Quantification Reviewer

Nucleic Acid Quantification Reviewer

MLS 420 | BSMLS 3F

1. WORKFLOW IN MOLECULAR BIOLOGY

Sample Collection → Nucleic Acid Extraction → Nucleic Acid Quantification → Amplification (PCR) → Gel Electrophoresis → Downstream Applications

Post-DNA Isolation steps: RNAse treatment, DNAse treatment, Gel Electrophoresis, Nanodrop Method

2. QUALITY CHECKING (Gel Electrophoresis)

Good quality DNA — band appears intact, clear crescent, minimal to no smudging

Bad quality DNA — band appears smudged or smeared → DNA is degraded

Quantity issues:

• LOW concentration → no bands appear

• HIGH/excess concentration → DNA does not migrate; stays in the well

• Bad quality DNA? → Repeat the ENTIRE extraction process

3. NUCLEIC ACID QUANTIFICATION

Purpose:

• Determines the average concentration of DNA/RNA before downstream experiments

• Tells you what dilutions are needed per experiment

PCR standard concentration requirement: 5–50 ng/mL

If concentration < 5 ng:

1. Perform concentration step first

2. If still insufficient → repeat entire extraction

If excess: Perform dilution

4. TWO PRINCIPLES OF QUANTIFICATION SPECTROPHOTOMETRY

• Based on Beer-Lambert's Law: Absorbance is directly proportional to concentration

• Formula: A = log₁₀ (I₀/I) = εcl

• Made up of two parts: Spectrometer (light source to sample) + Photometer (signal to numerical value)

• Nucleic acids are measured at 260 nm

Key relationships:

• Concentration ↑ → Absorbance ↑ (direct)

• Concentration ↑ → Transmitted light ↓ (inverse)

FLUOROMETRY

• Uses fluorescent dyes (fluorophores) that bind specifically to the target molecule

• Fluorophore only fluoresces if binding is successful

• Intensity of emitted light is directly proportional to concentration

• Fluorometry is 1,000–10,000× more sensitive than spectrophotometry

5. SPECTROPHOTOMETRY vs. FLUOROMETRY — COMPARISON TABLE

Key rule: BOTH provide concentration, but ONLY spectrophotometry provides purity ratios.

6. NANODROP METHOD

Type: UV Spectrophotometer
Volume needed: 1–2 µL (smaller than standard drop of 20–25 µL)
Reagents: None required — quick and easy

Principle: A 260 nm light shines on the sample; light absorbed is proportional to nucleic acids present.

Constant for dsDNA: A260 of 1 = 50 µg/mL

Formula:

Example problem:

• A₂₆₀ = 0.5, Dilution Factor = 10

• C = 0.5 × 50 µg/mL × 10 = 250 µg/mL

Advantages:

• Works with microvolumes

• Measures DNA & RNA at 260 nm, proteins at 280 nm, salt/organic contaminants at 230 nm

• Wide range (2 ng/µL – 15,000 ng/µL dsDNA) without needing dilution

7. DNA PURITY RATIOS (Nanodrop)

Possible contaminants by wavelength:

• A230 → Organic substances / salts

• A270 → Phenol

• A280 → Proteins

Optimal Ratios Table:

Note: pH affects ratios → use 10 mM Tris·Cl, pH 7.5

8. PRACTICE TEST INTERPRETATIONS

Sample #1 (Good): 260/280 = 1.98 Pure | 260/230 = 2.10 Pure | Conc. = 258.83 ng/µL → Too HIGH for PCR → Perform dilution

Sample #2 (Bad): 260/280 = 1.72 Impure (protein) | 260/230 = 0.21 Impure (salts) | Conc. = 53.78 ng/µL → Slightly high, but repeat entire process due to impurity

Sample #3 (Bad): 260/280 = 1.32 Impure (protein) | 260/230 = 0.71 Impure (salts) | Conc. = 22.43 ng/µL Good range — but repeat entire process due to impurity

9. DIPHENYLAMINE METHOD

• 2-deoxyribose of DNA + acid → ω-hydroxilevulinic aldehyde

• This reacts with diphenylamine → forms a blue colored complex

• Read at 595 nm

• Blue color intensity is directly proportional to DNA concentration

• Concentration is calculated by interpolating from a standard curve (X-axis: concentration, Y-axis: absorbance at 595 nm)

10. SPECTROPHOTOMETER TIPS (9 Rules)

1. Always clean the pedestal before use

2. Always measure your blank

3. Check for air bubbles

4. Mix samples before measuring

5. Be wary of low concentration samples

6. Re-blank every 30 minutes

7. Take measurements in at least duplicates

8. Always write down your results

9. Always clean the pedestal after use

QUICK MEMORY AIDS