PCR Optimisation, Genomic Contamination and a Low-Molecular-Weight Smear
A 2.4 kb cDNA fragment from a human gene is to be amplified both for cloning and for a semi-quantitative expression assay. Pair A consists of a 22-nucleotide forward primer with 10 G+C residues and a 20-nucleotide reverse primer with 12 G+C residues. The template is reverse-transcribed RNA that was not DNase-treated; the corresponding genomic locus carries a 700 bp intron between the exons where the primers anneal. Thirty-five cycles with Taq (assumed error rate 1 × 10⁻⁴ per base per cycle) give the expected 2.4 kb band, but also a faint band at 3.1 kb. When the same cDNA is diluted 1:10, the 2.4 kb band fades and a broad 40–60 bp smear becomes prominent. Cycle sequencing of the Taq product shows several point mutations, and one cloned fragment has lost an expected restriction site.
1. Calculate the Wallace-rule Tm of each primer in pair A, recommend an annealing temperature, and give extension times for Taq and for a proofreading polymerase (1 kb per 30 s) for this 2.4 kb product.
Read the worked answer
Wallace rule: Tm = 2(A+T) + 4(G+C).
- Forward, 22 nt with 10 G+C: A+T = 12 → Tm = 2(12) + 4(10) = 24 + 40 = 64 °C.
- Reverse, 20 nt with 12 G+C: A+T = 8 → Tm = 2(8) + 4(12) = 16 + 48 = 64 °C.
The pair is well matched (both 64 °C), which is what you want; a Tm difference above ~5 °C causes one primer to anneal poorly. A working annealing temperature is about 5 °C below the lower Tm, i.e. 58–59 °C, best set with a 56–62 °C gradient rather than assumed.
Extension: Taq processivity is usually taken as ~1 kb/min, so 2.4 kb needs 2.5 min (use 2.5–3 min for margin). A proofreading polymerase at 1 kb per 30 s needs ~1.2 min (use 1.5 min). Note that proofreading enzymes also prefer slightly higher annealing temperatures and a longer initial denaturation, and that the Wallace rule ignores salt and mismatches, so it is only a starting estimate.
2. Estimate the number of polymerase errors accumulated per molecule in the Taq product and decide whether this product is suitable for cloning and expression.
Read the worked answer
Each finished molecule has incorporated roughly one base per cycle per position, so the total bases polymerised per molecule ≈ product length × cycle number = 2400 × 35 = 84,000.
Expected errors ≈ 84,000 × 1 × 10⁻⁴ = ~8.4 per molecule. In practice every molecule in the population carries several mutations, and because errors arise early in cycling some molecules are far worse than the average.
That is not acceptable for cloning a 1.4–2.4 kb ORF that must be expressed: a single missense or nonsense mutation can abolish activity, and the loss of a restriction site in one clone is exactly the expected signature. Reducing to 20 cycles helps only proportionally (2400 × 20 × 10⁻⁴ ≈ 4.8 errors), which is still too many. The correct move is to re-amplify with a proofreading (3'→5' exonuclease) polymerase at ~1 × 10⁻⁶ per base per cycle, which gives ~0.08 errors per molecule, and to keep cycles as low as the yield allows. The Taq product remains fine for the semi-quantitative assay, where the exact sequence is irrelevant.
3. Explain the 3.1 kb band and the 40–60 bp smear, and describe the controls and redesign that would resolve both.
Read the worked answer
3.1 kb band: 2.4 kb + 0.7 kb = 3.1 kb, precisely the size expected if the template contains genomic DNA and the amplicon spans the 700 bp intron. Because the RNA was not DNase-treated, residual genomic DNA is present and is amplified by the same primers. A no-reverse-transcriptase control (all reagents minus RT) will show the 3.1 kb band if gDNA is the source; if the band appears there, it is gDNA, not cDNA. Fix: DNase-treat the RNA before reverse transcription, and place one primer across an exon–exon junction so that gDNA cannot be amplified at all.
40–60 bp smear at low template: this is primer-dimer and short mispriming product, which outcompetes the true amplicon when template is scarce. Evidence: it appears only on dilution and is far below any legitimate product. Controls: a no-template control must be clean, and melt-curve analysis should show a single peak for the 2.4 kb product. Fixes: hot-start polymerase, lower primer concentration (to ~0.2 µM), touchdown annealing, and redesign of the 3' ends so the primers are not mutually complementary. Do not simply shorten the extension time to hide the 3.1 kb band - that suppresses detection without removing the contaminating template.