In gel electrophoresis of linear DNA, how can the fragment size be estimated accurately during analysis?

Introduction / Context:
Agarose gel electrophoresis separates linear DNA fragments primarily by size, with mobility inversely related to the logarithm of fragment length. Accurate size estimation requires internal or parallel standards known as DNA ladders.

Given Data / Assumptions:

• We are analyzing linear DNA bands on an agarose gel.
• Staining (e.g., ethidium bromide, SYBR dyes) provides fluorescence but not absolute size.
• Standards such as HindIII-digested lambda DNA or commercially prepared ladders have known fragment sizes.

Concept / Approach:
To estimate fragment size, run a DNA ladder in a lane beside samples. Plot distance migrated versus log10(size) for standard bands to create a calibration curve, then read sample sizes by interpolation. Relying solely on fluorescence brightness, sedimentation coefficients, or run time is unreliable because they are influenced by concentration, gel composition, voltage, buffer, and conformation.

Step-by-Step Solution:
Load molecular weight standard (e.g., HindIII-digested lambda) in one lane.Run samples under the same conditions.Measure migration distances of ladder bands and construct a standard curve: distance vs. log(size).Measure migration of sample band; interpolate size from the standard curve.Report size with an appropriate error margin.

Verification / Alternative check:
Using different ladders (100 bp, 1 kb) tailored to expected size ranges cross-validates estimates. Including an internal standard band within the same sample lane further improves accuracy.

Why Other Options Are Wrong:
Fluorescence intensity: reflects DNA quantity and dye binding, not size.Sedimentation coefficient: pertains to ultracentrifugation, not agarose mobility.Run time: affects overall migration but cannot directly translate to size without standards.

Common Pitfalls:
Misreading bands due to gel smearing, running gels too hot (band smiling), or using an inappropriate ladder range for the fragment sizes of interest.

Final Answer:
By comparison with a molecular weight standard such as HindIII-digested phage lambda DNA