Molecular Biology Techniques — PCR, Blotting and Recombinant DNA Tools

Exam questions on this topic nearly always test which technique detects which molecule and what each step does. The core idea of the three blots is the same: separate molecules by size on a gel, transfer them to a membrane, then probe the membrane to light up the one you want.

The polymerase chain reaction uses Taq polymerase — a heat-stable DNA polymerase isolated from Thermus aquaticus — to synthesise DNA after thermal denaturation and primer annealing. Kary Mullis introduced PCR in 1985 and later received the Nobel Prize in Chemistry. A small amount of nucleic acid is placed in a tube and cycled in a thermal cycler.

• Why Taq? It stays active despite repeated exposure to high temperatures.
• Cycle number: efficiency falls after 30 to 40 cycles because reagents are depleted and by-products accumulate.
• Amplification power: PCR can make 10^6 to 10^9 copies from as little as 1 to 100 ng of template, often within a few hours.
• Visualisation: products are run on agarose gel and stained with ethidium bromide, then viewed under UV light.
• Inhibitors: proteinase K, phenol and EDTA can inhibit PCR, as can heparin, haemoglobin and ionic detergents — which is why heparinised or blood-rich samples can fail.

In real-time PCR the quantification cycle (Cq) is the cycle number at which fluorescence crosses a threshold. A lower Cq means more starting template. Results are often reported as ΔCq or ΔΔCq. Real-time PCR is more expensive than conventional PCR because of specialised instruments and reagents.

1. Extract and separate — for Southern blot, purified DNA is first digested with restriction enzymes; fragments (or RNA, or proteins) are run through a gel with an electric current; smaller pieces travel faster.
2. Transfer to a solid membrane (nylon or nitrocellulose for nucleic acids; PVDF commonly for proteins).
3. Probe — a DNA probe labelled with a radioactive, fluorescent or chemical tag (Southern/Northern), or an antibody (Western).
4. Wash and detect — only bound probe or antibody remains; one band appears for the target, and its thickness reflects the amount present.

In a Western blot, the membrane is first blocked (for example with skim milk), then incubated with a primary antibody specific to the target protein, then a labelled secondary antibody; bound antibody is detected by developing film or an ECL reaction. Because only the target is recognised, a single band should appear.

Restriction enzymes are named from the species and the order of isolation — for example EcoRI comes from E. coli strain RY13, enzyme 1. They arose from bacterial restriction–modification defence against bacteriophage: the enzyme cuts foreign DNA while the host's own sequences are protected, typically by methylation. In 1973–74 Cohen, Boyer and colleagues ligated EcoRI fragments into a plasmid and reintroduced it into E. coli (using a calcium chloride transformation), proving gene cloning was possible.

Later plasmids such as pBR322 (about 4 kb) carried two antibiotic-resistance genes: one for selecting transformed cells and one containing unique restriction sites, so that insertion into it destroys resistance and identifies recombinants (insertional inactivation). Newer vectors place a multiple cloning site within lacZ, giving blue–white colony screening: colonies with an insert have a disrupted β-galactosidase and are white; those without an insert stay blue.

A Western blot is a protein detection method with a fixed workflow, and exam questions often ask what a particular step is for. The sequence below follows the standard protocol described for cultured cells.

1. Cell lysis — cells are washed with cold buffer and lysed on ice in lysis buffer containing a protease inhibitor cocktail, so proteins are not degraded; the lysate is clarified by centrifugation at 4°C.
2. Protein measurement — concentration is measured (for example by spectrophotometer) so that an equal amount, about 50 µg per well, is loaded in each lane.
3. Sample preparation — sample buffer is added and the samples are heated at 100°C for 5 minutes.
4. Gel electrophoresis — proteins pass through a stacking gel and then a separating gel with a molecular-weight marker alongside; separation is by molecular weight, and the run ends when the dye front reaches the bottom.
5. Electrotransfer — the gel and a PVDF membrane are assembled as a sandwich with filter paper and sponges; the membrane lies between the gel and the positive electrode, and transfer is done on ice (about 4°C).
6. Blocking — the membrane is blocked, for example with 5% skim milk, so antibodies do not stick non-specifically.
7. Primary antibody — incubated overnight at 4°C; unbound antibody is washed off (TBST washes).
8. Secondary antibody — a labelled antibody against the primary antibody is added; after washing, bound antibody is revealed with an ECL reaction and film or imager.

Because the band thickness reflects the amount of target protein, running a standard alongside the samples lets the Western blot give a semi-quantitative estimate, as well as the size of the protein.

Unlike nuclear DNA, mtDNA replicates independently of the cell cycle (some molecules replicate repeatedly, others not at all), segregates randomly at cell division and has no recombination between maternal and paternal variants.

Real-time PCR can be reported as Cq, ΔCq or ΔΔCq, and accurate interpretation depends on the amplification efficiency, which ranges from 1 to 2 (a fold value of 2 represents 100% efficiency). Low efficiency means more cycles are needed to reach the threshold, producing a higher Cq.

• PCR contamination gives false positives; primer design must be precise.
• In real-time PCR, absence of amplification is not a fully reliable negative result.
• Serial Cq values can track recovery; a higher viral load gives a lower Cq.
• Western blot does not tell you the gene is transcribed — it shows the protein. Northern blot shows the transcript. Southern blot shows the gene structure (copy number, deletions, rearrangements).
• Southern blot was historically used to confirm PCR product identity (transfer of the product to a membrane and probing).
• Reverse transcriptase is needed whenever the starting material is RNA.

For related genetic concepts see Mendelian inheritance patterns and chromosomal syndromes; for laboratory identification of bacteria see stains and culture media.