ABSTRACT
A theoretical analysis is presented with experimental confirmation to conclusively demonstrate the critical role that annealing plays in efficient PCR amplification of GC-rich templates. The analysis is focused on the annealing of primers at alternative binding sites (competitive annealing) and the main result is a quantitative expression of the efficiency (eta) of annealing as a function of temperature (T(A)), annealing period (t(A)), and template composition. The optimal efficiency lies in a narrow region of T(A) and t(A) for GC-rich templates and a much broader region for normal GC templates. To confirm the theoretical findings, the following genes have been PCR amplified from human cDNA template: ARX and HBB (with 78.72% and 52.99% GC, respectively). Theoretical results are in excellent agreement with the experimental findings. Optimum annealing times for GC-rich genes lie in the range of 3-6s and depend on annealing temperature. Annealing times greater than 10s yield smeared PCR amplified products. The non-GC-rich gene did not exhibit this sensitivity to annealing times. Theory and experimental results show that shorter annealing times are not only sufficient but can actually aid in more efficient PCR amplification of GC-rich templates.
Subject(s)
Base Composition , DNA/chemistry , Polymerase Chain Reaction/methods , Models, TheoreticalABSTRACT
Cellulose-based composite materials containing xyloglucans or mannan-based polysaccharides have been shown to possess organisational features with many characteristics similar to primary plant cell walls. We have tested the effects of a typical alpha-expansin (CsExp1) on these composites using two different mechanical assays. We show that CsExp1 induces very rapid extension in composites containing tamarind xyloglucan under constant load. In contrast, expansin treatment had no effect in constant load extension assays using cellulose-only materials or in those carried out on composites containing glucomannan or galactomannan. We show that the effect of expansins is much smaller on composites made with short chain length xyloglucans than on those containing longer chains. In uniaxial extension tests we found that expansin could double the total extension (before failure) in xyloglucan composites and that the effects were again lower in composites containing shorter xyloglucans. We found no effect of expansin on uniaxial extensions with glucomannan or galactomannan. However, a significant effect of expansin on the uniaxial extension behaviour of cellulose-only material was observed. These experiments suggest that the target of CsExp1 in cell walls is probably the cellulose xyloglucan matrix, but that other (1-4) beta-glucan to (1-4) beta-glucan hydrogen bonded contacts can also serve as substrates.
