Molecular Biology Techniques Study Notes, Formulas & Summary

1. Chromatography

Chromatography is a set of laboratory techniques used to separate, purify, and identify the components of a complex mixture of biomolecules (proteins, nucleic acids, metabolites) based on their physical and chemical properties.

All chromatographic methods involve a stationary phase (a solid or liquid bound to a support) and a mobile phase (a liquid or gas that flows over or through the stationary phase, carrying the sample).

Common Types of Chromatography

Technique	Separation Principle	Stationary Phase	Application
Size-Exclusion (Gel Filtration)	Molecular size / shape	Porous polymer beads (e.g., agarose)	Separating large proteins from small proteins; desalting
Ion-Exchange (IEC)	Net electrical charge	Charged resin beads (cationic or anionic)	Purifying proteins by charge; separating amino acids
Affinity Chromatography	Specific biospecific binding	Ligands covalently bound to beads (e.g., antibodies)	Purifying specific proteins (e.g., His-tagged proteins)
High-Performance Liquid (HPLC)	Hydrophobicity (usually reversed-phase)	Micro-fine silica beads with hydrocarbon chains	Rapid, high-resolution separation and quantitation

graph TD
    A[Protein Mixture] --> B{Choose Separation Mode}
    B -->|By Size| C[Size-Exclusion: Large elutes first, small gets trapped in beads]
    B -->|By Charge| D[Ion-Exchange: Bind oppositely charged, elute with salt gradient]
    B -->|By Affinity| E[Affinity: Specific binding to ligand, elute with free ligand]

2. Electrophoresis

Electrophoresis separates charged biomolecules (proteins, DNA, RNA) in an electric field through a porous gel matrix. The rate of migration depends on the molecule's charge-to-mass ratio and the pore size of the gel.

Agarose Gel Electrophoresis (Nucleic Acids)

Used to separate DNA and RNA fragments.
DNA is negatively charged due to its phosphate backbone. It migrates toward the positive electrode (anode).
Separation is based solely on size: smaller fragments move faster through the agarose pores than larger ones.
Fragments are visualized using fluorescent dyes (e.g., ethidium bromide or GelRed) that intercalate between bases and glow under UV light.

SDS-PAGE (Proteins)

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis separates proteins by size. - SDS: An anionic detergent that denatures proteins and coats them with a uniform negative charge proportional to their mass. This masks the native charge of the protein. - Beta-Mercaptoethanol (or DTT): Reducing agents added to break disulfide bonds, fully unfolding the protein subunits. - Since all proteins now have the same negative charge-to-mass ratio, they migrate through the polyacrylamide gel toward the anode separated strictly by molecular weight (MW). Small proteins move faster. - Gels are stained with Coomassie Brilliant Blue to visualize bands.

3. Spectrophotometry

Spectrophotometry is a technique that measures how much light a chemical substance absorbs by measuring the intensity of light as a beam passes through a sample solution.

The Beer-Lambert Law

The concentration of a solute in solution is directly proportional to its absorbance:

$$A = \varepsilon \cdot c \cdot l$$

Where: - $A$ = Absorbance (dimensionless, measured as $\log(I_0 / I)$). - $\varepsilon$ = Molar absorption coefficient (or molar extinct coefficient, $\text{M}^{-1} \text{ cm}^{-1}$), specific to the solute at a given wavelength. - $c$ = Concentration of the solute ($\text{M}$ or $\text{mol/L}$). - $l$ = Path length of the cuvette (typically 1 cm).

Biomolecule Absorbance Signatures

Proteins: Absorb ultraviolet light strongly at 280 nm, primarily due to the aromatic side chains of Tryptophan and Tyrosine residues.
Nucleic Acids (DNA/RNA): Absorb UV light strongly at 260 nm due to the conjugated ring systems of the purine and pyrimidine bases.
Purity Ratio ($A_{260} / A_{280}$): Used to assess nucleic acid purity. A ratio of ~1.8 indicates pure DNA; ~2.0 indicates pure RNA. Lower ratios suggest protein contamination.

4. Polymerase Chain Reaction (PCR)

PCR is an in vitro technique used to amplify a specific target DNA sequence, producing millions of copies from a minute starting sample. It relies on a thermostable DNA polymerase (e.g., Taq Polymerase from Thermus aquaticus).

The Three Steps of a PCR Cycle

Each cycle doubles the amount of target DNA and is repeated 25–35 times.

Denaturation (~94–98°C): Heat breaks the hydrogen bonds between DNA strands, separating the double helix into single strands.
Annealing (~50–65°C): Temperature is lowered to allow synthetic DNA primers (forward and reverse) to bind complementarily to their target sequences on the single-stranded templates.
Extension (~72°C): Taq Polymerase synthesizes new complementary DNA strands in the 5' $\rightarrow$ 3' direction, extending from the primers.

graph LR
    A[Denaturation: 95°C] --> B[Annealing: 55°C]
    B --> C[Extension: 72°C]
    C -->|Repeat 30 cycles| D[Exponential DNA Amplification]

Applications of PCR

RT-PCR: Uses reverse transcriptase to convert RNA to cDNA, followed by PCR amplification. Used for viral detection (e.g., COVID-19).
qPCR (Quantitative/Real-Time PCR): Monitors amplification in real-time using fluorescent probes or dyes, allowing quantification of starting DNA template levels.
Forensics: DNA profiling/fingerprinting.
Cloning and Sequencing.

Molecular Biology Techniques — Study Notes