Lab4

Question 1

When is Agarose gel electrophoresis used?

Answer 1

To check wheter the PCR generated virus-specific amplification product, i.e the sample, is positive for the tested virus.

Question 2

Why is Agarose gel electrophoresis used?

Answer 2

For size separation and visualization of the PCR products.

Question 3

How is the amplified DNA molecules separated?

Answer 3

By applying an electric current to move the negatively charged molecules through an agarose mix.

Question 4

How can the DNA produced in the PCR be identified? (by the agarose G.E)

Answer 4

Comparing the size of the product amplified in the sample to the positive control and the molecule weight marker.

Question 5

What are the components of the Agarose gel?

Answer 5

• electrophoresis buffer
• agarose powder
• intercalating dye

Question 6

Electrophoresis buffer contains?

Answer 6

Ions that can carry the electric current through the gel.
Usually Tris-acetate-EDTA (TAE, contains the salts of acetic acid) or tris-borate-EDTA (TBE, contains the salts of boric acid) is used.

Question 7

What type of molecule is the Agarose?

Answer 7

synthetic polysaccharide

Question 8

What are the characteristics of the gel made from purified agarose?

Answer 8

relatively large pore size, making them appropriate for separation of DNA fragments larger than 100 base pairs.

Question 9

Difference between shorter and longer molecules in the agarose gel, at the same voltage:

Answer 9

Shorter molecules move faster and therefore travel further than longer molecules in the same timer interval. this is because shorter molecules migrate more easily trough the pores of the gel.

Question 10

Gel concentration for short and long DNA products

Answer 10

• For shorter DNA products (100-300 bp) more concetrated gel is needed (1.5-2%).
• for longer DNA products (>800 bp) less concentrated gel is needed (0.5,1%)

Question 11

How can DNA be visualized in the agarose gel?

Answer 11

by using an intercalating dye (mixed in the gel), which fluoresces under ultraviolet light.

Question 12

What types of buffers are used in the loading of the PCR products?

Answer 12

TAE and TBE

Question 13

What is the functions of the TAE and TBE buffers?

Answer 13

provide ions that carry the current, and to maintain the pH at a relatively constant value.

Question 14

What does the Loading buffer contain?

Answer 14

A dense compound (e.g sucrose), that increases the density of the sample so that the DNA sinks to the bottom of the well.

Question 15

What are the color dyes used in the loading? and functions

Answer 15

Xylene Cyanol and Bromophenol blue, used to visualise the progress of the electrophoresis, as they migrate together with the PCR products in the gel.

• Xylene cyanol (light blue color) migrates together with the large fragments
• Bromophenol blue (dark blue) migrates together with the smaller fragments.

Question 16

What current is used for optimal resolution in electrophoresis?

Answer 16

10 V/cm electric current.

- the distance in cm refers to the distance between electrodes.

Question 17

What happens during electrophoresis?

Answer 17

the negatively charged DNA migrates in the gel from the negative pole to the positive pole

Question 18

After the end of the electrophoresis, the gel is viewed with=

Answer 18

UV transilluminator.

Question 19

What is a molecule weight marker?

Answer 19

mixture of different size DNA fragments

Question 20

What happens during Evaluation?

Answer 20

The size of the amplified DNA products are compared to a molecule weight marker

Question 21

How can we declare a PCR sample positive?

Answer 21

The size of the DNA product amplified mys be what is expected by the position of the primers and the same as the size of the product in the positive control.

Question 22

How are the viruses investigated indirectly?

Answer 22

the detection of the antibodies produced by the immune system provides information about the virus.

Question 23

What is the advantage of the indirect virus investigation?

Answer 23

• is that antibodies are present for a longer period in the blood.
• therefore infections that occurred weeks, months or even years before can be investigated.
• there is a higher chance of diagnosis of a former virus infection

Question 24

What are the disadvantage of the indirect virus investigation?

Answer 24

• the methods used usually cannot differentiate the maternally or vaccine-derived antibodies, or seroconversion.
• therefore in the interpretation of the result, maternal immunity and immunisation should be taken into consideration.

Question 25

What is the aim of serological investigation?

Answer 25

either individual investigation or a survey at herd level for the identification of the virus (or viruses) causing infections.

Question 26

which methods can be used for the detection of antibodies in blood or body fluids (milk),?

Answer 26

• virus neutralisation test

- haemagglutination inhibition test

Question 27

What happens when a host is infected with a virus?

Answer 27

The immune system starts to produce antibodies to eliminate the infection

Question 28

How can the increase of the antibody-level be detected?

Answer 28

In a virus neutralisation test by investigation of subsequent sampling.

Question 29

When are the 1st and 2nd serum sample taken in the paired sera investigations?

Answer 29

• 1st serum sample is taken at onset of the clinical signs (about 3-4 day of the infection)
• 2nd serum sample is taken 10-14 days later.

The difference (at least fourfold titre, i.e 2 dilution increase) in the antibody-tire of the samples proves the immune response (seroconversion) against a certain virus

Question 30

What is virus neutralisation?

Answer 30

the loss of infectivity through reaction of the virus with a specific antobody.

Question 31

The neutralisation (i.e presence of virus-specific antibodies in the serum) can be detected by?

Answer 31

the lack of reactions such as CPE, plaque formation or disease in animals.

Question 32

Which method is used for antibody detection and the determination of the antibody-level of the blood sample?

Answer 32

Constant virus serum dilution method.

Question 33

The serum of the samples contain?

Answer 33

Heat-sensitive blocking factors and complement, so they should be inactivated at 56*C for 1 h.

Question 34

What happens in incubation for 1h at 37*C?

Answer 34

the antibody neutralise viruses.

Question 35

What is VNT?

Answer 35

• Virus neutralising titre, the greatest dilution of the serum where there is 50% CPE. =the amount of blodking antibodies was sufficient.
• CNT indicates the protection of the animals as well

Question 36

If the negative control is negative (healthy cell culture) it can be used for?

Answer 36

the recognition of the virus-caused CPEs in the infected cell cultures of the virus control and the assay wells of the plate.

Question 37

The virus neutralisation titre is calculated by?

Answer 37

finding the highest dilution of the serum where CPE occured in at least one of the two inculated cell cultures.

Question 38

What are the dilutions used in the virus neutralisation test?

Answer 38

• 10^-1 (10x dilution)
• 10^-2 (100x dilution)
• 10^-3 (1000x dilution)
• 10^-4 (10000x dilution)

Question 39

Each of the serum (assay) wells contains:

Answer 39

• cell culture
• virus (constant)
• sera (diluted)
• cell culturing media

Question 40

Each well of the virus control contains:

Answer 40

• cell culture
• Virus (10-fold dilution)
• cell culturing media

Question 41

Wach well of the cell control contains:

Answer 41

• cell culture

- cell culturing media