Genetic engineering 1

Question 1

What is immunocytochemistry?

Answer 1

• Localisation of specific antigens in cells (WITHOUT extracellular maxtrix)

Question 2

3 ways of homogenating tissue?

Answer 2

1) Repeated freeze/ thaw
2) Mechanical devices
3) Detergents - dissolve the lipid bilayer

Question 3

How study RNA in situ?

Answer 3

In situ hybridistaion

Question 4

What kind of gel is used in electrophoresis

Answer 4

Porous - acts like a sieve, sorting DNA/RNA according to size
Usually agarose

Question 5

How does DNA/RNA move through agarose in electrophoresis?

Answer 5

• Towards the positive anode as DNA/RNA is negatively charged due to the phosphate groups
• Smaller fragments move faster as less impeded by the gel

Question 6

How study proteins in situ?

Answer 6

Immunocytochemistry

Immunocytohistochemistry

Question 7

How is a specific DNA/RNA fragment detected at the end of electrophoresis?

Answer 7

• By nucleaic acid hybridisation
• Synthesise strand complementary to target with a radioactive or fluorescent marker attached
• DNA hybridises to target
• Stability of the probe depends upon the degree of match (amount of H bonds that can form)

Question 8

What does in-situ hybridiation detect and why is this important?

Answer 8

• Presence and quantity of mRNA expression in a cell

- Important because mRNA expression is different at different times and in different cells

Question 9

What is immunohistocytochemistry?

Answer 9

• Localisation of specific antigens in tissues (WITH extracellular maxtrix)

Question 10

How study a protein homogenate?

Answer 10

Western blotting

Question 11

Advantages of studying in situ?

Answer 11

• Tissue/ subcellular istribution
• Can infer function
• Changes easily observed

Question 12

What is the cell blotting technique?

Answer 12

1) Size separation in a gel
- By electrophoresis
- Fist must fragment DNA (not RNA) using restriction enzymes

2) Transfer out of gel into a membrane (this is the blotting part)
3) Detection on a membrane

Question 13

What does a protein function relate to?

Answer 13

Its location within the cell

Question 14

How study a DNA homogenate?

Answer 14

Southern blotting

Question 15

Disadvantages of studying in situ?

Answer 15

• Limited/ compromised by reagents and resolution

- Requires tissue processing (staining/ fixing)

Question 16

How detect proteins in western blotting?

Answer 16

• Antigen- antibody interactions
• Primary antibody probe binds to a specific target antigen on a protein
• Secondary antibody (which is labelled) binds to the primary antibody

Question 17

How study DNA in situ?

Answer 17

Chromosome painting/ spreads

Question 18

What are the 4 things which affect migration of fragments in electrophoresis?

Answer 18

1) DNA size

2) Gel concentration
- Higher concentration, DNA moves slower

3) DNA shape
- Compacted moves faster
- Super-coiled move faster than Linear DNA
- Circular DNA moves slowest

4) Gel type
- Agarose for base pairs 100-20,000bps
- Polyacrylamide for 10-700bps

Question 19

Disadvantages of studying an homogenate?

Answer 19

• Requires a larger sample of tissue

- Can’t see distribution or intensity

Question 20

What must be done to proteins before electrophoresis?

Answer 20

Negative charge must be applied as the charge on proteins is dependent on the side chains

Question 21

How study a RNA homogenate?

Answer 21

Northern blotting

Question 22

Advantages of studying an homogenate?

Answer 22

• Can infer quantification
• Can infer size
• Isolation

Question 23

What does western blotting allow scientists to identify?

Answer 23

The presence, size and relative abundance of a PROTEIN