Q.7 The method used to analyse larger DNA fragments (> 1 Mbp) is
1. polyacrylamide gel electrophoresis
2. Isoelectric focusing
3. agarose gel electrophoresis
4. pulsed-field gel electrophoresis
DNA Fragment Analysis: PFGE for Large Fragments Explained
Separating DNA fragments exceeding 1 megabase pair (Mbp) requires specialized electrophoresis techniques beyond standard methods. The correct answer is 4. pulsed-field gel electrophoresis (PFGE), designed specifically for megabase-scale genomic DNA.
This technique revolutionized genome mapping and microbial typing by overcoming limitations of conventional gels. Here’s a breakdown of why PFGE excels and why other options fail.
Why Pulsed-Field Gel Electrophoresis (PFGE) Works for >1 Mbp DNA
PFGE applies alternating electric fields in different directions, allowing massive DNA molecules to reorient and migrate through agarose pores at size-dependent rates. Large fragments (>50 kb, up to 10 Mbp) “snake” through the gel—smaller ones realign faster than giants.
Developed in 1984 by Schwartz and Cantor, it’s essential for intact chromosomal analysis, bacterial strain typing (e.g., PulseNet for outbreaks), and yeast artificial chromosome (YAC) sizing. Routine agarose fails here as megabase DNA gets stuck or comigrates.
Comparison of All Electrophoresis Options
Each method suits different DNA sizes and principles—PFGE uniquely handles megabases.
| Option | Technique | DNA Size Limit | Mechanism | Why Not for >1 Mbp? |
|---|---|---|---|---|
| 1 | Polyacrylamide gel electrophoresis (PAGE) | Up to ~1 kb | Dense matrix sieves small ss/dsDNA/RNA | Pores too small; large DNA can’t enter |
| 2 | Isoelectric focusing (IEF) | Proteins/small peptides | pI-based migration in pH gradient | Not for DNA size separation; charges proteins |
| 3 | Agarose gel electrophoresis | Up to ~50 kb | Porous agarose for routine plasmids/genomic digests | Large fragments comigrate or reptate uniformly |
| 4 | Pulsed-field gel electrophoresis (PFGE) | 50 kb–10 Mbp | Pulsed fields force reorientation | Correct: Resolves megabases via directional shifts |
Practical Applications and Limitations
PFGE shines in genomics: separate restriction-digested chromosomes, map physical maps, or fingerprint pathogens. Labs embed cells in agarose plugs to avoid shearing, then run 24-48 hour gels with ramped pulse times.
Limitations include long run times and equipment needs, but it’s gold-standard for intact genomes. For smaller fragments, stick to agarose or PAGE.
Correct Answer: 4. pulsed-field gel electrophoresis
