practicals

Question 1

what are the 3 steps of PCR?

Answer 1

1. denaturation (DNA is heated to 95 degrees)
2. primer attachment(annealling) (primer anneals to template strands)
3. elongation (temperature to 72 degrees and polymerase can do its work)

Question 2

Our normal DNA polymerase is not functional at a high temperature of 95 degrees. How is this fixed?

Answer 2

Taq polymerase, from certain bacteria can withstand the high temperature and still replicate our DNA.

Question 3

What is the difference between real-time PCR and conventional PCR?

Answer 3

With real-time PCR, you monitor the amplification during PCR. conventional is only after the PCR procedure.

Question 4

How can we monitor real-time PCR?

Answer 4

• fluorescent dye
• specific DNA probes

Question 5

What are the advantages and disadvantages of specific DNA probes and fluorescent dye?

Answer 5

1. fluorescent dye can bind to any DNA probe, thus not only our own. DNA probes are more specific.
2. fluorescent dye is more affordable than the DNA probes

Question 6

What is the melting curve analysis?

Answer 6

when 50% of the DNA bonds are hybridised at a certain temperature, that temperature is the melting temperature.

Question 7

How do you check for contamination?

Answer 7

negative control:
- agarose gel
- fluorometer
NTC

Question 8

How do you check for PCR inhibition?

Answer 8

• internal amplification control
• Kinetic outlier detection (KOD)
• positive sample (with target DNA)

Question 9

The melting curve analysis consists of 2 peaks, but only one is the specific product you target. what is the other one?

Answer 9

The primer dimer, this is around 72 degrees, thus the first peak you see on the left. the second peak is the specific product.

Question 10

If a B-cell follicle has a gemrminal center, what does it mean?

Answer 10

It is the secondary follicle that is activated. The germinal center is the proliferation zone of the B-cells. When antigens are presented by the follicular dendritic cells, the B-cells undergo hypermutation and selection affinity

Question 11

Where are the T-cell follicles located?

Answer 11

between the B-cell follicles

Question 12

What is the anatomial name of the tonsils in the upper airway?

Answer 12

Waldeyer’s tonsillar ring

Question 13

In histology, certain small rings of cells are found near the b-cell and t-cell follicles. What are they and what is their function?

Answer 13

HEV: high endothelial venules. they are specialised blood vessels mediating lymphocyte trafficking to lymphocytes