DNA Technology Part 3 - (Week 10)

Question 1

What are reporter genes?

Answer 1

Genes whose product can be easily detected & monitored

Question 2

What are reporter genes most often used for?

Answer 2

• Often used as an indication of whether a certain gene has been taken up by or expressed in the cell/organism (expression is quantifiable)
• Usually attached to regulatory sequence of another gene of interest (transcriptional reporters) or fused with the gene of interest

Question 3

Give at least one other use of reporter genes

Answer 3

To measure:
- Promoter activity
- Protein expression
etc.

Question 4

What is ‘transformation’ in gene cloning?

Answer 4

Where the recombinant plasmid is transferred into the host cell

Question 5

Give at least one method in which ‘transformation’ occurs in gene cloning

Answer 5

Method 1:
Heat shock in Ca rich solution (chemically competent cell). This causes the membrane to become permeable to the plasmids
Method 2:
Electroporation - Small electric current causes bacterial membrane to become permeable

Question 6

What is gel electrophoresis?

Answer 6

A technique used to check if the recombinant DNA plasmid has been taken up by host cell - separates a mixture of nucleic acids based on size

Question 7

Describe the 5 steps of gel electrophoresis

Answer 7

1. DNA samples containing fragments of different size are placed in wells in an agarose gel
2. An electrical current is passed through the gel
3. All DNA fragments move toward the positive pole; small fragments migrate faster than large ones. After electrophoresis, fragments of different sizes have migrated different distances
4. A dye specific for nucleic acids is added to the gel
5. DNA fragments appear as bands on the gel

Question 8

What is a hybridisation (DNA/RNA) probe?

Answer 8

It is a fragment of DNA/RNA of variable length which can be radioactively labelled

Question 9

What does a DNA/RNA probe do?

Answer 9

It can be used in DNA or RNA sample to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe

Question 10

Describe the steps when using DNA probes to check the presence of a specific gene

Answer 10

1. Isolate DNA from body fluid sample
2. Denature DNA sample & combine with DNA probes. Probes are complementary to the gene of interest & labelled w/ a molecular beacon (e.g. fluorescent probe)
3. DNA probes will bind to the gene of interest if it is present in the DNA sample

Question 11

Give an example of the applied use of recombinant DNA technology used today

Answer 11

Recombinant human insulin - is available in larges amounts & provide reliable supplies of the hormone worldwide at affordable costs