3.8.4.2 DNA Profiling & Medical Diagnosis

• Explain how electrophoresis is used to separate DNA fragments. (5 marks)

  • DNA fragments are placed into wells in an agarose gel. (1)
  • The gel is submerged in a buffer solution, and an electric current is applied. (1)
  • DNA is negatively charged due to phosphate groups, so it moves towards the positive electrode. (1)
  • Smaller DNA fragments move faster and travel further through the gel. (1)
  • The fragments are stained or visualized under UV light to compare banding patterns. (1)
• DNA profiling (also called genetic fingerprinting) is a technique used to identify individuals based on their unique DNA sequences.
• The process of DNA profiling:

  • Step 1: Extract DNA – DNA is obtained from a sample (e.g., blood, saliva, hair).
  • Step 2: Use PCR (Polymerase Chain Reaction) to Amplify DNA – This makes millions of copies of the target DNA.
  • Step 3: Use Gel Electrophoresis to Separate DNA Fragments
• The first step of DNA profiling is to obtain DNA from a biological sample.

  How is the DNA extracted?

  • Cells are broken open (lysis step) using a detergent or enzyme to release DNA from the nucleus.
  • Proteins and other cellular components are removed using chemicals or centrifugation.
  • The purified DNA is collected in a buffer solution for analysis.
• PCR (Polymerase Chain Reaction) is used to amplify DNA. This is the second step of DNA profiling.

  It consists of three key steps:

  • Denaturation (95°C):
  • DNA is heated to break hydrogen bonds between strands, separating it into two single strands.
  • Annealing (50-65°C):
  • Mixture is cooled so primers can bind (anneal) to complementary regions on strands.
  • Extension (72°C):
  • DNA polymerase adds nucleotides via complementary base pairing to build a complementary strand
• The PCR (Polymerase Chain Reaction) mixture contains the DNA sample, primers, free nucleotides and DNA polymerase.
• The DNA polymerase used in PCR is called Taq polymerase. It is from bacteria that live in hot springs.
• Primers are short DNA sequences that bind to the 3' end of the template strand and provide a starting point for addition of nucleotides by DNA polymerase.
• Nucleotides are in the PCR reaction mixture in order to synthesise the complementary strand of DNA.
• Once DNA has been amplified, it must be separated by size to create a DNA profile. This is done using gel electrophoresis.

  • DNA samples are loaded into wells at one end of an agarose gel.
  • The gel is placed in a buffer solution, and an electric current is applied.
  • DNA is negatively charged, so it moves toward the positive electrode.
  • Smaller DNA fragments move faster through the gel than larger fragments.
  • This creates a distinct banding pattern unique to each individual.
• DNA probes are short, single-stranded DNA molecules that are designed to be complementary to a sequence to be detected.

  • The DNA labelling of fragments either uses radioactive isotopes or a fluorescent dye
• DNA probes can be used to screen patients for heritable conditions, drug responses or health risks.