Dai ZM, Zhu XJ, Chen Q, Yang WJ.

J Biotechnol. 2007 Feb 20;128(3):435-43. doi:10.1016/j.jbiotec.2006.10.018

PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.

PCR amplification of a GeneRuler 1kb DNA Ladder (Fermentas) on which adatpers were ligated. Investigates “parameters which affect ITR self-annealing: (i) the length of PCR products [...] (ii) Primer concentration [...] (iii) The ratio of ITR length to primer length [and] (iv) [annealing temperature]”. Uses a 3′ phosphate to prevent the template switching oligonucleotide to prime a first strand cDNA.

“The 10 µl final [RT] reaction mixture contained 50 mM Tris–Cl (pH 8.3 at 25°C), 75 mM KCl,6 mM MgCl2, 2 mM MnCl2, 0.2 mg/ml BSA, 10 mM DTT, 1 mM dNTPs, 1µM TS-oligo, 1µM oligo(dT) adaptor (, 10 U RNase Inhibitor, and 150 U SuperScript II [and] was incubated at 42 °C for 1 h, followed by 45 °C for 30 min, and 50 °C for 10 min.”

“Using [...] the shortest adaptor, whose length is equal to [the PCR primer], short fragments corresponding to 0.25 kb were efficiently amplified, while longer fragments (>3.5 kb) could not be distinguished from the background. Using [...] the longest adaptor, whose length is more than twice [the PCR primer], fragments shorter than 1.5 kb were suppressed, while fragments up to 10 kb were efficiently amplified.” [My comment: this analysis does not take into account the overall reduction of PCR efficiency with increasing and the fact that mass of shortest fragments were also lower in the GeneRuler ladder. This also contributes to the disappearance of these bands on the electrophoresis pictures. This might explain why the high-length fragments could not resolve in the amplifications with highest yields.]

“Lower [annealing temperature] enhanced the PS-effect under any condition. [Annealing temperature] greatly affected the PS-effect when the primer concentration was low (P2 = 0.04M). [The] average length of the products was longer at lower [annealing temperature]. [...] From 50 to 61.2°C), longer products (up to 10 kb) wer eefficiently amplified. When [annealing temperature] was higher (>65.9°C), much shorter products (<4 kb) were efficiently amplified and longer products disappeared. [Annealing temperature] also affected the PS-effect when primer concentration was 15-fold higher [...] The average product length between higher (70◦C) and lower (60◦C) [annealing temperature] was significantly different, regardless of adaptors used.”

“[Only] with the longest adaptor [...] we were able to amplify products in a wide range (from 0.25 to 10 kb) simultaneously with a slight over-representation of longer molecules. [To] representatively amplify a complex sample, relatively long ITR (compared to primer length), high [annealing temperature], and high concentratio nof primer should be used.”