Genetic Engineering

Question 1

Homogenate visualisation of DNA

Answer 1

southern blotting

Question 2

Homogenate visualisation of RNA

Answer 2

Northern Blotting

Question 3

Homogenate visualisation of protein

Answer 3

Western blotting

Question 4

In situ visualisation of DNA

Answer 4

chromosomal painting/chromosomal spreads

Question 5

In situ visualisation of RNA

Answer 5

In situ hybridisation

Question 6

In situ visualisation of proteins

Answer 6

Immunochemistry/immunohistochemistry

Question 7

Advantages of homogenate visualisation

Answer 7

1. Easier to quantify it
2. You know the size of the macro-molecule
3. Easier to isolate the macro-molecules

Question 8

Disadvantages of homogenate visualisation

Answer 8

Requires more of the tissue/sample to see the cells well as they are not that sensitive.

If you have a limited amount of tissue, this becomes an issue

Question 9

Advantages of in situ hybridisation

Answer 9

1. You can know the tissue/cell structure and tissue/subcellular distribution (which is not possible in homogenate as the cell structure is destroyed)
2. From the above info, you can infer the function of the cell
3. You can then identify changes/infer changes in these cells and the effects of diseases

Question 10

Disadvantage of in situ hybridisation

Answer 10

Requires tissue processing (preserving the tissue)

Question 11

Outline the 3 stages of the blotting technique used to visualise DNA and RNA only

Answer 11

1. Size sorting in gel
2. Transfer out of the gel into a membrane
3. Detection on a membrane

Question 12

How are cells/tissues of interest hypogenised?

Answer 12

• Repeated freeze/thaw
• Mechanical devices (e.g. pestle and mortar)
• Detergents

Question 13

Outline step 1 (size sorting in gel)

Answer 13

• DNA is loaded into a well within a pre-cast gel
• The gel is a porous matrix that acts like a sponge
• The DNA is forced through the gel by an electric field, as DNA is -ve (due to the phospohate group) it makes its way to the +ve electrode
• The migration speed depends on the size, larger DNA fragments migrate slower

Question 14

Outline stage 2 (transfer out of the gel into membrane) - BLOTTING

Answer 14

• Transfer the sorted DNA out from the gel into a specialised membrane (this is known as blotting)
• Could use a capillary action or electric field

Question 15

Outline stage 3 (detection by a labelled probe)

Answer 15

-For DNA and RNA, the detection method is the same. Via nucleic acid detection

Question 16

Detection of RNA or DNA

Answer 16

Specific base pairings of 2 complementary single strand molecules to form a double stranded molecule

Synthesis of a complimentary strand with an attached marker (e.g. radioactive or fluorescent tag)

The stability of hybridisation depends on the degree of match between the 2 fragments (target and probe DNA base sequence)

Question 17

Detection proteins via westernblotting

Answer 17

Relies on principle of specific antigen-antibody interaction

Western blotting identifies the presence, size and relative abundance of a protein

Question 18

In-situ detection of RNA

Answer 18

locates RNA within a tissue

detects and quantifies the mRNA expression

Question 19

In situ detection of protein

Answer 19

antigen-antibody interactions