STRAND A

Question 1

no. H bonds between CG

Answer 1

3

Question 2

no. H bonds between AT

Answer 2

2

Question 3

primase function

Answer 3

synthesizes RNA primers

Question 4

PCR benefits

Answer 4

sensitive
robust
cheap
rapid
specific

Question 5

PCR tube contents

Answer 5

template (ds DNA)
2 primers
polymerase
dNTP’s
Magnesium
buffer (8-9.5)

Question 6

3 regions of Taq polymerase

Answer 6

synthesis
proof-reading
primer removal

Question 7

Taq polymerase characteristics

Answer 7

heat-stable
3 regions
accurate DNA copying

Question 8

implications of too long of a primer in PCR

Answer 8

slow hybridization

Question 9

implications of too short of a primer in PCR

Answer 9

not specific

Question 10

primer size range in PCR

Answer 10

18-24 bp

Question 11

primer characteristics in PCR

Answer 11

oligonucleotide/ ssDNA
start/ finish with G/C pairs
Tm = 50-60 degrees C (5 degrees between pairs)
3’ comp to template

Question 12

Magnesium role in PCR

Answer 12

non-protein co-factor allowing catalysis for enzymatic activity of DNA polymerase

Question 13

potassium ions role in PCR

Answer 13

promote annealing

Question 14

PCR process 3 stages

Answer 14

denaturation
annealing
elongation

Question 15

PCR 1st cycle

Answer 15

1 strand synthesis
(boil, anneal and extend w polymerase/ dNTPS)

Question 16

PCR 2nd cycle

Answer 16

synthesis of 2 strands
(boiling and annealing different primer comp to new/original DNA strand, polymerase extends)

Question 17

final PCR cycles

Answer 17

simultaneous synthesis of both strands
30 repeats

Question 18

PCR product detection

Answer 18

molecular weight markers
PCR products
primers
template
(agarose gel w intercalating dye)

Question 19

uses of PCR

Answer 19

DNA manipulation/ quantification/ amplification
genetic disease diagnosis
pathogen detection
ancient DNA
gene function study
knock-out genes
biotechnology

Question 20

reverse transcriptase PCR

Answer 20

1. RNA converted to cDNA via reverse transcriptase
2. amplification via PCR

Question 21

RNA sources in reverse transcriptase PCR

Answer 21

gene expression
RNA virus

Question 22

reverse transcriptase PCR ingredients

Answer 22

reverse transcriptase
dNTPs
buffer
primer
RNA template

Question 23

end-point vs qPCR
age?
price?
precision

Answer 23

qPCR newer (1996 vs 1983), more expensive, more precise

Question 24

end-point vs qPCR quantification

Answer 24

end-point semi-quantitative, measuring densitometry
qPCR amount proportionate to template amount
end-point measurement at end (plateau) and qPCR continuous (exponential phase) measurement

Question 25

end-point PCR uses

Answer 25

cloning genotyping sequencing

Question 26

qPCR uses

Answer 26

gene expression quantification microarray verification quality control assay validation SNP genotyping copy number variation viral quantification siRNA/ RNA experiments

Question 27

THERMOS LIGHTCYCLER

Answer 27

thermal cycler incorporating fluorometer for detection and quantification of PCR products

Question 28

master mix

Answer 28

pre-measured solution at optimal concentration for each reagent

Question 29

2 fluorescence reagents

Answer 29

SYBR green TAQman

Question 30

SYBR green

Answer 30

binds to groove of dsDNA

Question 31

TAQman

Answer 31

probes with fluorescence reporter and quencher probe hybridizes as FUP/RUP anneal and extend DNA pol cleaves probe and fluorescence increases

Question 32

3 phases of PCR logarithmic standard curve

Answer 32

exponential linear plateau

Question 33

Ct

Answer 33

cycle threshold no. cycles required for the PCR to reach the threshold/ exceed background level.

Question 34

Ct level indication

Answer 34

lower Ct value, higher amount of cDNA (starting material)

Question 35

reference gene

Answer 35

control to normalize gene expression levels, constant gene expression, unaffected by experimental factors

Question 36

house-keeping gene

Answer 36

normalizes mRNA levels between samples to for sensitive comparison ^reliability and reproducibility of experimental results

Question 37

PCR standard curve

Answer 37

semi-log regression line plot of Ct value vs log of nucleic acid input

Question 38

reference gene examples for PCR

Answer 38

Beta actin GAPDH Albumin 18S rRNA TATA sequence binding protein

Question 39

3 examples of clinical applications of PCR

Answer 39

Genotyping patient genotyping pathogen phenotyping disease

Question 40

PCR patient genotyping components

Answer 40

genetic trait diagnosis carrier detection tissue matching (HLA typing) predicting response to drugs

Question 41

DNA sources for patient phenotyping

Answer 41

blood, hair, buccal smear, amniotic fluid cells

Question 42

2 PCR based techniques for genotyping

Answer 42

PCR-RFLP (Restriction Fragment polymorphism) ARMS-PCR (Amplification Refractory Mutation System)

Question 43

PCR-RFLP process

Answer 43

1. amplify substrate to 2 strands of dsDNA 2. add the restriction enzyme 3. analysis with electrophoresis

Question 44

clinical example of PCR-RFLP

Answer 44

Diagnosis of Sorsby's Fundus dystrophy degenerative eye disease leading to blindness autosomal dominant TIMP3 mutation (tissue inhibitor of metalloproteinase 3) introduces premature stop codon

Question 45

PCR RFLP positives

Answer 45

cheap easy design microindel/SNP application simple resources commonly used techniques

Question 46

PCR RFLP negatives

Answer 46

only possible with known restriction site some RE expensive requires single nucleotide polymorphism time-consuming not suitable for high-throughput

Question 47

clinical example of ARMS-PCR

Answer 47

diagnosis of cystic fibrosis mutation in CFTR gene leading to Cl- transport imbalance across PM F508 common mutation

Question 47

ARMS-PCR

Answer 47

use of allele specific primers to detect alelic variants

Question 48

RFLP vs ARMS primers? dependents? alternatives?

Answer 48

RFLP has locus-specific primers vs ARMS allele specific primers RFLP relies on presence/ absence of restriction site vs ARMS relies on PCR stringency ARMS has tetra primer alternative w non-allele specific primers in addition

Question 49

DNA sources for pathogen phenotyping

Answer 49

blood sputum urine faeces skin swab tissue biopsy

Question 50

pathogen phenotyping influence

Answer 50

patient management and infection control measures

Question 51

microscopy disadvantages to PCR

Answer 51

less sensitive (high levels required) difficult to distinguish strain/ species

Question 52

culture disadvantages to PCR`

Answer 52

not all organisms can be cultured (PCR doesn't require culture) takes weeks (rather than hours) less specific

Question 53

patient antibody response disadvantages to PCR

Answer 53

May not illicit strong response whereas PCR not dependent on immune response

Question 54

pathogen phenotyping clinical example

Answer 54

TB smear test w acid fast stain > dependent on bacterial load/ quality/ expertise culture for mycobacteria then molecular testing

Question 55

disease phenotyping technique

Answer 55

RT-PCR

Question 56

clinical example of disease phenotyping

Answer 56

HIV viral load measurement with RT-PCR