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, and evaluate its role within the wider context of genetic‑technology techniques (Cambridge International AS & A Level Biology 9700, Topic 19).

Why Gel Electrophoresis Is Used in Genetics

Gel electrophoresis provides a rapid, inexpensive visual check of DNA size and purity. It is essential for:

• Confirming that a restriction‑enzyme digest has produced the expected fragment pattern (RFLP analysis).
• Verifying the length of a PCR product before sequencing, cloning or quantitative analysis.
• Screening recombinant colonies for the presence of an insert of the correct size.
• Assessing DNA integrity in forensic or diagnostic samples.

Overview of Core Genetic‑Technology Techniques

What Is Gel Electrophoresis?

Gel electrophoresis is a laboratory technique that separates charged molecules in an electric field. DNA carries a uniform negative charge because each nucleotide contributes one phosphate group; therefore, separation of DNA fragments in a gel is governed almost entirely by their length (size) and by the resistance offered by the gel matrix.

Key Components of the Apparatus

Principle of Separation

• Each base pair contributes a fixed negative charge; the total charge on a fragment is therefore proportional to its length, but the charge‑to‑mass ratio is very close to constant for all fragments¹.
• The electric force on a fragment is F = qE, where q is the charge and E is the electric field strength.
• Migration speed (electrophoretic mobility, μ) is set by the balance between this force and the frictional resistance of the gel matrix:

  μ = v/E ≈ 1 / (friction coefficient)

  The friction coefficient increases with higher agarose % (smaller pores) and with larger fragment size or irregular shape.
• For a given gel, voltage and run time, the distance migrated (d) is related to fragment length (L) by the empirical linear relationship:

  d = k · log₁₀(L) + c

  where k (slope) and c (intercept) depend on gel concentration, voltage and buffer composition.

Practical Planning Checklist (AO3)

1. Hypothesis / aim – e.g., “The restriction enzyme EcoRI will cut the plasmid into fragments of 1.2 kb and 2.8 kb.”
2. Variables
   • Independent: enzyme type, incubation time.
   • Dependent: fragment pattern on the gel.
   • Controlled: DNA amount, buffer, temperature, gel concentration.
3. Controls
   • Negative control – DNA not exposed to enzyme.
   • Positive control – DNA of known size (ladder).
4. Safety & waste disposal – list of hazardous reagents, PPE, disposal routes for ethidium bromide or SYBR® Safe.
5. Data‑recording table – include columns for sample ID, volume loaded, lane number, distance migrated (mm), calculated size (bp), observations.

Step‑by‑Step Procedure

1. Prepare the gel
   • Weigh agarose (e.g., 1 % w/v for fragments 500 bp–10 kb).
   • Mix agarose with the appropriate buffer, heat until dissolved, and cool to ~60 °C.
   • Pour into the casting tray with a comb in place; allow to solidify (≈20 min).
2. Set up the electrophoresis chamber
   • Place the gel in the chamber and cover with enough running buffer to submerge the gel and fill the wells.
   • Connect the power supply (anode positioned opposite the wells).
3. Load the samples
   • Mix each DNA sample with loading dye (glycerol for density, tracking dye for visual monitoring).
   • Using a micropipette, add the mixture to the wells without introducing air bubbles.
   • Load a DNA ladder in one lane for size reference.
4. Run the gel
   • Apply a constant voltage (commonly 100 V).
   • Allow migration until the tracking dye has moved about two‑thirds of the gel length (typically 30–45 min).
5. Stain and visualise
   • Stain the gel with ethidium bromide (handle with care) or a safer alternative such as SYBR® Safe.
   • Place the gel on a UV transilluminator, photograph the band pattern, and record distances.

Quantitative Size Estimation (Worked Example)

Given: 1 % agarose gel, 100 V, 35 min run. DNA ladder fragments: 500 bp, 1000 bp, 2000 bp, 4000 bp. Measured distances from the well are shown below.

1. Plot **distance (y‑axis)** against **log₁₀(fragment size) (x‑axis)**. The points lie on a straight line.
2. Determine the linear equation (e.g., d = –12 · log₁₀(L) + 73).
3. For an unknown band that migrated 38 mm:

   38 = –12 · log₁₀(L) + 73 → log₁₀(L) = (73 – 38)/12 = 2.92 → L ≈ 10^2.92 ≈ 830 bp.

Mark‑scheme tip: Give the final size to two significant figures unless the question specifies otherwise, and state the formula used.

Data Analysis & Evaluation

• Constructing the plot – Use graph paper or spreadsheet software; draw a best‑fit straight line and record the slope (k) and intercept (c) with appropriate units.
• Calculating fragment size – Rearrange the linear equation to log₁₀(L) = (d – c)/k and then take the antilog.
• Assessing reliability
  • Repeat the run with a second ladder to check consistency.
  • Measure each band three times and use the mean distance.
  • Report the standard deviation or an estimated uncertainty.
• Common sources of error
  • Uneven gel thickness or polymerisation.
  • Temperature gradients causing uneven migration.
  • Inaccurate distance measurement (parallax error).
  • Diffusion of bands during staining or over‑long runs.
• Improving resolution
  • Adjust agarose concentration (higher % for small fragments, lower % for large fragments).
  • Use a lower voltage for long runs to reduce heat.
  • Choose TBE buffer for sharper bands when high resolution is required.
  • Consider polyacrylamide gels for fragments < 200 bp.

Factors Influencing Resolution

• Agarose concentration – Higher % → smaller pores → better separation of 100–1000 bp; lower % → larger pores → better for >5 kb.
• Voltage – Higher voltage shortens run time but generates heat, leading to band smearing.
• Buffer choice – TBE gives sharper bands (lower conductivity); TAE provides higher conductivity and is useful when the gel will be used for downstream enzymatic reactions.
• Run time – Too short = incomplete separation; too long = diffusion and loss of resolution.

Link to Other Genetic‑Technology Techniques

• Restriction‑enzyme digests – Gel electrophoresis confirms that the expected fragment sizes have been produced.
• Polymerase‑chain reaction (PCR) – Verifies the size of the amplified product before sequencing or cloning.
• Cloning – Screening of transformants by electrophoresis identifies colonies carrying inserts of the correct length.
• DNA sequencing preparation – Purified PCR products of the correct size are excised from a gel before Sanger or NGS.
• CRISPR‑Cas editing – Detects small insertions/deletions by size shift on a high‑resolution gel.

Safety & Ethical Considerations

• Ethidium bromide is a mutagenic intercalating agent; wear gloves, use a dedicated staining area, and dispose of waste in a labelled container.
• Safer dyes (e.g., SYBR® Safe) reduce health risks but still require UV‑protective goggles during visualisation.
• Agarose and buffer solutions should be disposed of according to the school’s chemical‑waste protocol.
• Data integrity – Gel images must not be altered; all band interpretations should be recorded accurately because results may inform diagnostic or forensic decisions.
• Ethical use of human DNA – Obtain informed consent, anonymise samples, and follow data‑protection regulations.

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 (RFLP analysis).
• Detecting CRISPR‑induced indels.

¹ In practice the charge‑to‑mass ratio is very close to constant; minor deviations (e.g., end‑effects on very short fragments) are negligible for routine sizing.