Genetic Engineering

Question 1

Are the three types of blotting done in situ or homogenate?

Answer 1

Homogenate

Question 2

What is southern blotting used for visualising?

Answer 2

DNA

Question 3

What is western blotting used for visualising?

Answer 3

Proteins

Question 4

What is northern blotting used for visualising?

Answer 4

RNA

Question 5

What is an in situ method for visualising RNA?

Answer 5

In situ hybridisation

Question 6

What is a method to visualise protein in homogenate?

Answer 6

Western blotting

Question 7

What is used to visualise DNA in situ?

Answer 7

Chromosome painting/Chromosomal spreads

Question 8

What is used to visualise protein in situ?

Answer 8

Immunocytochemistry/Immunohistochemistry

Question 9

What are the advantages of visualising in homogenate?

Answer 9

• Quantification
• Size
• Isolation

Question 10

What are the disadvantages of visualising in homogenate?

Answer 10

• Requires more tissue

Question 11

What are the advantages of visualising in situ?

Answer 11

• Tissue distribution
• Function
• Changes in above

Question 12

What are the disadvantages of visualising in situ?

Answer 12

• Requires tissue processing

- Limited and compromised by reagents and resolution

Question 13

3 Steps of visualisation and detection of DNA, RNA and protein in homogenates

Answer 13

1. Gel electrophoresis
2. Blotting (transfer out of gel into a nylon membrane)
3. Detection by probing

Question 14

What is a gel?

Answer 14

A gel is a porous matrix that acts like a sieve. Pores are present between the gel particles

Question 15

Gel electrophoresis

Answer 15

DNA is loaded into a well on negative side
Electric current through gel and due to negatively charged phosphate moves towards positive electrode
Smaller molecules move further in the set time
Compare bands with DNA standard

Question 16

Is there a proportion between the distance between the bands and the size of the fragment?

Answer 16

No, distance between bands is not proportional the size of fragments. Based on a logarithmic scale

Question 17

Four factors affecting DNA migration

Answer 17

• Gel concentration
• DNA shape
• DNA size
• Gel type: agarose, polyacrylamide

Question 18

Which gel type is most common?

Answer 18

Polyacrylamide

Question 19

Which gel would be more appropriate for DNA with 15,000 base pairs?

Answer 19

Agarose

Question 20

What are the optimum base pair ranges for each gel type?

Answer 20

Agarose: 100-20,000bp
Polyacrylamide:10-700bp

Question 21

Which gel has higher resolution?

Answer 21

Polyacrylamide

Question 22

What are the two ways of carrying out blotting?

Answer 22

• Blotting by capillary action

- Blotting by an electric field

Question 23

How would you detect DNA/RNA after gel electrophoresis?

Answer 23

Hybridisation

Labelled with marker

Question 24

How would you detect protein after gel electrophoresis?

Answer 24

Antibody staining

Question 25

Where is the DNA polymerase that we study originally from?

Answer 25

T7 in E. coli

Question 26

When does exonucleolytic proofreading take place?

Answer 26

When an incorrect base pair is added

Question 27

Which way does the proofreading exonuclease proofread?

Answer 27

3'-->5'

Question 28

Does DNA polymerase need a primer?

Answer 28

Yes

Question 29

What is the chain termination an example of?

Answer 29

DNA sequencing

Question 30

What is PCR used for?

Answer 30

To amplify DNA. Proceeds on an exponential scale 2^#of cycles

Question 31

What do you need for a PCR?

Answer 31

- RNA primers - Free DNA nucleotides - 1 copy of DNA sequence to be amplified (template) - DNA polymerase - Buffer solution

Question 32

3 steps in PCR

Answer 32

1. Heat to 95ºC to denature dsDNA to ssDNA 2. Cool to ⁓60ºC for annealing of primers to template DNA 3. Heat to 72ºC so that DNA polymerase can extend the primers to the end of the template Cycle repeats but with the products as new templates as well

Question 33

How can PCR be used to manipulate DNA sequences?

Answer 33

- Primers can be used to insert single point mutations by lowering temperature for annealing to allow imperfect binding between primer and DNA sequence - Tailed primers can be used to insert restriction enzyme sites to produce 'sticky ends' for DNA engineering