6.1.3 (e)

Question 1

Describe the process of DNA profiling

Answer 1

1. DNA extraction: DNA is extracted from a tissue sample (which has cells containing and individual’s genome)
   
   1. DNA must be amplified by PCR
2. DNA digestion: The DNA is digested by cutting it into smaller fragments using restriction endonucleases
   
   1. Restriction endonucleases: An enzyme that cuts DNA at specific base sequences called recognition sites
   2. The active site recognises particular nucleotide sequences (known as recognition sites)
   3. The enzyme cuts the DNA at these points leaving VNTRs intact – as they are needed to create a DNA profile
3. DNA separated by gel electrophoresis:
   
   1. Smaller DNA fragments move faster and further than larger DNA fragments
   2. The fragments are transferred onto a nylon membrane by Southern Blotting
4. Hybridisation: Fluorescent/radioactive DNA probes are added in excess to the nylon membrane.
   
   1. The DNA probes complementary base pair to known DNA sequences
   2. The probes identify microsatellite regions

Question 2

How is the DNA profile examined?

Answer 2

1. Examining evidence – fragments give a unique pattern of bars per individual
   
   1. If radioactive DNA probes were used, an X-ray of the membrane can be examined
   2. If fluorescent DNA probes were used, the membrane can be placed under UV light to allow the tags to glow

Question 3

Describe the process of gel electrophoresis

Answer 3

1. Place DNA fragments into wells in agarose gel strips (load DNA) at the cathode (that have buffers to maintain pH)
   
   1. In the 1^st and last wells, there are fragments of known length for reference for fragment sizing
2. An electric current is passed over the electrophoresis plate at the cathode and since DNA is negatively charged (phosphate groups) it moves towards the anode (+ve charge)
3. Rate of fragment movements depends on mass/length of fragments
   
   1. The gel acts as resistance to molecular movement, smaller fragments move faster than longer ones
4. Once the smallest fragment reaches the anode, the current is switched off
5. The gel is placed in an alkaline buffer solution to denature DNA fragments to expose the bases
6. SOUTHERN BLOTTING

Question 4

Describe the process of southern blotting?

Answer 4

1. The alkaline solution is transferred to a nylon membrane that’s placed over the gel
2. The membrane is covered in dry absorbent paper, drawing the alkaline solution with DNA out
3. The fragments of DNA are transferred to the membrane as they cannot move through it, in the same positions as on the gel
4. They are then fixed in UV light or heat

Question 5

What are DNA probes?

Answer 5

• DNA fragments separated in electrophoresis are not visible
• Need to be labelled to identify desired gene/nucleotide sequence

A DNA probe:

• Short DNA sequence
• Single stranded
• Has specific nucleotide sequence complementary to the desired sequence
• Is labelled
  
  • Either radioactively
  • Fluorescently

Question 6

How are DNA profiles compared?