Q.69 The gradient used in ultracentrifugation for fractionation of DNA is:
- CsCl
- NaCl
- Glucose
- Fructose
CsCl is the gradient used in ultracentrifugation for DNA fractionation.
Cesium chloride (CsCl) equilibrium density gradient centrifugation separates DNA by buoyant density (1.66-1.77 g/cm³), enabling GC-rich vs AT-rich DNA isolation and Meselson-Stahl replication studies.
Option Analysis
CsCl
High solubility (saturates ~1.9 g/cm³ at 150,000g), forms linear gradient spontaneously. DNA bands isopycnally per base composition (ρ = 1.660 + 0.098 × GC mole fraction). Gold standard for plasmid prep, isotope labeling. Correct.NaCl
Low density (~1.2 g/cm³ saturated), insufficient range for DNA separation (1.66+ g/cm³). Used for salting out, not gradients. Incorrect.Glucose
Rate-zonal sucrose/glucose gradients separate by sedimentation coefficient (size/shape), not buoyant density. Dissipates under ultracentrifugation. Incorrect for DNA fractionation.Fructose
Rarely used; similar viscosity/density issues as glucose. No historical/standard application for nucleic acid density gradients. Incorrect.Gradient used in ultracentrifugation for fractionation of DNA leverages cesium chloride’s unique physicochemical properties enabling isopycnic separation by buoyant density—fundamental to molecular cloning and replication studies.
CsCl Physics and Mechanism
Initial ~1.72 g/cm³ solution redistributes under 100,000-400,000g forming 1.55-1.85 g/cm³ gradient over 36-72 hours. DNA migrates to ρ = 1/¯v position (¯v = partial specific volume ~0.60 cm³/g). GC pairs bind more Cs+ ions, increasing density 0.098 g/cm³ per 100% GC rise.
Historical Significance
Meselson-Stahl (1958) used CsCl to resolve 15N-14N hybrid DNA bands, proving semiconservative replication. UV absorbance scanning (260 nm) profiles hybrid (1.74 g/cm³) vs light (1.70) vs heavy (1.77) DNA post-generations.
Applications Table
Application CsCl Advantage Alternatives Plasmid supercoiling CCC > OC > linear by EtBr binding Column kits GC content analysis Direct ρ-GC calibration Melting temp Viral DNA/RNA sep Ribosomal RNA (1.58) vs DNA (1.7) Metrizamide Isotope labeling 3H/32P/13C/15N resolution Pulsed-field agarose Modern Context: Ethidium bromide/CsCl remains laboratory standard despite column chromatography convenience; superior for preparative megabase DNA isolation.
