8-4-11-gel-electrophoresis

Gel electrophoresis

• Gel electrophoresis is a technique used widely in the analysis of DNA, RNA and proteins

• During electrophoresis, the molecules are separated according to their size/mass and their net (overall) charge

  • Positively charged molecules will move towards the cathode (negative pole), whereas negatively charged molecules will move towards the anode (positive pole)

  • DNA is negatively charged due to the phosphate groups, and so when placed in an electric field, the molecules move towards the anode

• Different-sized molecules move through a gel (agarose for DNA and polyacrylamide – PAG for proteins) at different rates

  • The tiny pores in the gel result in smaller molecules moving quickly, whereas larger molecules move slowly

• Different restriction enzymes cut the DNA at different base sequences

  • Therefore, scientists use enzymes that will cut close to the variable number tandem repeat (VNTR) regions

Apparatus

• Agarose gel

• Electrophoresis tank

• Electrolyte solution

• Micropipette

• Electrodes

• DNA sample

• DNA standard

• Probes

• Nitrocellulose

• A dye

Method

Preparation of DNA samples

1. Collect DNA

   • Obtain DNA from a biological source (e.g. hair root, saliva, skin cells)

2. Prepare the DNA for analysis

   • Purify the DNA sample

   • Amplify the DNA using the polymerase chain reaction (PCR)

   • Use restriction endonucleases to cut the DNA into fragments at specific sequences

Preparing the gel and loading the samples

3. Prepare the gel

   • Create a gel with wells at one end. The pore size of the gel can vary, which affects how fast DNA fragments move through it

4. Place the gel in a tank

   • Submerge the gel in an electrolyte buffer solution

5. Load the DNA samples

   • Use a micropipette to carefully load DNA samples into the wells

   • Ensure a DNA ladder is loaded into the first well for comparison

Running gel electrophoresis

6. Connect the electrodes

   • Attach the negative electrode near the wells (where DNA is loaded)

   • Connect the positive electrode (anode) at the opposite end of the gel

7. Apply an electric current

   • The negatively charged DNA fragments will migrate through the gel towards the positive electrode

   • Smaller DNA fragments move faster and travel further than larger fragments, separating based on size

Detection and visualisation

8. Transfer DNA to membrane

   • Since DNA fragments are not visible, transfer them onto absorbent paper or a nitrocellulose membrane using blotting

   • Heat the membrane to denature the DNA, separating it into single strands

9. Add DNA probes

   • Apply single-stranded DNA probes that are complementary to the target VNTR (Variable Number Tandem Repeat) regions

   • Probes are tagged for detection, using either:

     • a radioactive label (e.g. phosphorus isotope) that darkens X-ray film, or

     • a fluorescent dye (e.g. ethidium bromide) that glows under UV light

10. Visualise and interpret the results

    • Develop an X-ray image or expose the membrane to UV light to reveal a pattern of bands

    • Compare the banding pattern to a control sample or reference to draw conclusions (e.g. for DNA profiling or sequencing)

Limitations

• The measurements are not precise and must be compared to a standard to gather data

• Electrophoresis requires a lot of sample and therefore depends on PCR to work correctly to amplify DNA fragments