Biotechnology and molecular methods: study guide

Key ideas

Molecular methods turn DNA into numbers: qPCR gives expression changes, restriction digests give fragment sizes, gels give sizes by migration, and PCR gives copy numbers. Each has a short calculation.

• qPCR: ΔCt = Ct(target) − Ct(reference); ΔΔCt = ΔCt(treated) − ΔCt(control); fold change = 2^(−ΔΔCt). A lower Ct means more starting template.
• A restriction enzyme cutting a linear DNA at k sites gives k + 1 fragments, and a circular one gives k. Fragment sizes are the gaps between cut sites and ends.
• On a gel, log₁₀(size) is linear with the distance run, so a band’s size is found by interpolating between markers on a log scale.
• PCR runs denaturation (about 95 °C), annealing (about 55 °C) and extension (about 72 °C) in each cycle, and ideal copies are N₀ × 2ⁿ.

Key equations

• fold change = 2^(−ΔΔCt)
• copies = N₀ × 2ⁿ
• fragments (linear) = cuts + 1
• log₁₀(size) falls linearly with distance

Worked example

The reference gene has Ct 20 in both samples. The target has Ct 26 in the control and 24 in the treated sample, so ΔCt = 6 in the control and 4 in the treated, ΔΔCt = −2 and fold change = 2² = 4. A linear 10 kb molecule cut at 2 kb and 7 kb gives 2, 5 and 3 kb fragments, so the largest is 5 kb. Starting with 10 copies and 20 ideal cycles gives 10 × 2²⁰ ≈ 1.0 × 10⁷ copies.

Common mistakes

• Using 2^ΔΔCt instead of 2^(−ΔΔCt), which inverts the fold change.
• Interpolating a gel linearly in base pairs instead of on a log scale.
• Counting k fragments for k cuts of a linear molecule. It gives k + 1.

Practice

Test yourself with practice questions on this topic: Biotechnology and molecular methods