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.
Highlights
The polymerase chain reaction (PCR) has become a core molecular biology technique due to its powerful capability to amplify large quantities of specific segments of DNA from very limited amounts of template in vitro[1,2]
Many modifications based on basic PCR to enhance the quality of amplifications except sophisticated optimizations were reported previously, but most of these entail the use of an additive in the reactions
We describe the use of thiol-modified primers to enhance PCR sensitivity and yield
Summary
The polymerase chain reaction (PCR) has become a core molecular biology technique due to its powerful capability to amplify large quantities of specific segments of DNA from very limited amounts of template in vitro[1,2]. PCR amplification cannot occur robustly in certain experimental conditions, and there are a large number of modifications to the basic PCR assay designed to enhance its specificity, efficiency, yield and fidelity. One of these entails the use of specialized DNA polymerases, such as hot-start and high-fidelity DNA polymerase. For PCR reactions using hot-start DNA polymerase, amplification does not begin until the reaction reaches the annealing temperature. This helps to prevent primer/template mispriming and the formation of primer dimers, leading to greatly improved specificity and sensitivity[5]. Such, considerable optimization is required to identify the strategy best suited for amplification under different PCR conditions
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call