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In multiplex PCR, as more loci are simultaneously amplified, the pool of enzyme and nucleotides becomes a limiting factor and more time is necessary for the polymerase molecules to complete synthesis of all the products. Extension time will play an important role in adjusting the outcome of the PCR reaction. This is illustrated in the experiments depicted in two figures below. In one experiment, multiplex mixtures A-D (see also fig. 1) were amplified using PCR programs with 1 and 2 minutes extension times, respectively. Higher yields of PCR products were obtained in all four mixtures when the longer extension time was used. Optimal amplification of all loci will require further adjustments in other factors influencing the reaction (buffer concentration, amount of individual primers). A somewhat lower reproducibility of the results between Fig 11 and Fig 25 was most probably due to a combination of small pipetting differences and the fine balance between buffer, dNTP and MgCl2 concentration (see those topics). Within the same experiment, however, results were reproducible and the effect of various parameters could be studied (Fig. 25).
In the other experiment (Fig. 26) increasing the extension time in the multiplex PCR increased the amount of longer products, at the "expense" of the shorter ones.
Fig. 25. Multiplex PCR of mixtures A-D comparing PCR programs with 2 (green) and 1 (yellow) minute extension time at 54° C annealing temperature. Comparison of equivalent lanes shows an improvement in yield when extension time is 2 minutes. Some faint unspecific bands appear, possibly due to the low buffer concentration (1x).
Fig. 26. Same multiplex mixture was amplified on PCR programs differing only in their extension time (1 and 4 minutes). Shorter amplification products are preferentially amplified with short extension times (1 minute) whereas the longer products become more visible as the extension time increases (arrows). At the same time, at 4 minutes, the shorter products lose much of their intensity. Reactions in lanes 1a and 1b are identical (different DNA templates only).
Figure 11 illustrates the influence of the extension temperature. Equimolar primer mixtures A-D were amplified using two different PCR programs, one at 65o C (yellow lanes) and the other at 72o C (green lanes) extension temperature. In general, there is a higher yield of PCR products for A, B and D when program A was used. This shows that the 72o C extension temperature, negtively influenced amplification of some loci (pink arrows),while also making some unspecific products visible (yellow arrows). It is likely that, for the short PCR products used in these examples (below 500 bp), the higher annealing temperature is probably detrimental to the stability of the DNA helix, so less strands of DNA have the chance to become "copied" by the polymerase after annealing.
Fig. 11 (duplicate).Example of the influence of extension temperature. Multiplex PCR with mixtrues A-B using two different PCR programs. Reactions on the right side (green) were performed in identical cycling conditions with Fig. 9, whereas reactions on the left side (yellow) were performed using cycling conditions in which extension temperature was dropped from 72 o C to 65 o C. Reaction worked more efficiently with the lower extension temperature (pink arrow show missing products, yellow arros show unspecific products).