describe and explain how gel electrophoresis is used to separate DNA fragments of different lengths

Principles of Genetic Technology – Gel Electrophoresis

Learning Objective

Describe and explain how gel electrophoresis is used to separate DNA fragments of different lengths.

What is Gel Electrophoresis?

Gel electrophoresis is a laboratory technique that exploits the movement of charged molecules in an electric field to separate them based on size, shape, and charge. For DNA, which is uniformly negatively charged due to its phosphate backbone, separation is primarily a function of fragment length.

Key Components of the Apparatus

Step‑by‑Step Procedure

1. Prepare the gel:

   • Weigh agarose (e.g., 1 % w/v for fragments 500 bp–10 kb).
   • Heat in buffer until dissolved, cool to \overline{60} °C, and pour into the casting tray with a comb in place.
   • Allow the gel to solidify (≈20 min).

2. Set up the electrophoresis chamber:

   • Place the solidified gel into the chamber.
   • Cover with running buffer, ensuring wells are filled.
   • Connect the power supply (anode at the opposite end from the wells).

3. Load samples:

   • Mix each DNA sample with loading dye.
   • Carefully pipette the mixture into individual wells.
   • Load a DNA ladder in one well for size reference.

4. Run the gel:

   • Apply a constant voltage (e.g., 100 V).
   • Allow migration until the dye front has moved \overline{2}/3 of the gel length (≈30‑45 min).

5. Visualise DNA:

   • Stain the gel with ethidium bromide or a safer alternative (e.g., SYBR® Safe).
   • Observe bands under U \cdot illumination.

How Separation Occurs

DNA fragments experience a force \$F = qE\$, where \$q\$ is the net negative charge (proportional to fragment length) and \$E\$ is the electric field strength. Because charge and mass increase together, the electrophoretic mobility \$\mu\$ depends mainly on the frictional resistance offered by the gel matrix:

\$\mu = \frac{v}{E} \approx \frac{1}{\eta} \cdot \frac{1}{\text{size}}\$

In practice, the migration distance \$d\$ after a fixed time is inversely related to the logarithm of fragment length \$L\$:

\$d = k \, \log_{10}\!\left(\frac{1}{L}\right) + c\$

where \$k\$ and \$c\$ are constants determined by gel concentration, voltage, and buffer composition. Smaller fragments encounter fewer pores and therefore travel farther than larger fragments.

Interpreting the Results

• Compare the position of each sample band with the DNA ladder.
• Estimate fragment size by interpolating between ladder bands on a semi‑log plot (distance vs. log size).
• Multiple bands from a single lane indicate heterogeneous fragment sizes (e.g., restriction digests).

Factors Influencing Resolution

• Agarose concentration: Higher % agarose → smaller pores → better resolution of small fragments; lower % → better for large fragments.
• Voltage: Higher voltage speeds runs but can cause band smearing due to increased heat.
• Buffer composition: Ionic strength affects current; TBE provides sharper bands than TAE for high‑resolution work.
• Run time: Insufficient time yields incomplete separation; excessive time can cause diffusion.

Common Applications in A‑Level Biology

• Checking the success of restriction enzyme digests.
• Verifying PCR product size.
• Assessing DNA purity and integrity before sequencing.
• Genotyping by comparing allele‑specific fragment patterns.