilization
4. hybridization
5. detection
11
Q
limitations of southern blot based on RFLP method
A
- labour intensive
- time consuming / slow turn around time
- require operator skills / not automated
- use radioactive isotopes
12
Q
steps of PCR
A
1. denature DNA (95*C)
2. anneal primers (55*C)
3. extension - Taq adds nucleotides (72*C)
13
Q
30 cycles of PCR can produce...
A
1 billion PCR products (new strands)
14
Q
PCR allows us to look at target gene without...
A
highly sensitive probes (because there's so many!)
15
Q
turn-around time of PCR-RFLP
A
1-2 days
16
Q
process of PCR-RFLP
A
1. PCR reaction
2. Restriction enzyme digestion
3. Gel electrophoresis
17
Q
limitations with PCR-RFLP
A
- based on known sequence (get right primer & restriction enzymes)
- risk of contamination (will amplify mistakes)
- miss heterozygous large insertion/deletion
18
Q
what technique is used to examine multiple mutations associated with a disease?
A
multiplexed PCR-RFLP
19
Q
multiplexed PCR-RFLP
A
- use multiple primers to amplify several DNA fragments in 1 run
- genotype based on electrophoresis pattern
20
Q
what disease is genotyped using multiplexed PCR-RFLP?
A
Hereditary hemocromatosis (HH)
21
Q
which gene is affected in those with HH?
A
HFE
22
Q
mutations of CF
A
many mutations in the disease-causing gene
23
Q
effect of CF / why is it named this way?
A
scarring (fibrosis) and cyst formation in the pancreas
24
Q
what is the most common life-limiting autosomal recessive disease among Caucasians?
A
CF
25
Q
what type of disease is CF (genetics)
A
autosomal recessive
26
Q
incidence of disease
A
1 in 2500
27
Q
incidence of carrier for CF
A
1 in 25
28
Q
what is the affected gene in CF?
A
cystic fibrosis transmembrane conductance regulator (CFTR)
29
Q
CFTR codes...
A
protein which is responsible for transport of Cl- ions across membrane (lungs, pancreas, liver, digestive tract, reproductive tract, skin)
30
Q
CFTR is a ___ transporter
A
ABC (ATP-binding cassette)
31
Q
what happens if there is defective CFTR?
A
thick, viscous, mucus secretions
32
Q
onset of CF symptoms happen...
A
at birth or later in childhood
33
Q
symptoms of CF
A
- frequent lung infection (due to mucus secretions)
- poor growth (malnutrition due to mucus in GI)
- infertility
- diabetes
34
Q
CFTR has __ exons and span more than __ kb
A
27
230
35
Q
CFTR gene is located on chromosome __
A
7
36
Q
how many mutations are there for the CFTR gene?
A
more than 1600
37
Q
purpose of genetic screening
A
diagnose affected patients and identify carriers
38
Q
how many mutations CFTR mutations are screened for? why were they picked?
A
25; >0.1% frequency in US population
39
Q
what are the most common mutations in CFTR?
A
F508: deletion for Phe at position 508
40
Q
Platforms for CFTR mutations
A
- Tag-It mutation detection system
- Denaturing high performance liquid chromatography (DHPLC)
- non-PCR based technologies (Third Wave INVADER)
41
Q
Tag-It mutation detection system
A
- bead-based microarray platform
- many primers that recognize specific sequences (mutations)
- catch about 50 mutations
- count beats to see if wild type or mutatnt
42
Q
DHPLC
A
- adjust temp
- mismatches separate at lower temp than the right match
43
Q
DNA sequencing
A
determines precise order of nucleotides in a DNA molecule
- any method/technology that determines the order of the bases
44
Q
4 basic sequencing methods
A
1. chemistry reaction
2. technology
3. labeling strategy
4. sequencing detection
45
Q
2 chemistry reactions used to sequence
A
1. enzymatic dideoxy (Sanger)
2. chemical (Maxam-Gilbert)
46
Q
2 technologies used to sequence
A
1. chain termination
2. thermal cycle
47
Q
2 labeling strategies used to sequence
A
1. primers
2. dideoxynucleotides
48
Q
2 sequence detection methods used to sequence
A
1. radioactive
2. fluorescent
49
Q
what is the gold standard for mutation detection & confirmation (sequencing)?
A
Sanger sequencing
50
Q
Sanger sequencing
A
- use DNA Pol to make a copy of ssDNA template at the 3' end of a primer
- use PCR
- terminate by adding 2', 3'-dideoxynucleotides
- analyze fragment using gel/capillary electrophoresis
51
Q
benefits of Sanger DNA sequencing
A
- automated (capillary electrophoresis)
- CCD laser detector
- multicolour fluorescent labeling & detection
- many applications for data (sequencing, fragment analysis, genotyping & SNP discovery, microsatellite analysis)
- software for data collection & analysis
52
Q
how does Sanger sequencing method terminate?
A
Add 2,3-dideoxyribose dideoxynucleodies
-li H instead of OH at C3
- won't H bond with the next nucleotide, so chain stops
53
Q
limitations of Sanger sequencing
A
- miss heterozygous large insertion/deletion
- cost, labour, turn-around time
- difficult to interpret variants with unknown clinical significance (variation in genome or harmful mutation?)
54
Q
clinical applications of DNA sequencing
A
- detect mutations
- confirm mutations detected by other methods
- genotyping (histocompatibility typing, resistance testing)
55
Q
histocompatibility typing
A
used for transplant patients to see if organ is compatible