Experiment 04UV Spectrophotometry • B.Sc. / M.Sc.
Spectrophotometric Determination of DNA Concentration, Purity & Hyperchromicity
🎯 Aim: To record the UV absorbance spectrum ($220–320,text{nm}$) of purified DNA samples using quartz cuvettes, calculate nucleic acid concentration, determine the $A_{260}/A_{280}$ and $A_{260}/A_{230}$ purity ratios, and evaluate hyperchromic shift upon thermal melting ($T_m$).
🔬 Theoretical Principle & Optical Equations
Purine (adenine, guanine) and pyrimidine (cytosine, thymine, uracil) aromatic rings contain conjugated $pi$-electron resonance systems that absorb ultraviolet radiation strongly with a characteristic absorption peak centered at $lambda_{max} = 260,text{nm}$:
- Beer-Lambert Quantitative Standards:
$$text{dsDNA Concentration } (mutext{g/mL}) = A_{260} times 50 times text{Dilution Factor}$$
$$text{ssRNA Concentration } (mutext{g/mL}) = A_{260} times 40 times text{Dilution Factor}$$
$$text{ssOligo Concentration } (mutext{g/mL}) = A_{260} times 33 times text{Dilution Factor}$$ - Purity Assessment Criteria:
- $A_{260}/A_{280} = 1.80–1.85$: Highly pure double-stranded DNA. Ratios $< 1.60$ indicate significant contamination with aromatic amino acid residues of proteins or organic phenol.
- $A_{260}/A_{230} = 2.00–2.20$: Secondary purity index. Ratios $< 1.80$ indicate residual guanidinium isothiocyanate salts, EDTA, or polysaccharide carryover.
- $A_{320}$: Light scattering index. Clean DNA displays $A_{320} approx 0.00$. Any absorbance indicates particulate turbidity. Corrected $A_{260} = A_{260} – A_{320}$.
- Hyperchromicity: In double-stranded helical DNA, aromatic rings are stacked tightly inside the helix, restricting $pi$-electron orbital excitation (hypochromism). Upon thermal denaturation above melting temperature ($T_m$), the unstacking of bases produces a dramatic $30–40%$ increase in absorbance at $260,text{nm}$ (hyperchromic shift).
Figure 4.1: UV Absorption Spectra & A260/A280 Purity Ratio Mechanics
📊 Representative Spectrophotometric Measurement Matrix
| Sample Code | A260 (OD) | A280 (OD) | A260 / A280 | A260 / A230 | Calculated Conc. (μg/mL) | Purity Diagnostics |
|---|---|---|---|---|---|---|
| Sample 1 (Genomic) | 0.450 | 0.245 | 1.84 | 2.12 | $0.450 times 50 times 50 = 1,125,mutext{g/mL}$ | Highly Pure DNA; Free of Protein |
| Sample 2 (Crude Prep) | 0.520 | 0.385 | 1.35 | 1.40 | — | Severely Contaminated with Protein |
| Sample 3 (Plasmid Miniprep) | 0.180 | 0.098 | 1.84 | 2.05 | $0.180 times 50 times 10 = 90.0,mutext{g/mL}$ | Cloning Quality Pure Plasmid |
💬 Viva-Voce Questions & Model Answers
Q1: Why must quartz cuvettes be used instead of standard glass or plastic cuvettes?
Standard optical glass and polystyrene plastic absorb UV radiation strongly below $300,text{nm}$, producing total light opacity at $260,text{nm}$. Fused quartz is optically transparent throughout the deep UV spectrum down to $190,text{nm}$.
Q2: Why does an A260/A280 ratio of 2.0 indicate RNA contamination in a DNA preparation?
Pure RNA has an intrinsically higher $A_{260}/A_{280}$ ratio ($2.0–2.1$) than pure DNA ($1.8$) because of the higher molar extinction coefficient of uracil compared to thymine and higher single-stranded hyperchromicity. A DNA sample with a ratio near $2.0$ reflects significant RNA contamination.
Q3: How does GC content affect the melting temperature (Tm) of DNA?
Guanine-Cytosine (GC) pairs contain three hydrogen bonds, compared to two in Adenine-Thymine (AT) pairs, and exhibit stronger base-stacking thermodynamics. Therefore, DNA molecules with higher GC content require significantly higher thermal energy to melt, directly elevating $T_m$.