Enhancement of PCR Sensitivity and Yield Using Thiol-modified Primers

Abstract

Various additives can enhance the quality of PCR amplification, but these generally require considerable optimization to achieve peak performance. Here, we demonstrate that the use of thiol-modified primers can enhance both PCR sensitivity and yield. In experiments with V. parahaemolyticus genomic DNA, this primer modification enhances PCR sensitivity by more than 100-fold, with accompanying improvements in amplicon yield. Then, an artificial plasmid with the same primer binding regions and different internal amplification sequence was designed. The result showed that the amplification also be improved by using the same thiol-modified primers. It indicated the enhancement was not caused by the effect of the thiol-modified primers on the second structure of amplification sequence. Subsequent experiments demonstrate that the effects of this modification are potentially due to altered interaction between the primers and proteins in the reaction mixture. Amplification with thiol-modified primers was strongly inhibited by the presence of extraneous proteins relative to standard DNA primers, which indicates that thiol-modified primers may be inhibited due to interaction with these proteins. In contaminant-free reactions, however, the thiol-modified primers might interact more strongly with DNA polymerase, which could in turn improve PCR amplification.

Figure 1

Sensitivity of a PCR assay for Vibrio parahaemolyticus genomic DNA using standard primers V451-r/f (lanes 1–9) and thiol-modified primers V451S-r/f (lanes 11–19). Lane M: Marker; Lanes 1–9 & 11–19: reactions containing the following amounts of their respective template: 50 ng, 5 ng, 500 pg, 50 pg, 5 pg, 500 fg, 50 fg, 5 fg, 0.5 fg. Lane 10 & 20: negative control without DNA template.

Figure 2

The yield of PCR amplification for V. parahaemolyticus genomic DNA using standard primers V451-r/f (lanes 2–6) and thiol-modified primers V451S-r/f (lanes 8–12). Lanes 2–6 & 8–12: reactions with 500 pg of V. parahaemolyticus genomic DNA. Lanes 1 & 7: negative control without DNA template.

Figure 3

PCR amplification of artificial plasmids using V451-r/f (A), V451S-r/f (B), V451-r & V451S-f (C) and V451S-r & V451-f (D). Lane M: Marker; Lanes 1–10: reactions with 190 pg, 19 pg, 1.9 pg, 190 fg, 19 fg, 1.9 fg, 190 ag, 19 ag, 1.9 ag, and 0.19 ag of plasmid DNA template. Lane 11: negative control without DNA template.

Figure 4

PCR amplification of artificial plasmids using standard V451-r/f primers (A,C,E) and thiol-modified V451S-r/f primers (B,D,F) in the presence of LB broth (A,B: 1 μL; C,D: 2 μL; E,F: 4 μL). Lane M: Marker. Lanes 1–9: reactions with 190 pg, 19 pg, 1.9 pg, 190 fg, 19 fg, 1.9 fg, 190 ag, 19 ag, and 1.9 ag of DNA template; Lane 10: negative control without DNA template.

Figure 5

PCR amplification of artificial plasmids using standard V451-r/f primers (A) and thiol-modified V451S-r/f primers (B) with 2 μL of 10% BSA. Lane M: Marker; Lanes 1–8: reactions with 190 pg, 19 pg, 1.9 pg, 190 fg, 19 fg, 1.9 fg, 190 ag, and 19 ag of DNA template. Lane 9: negative control without DNA template.

Figure 6

Sensitivity of a PCR assay for Salmonella genomic DNA using standard Sa375-r/f primers (A) and thiol-modified Sa375S-r/f primers (B). Lane M: Marker. Lanes 1–7: reactions with 2 μL of 70 ng/μL, 7 ng/μL, 700 pg/μL, 70 pg/μL, 7 pg/μL, 700 fg/μL, 70 fg/μL DNA template. Lane 8: negative control without DNA template.

Figure 7

Schematic diagram of thiol-modified primer.

Figure 8

Schematic diagram of the procedure for constructing an artificial plasmid with the same primer-binding regions.