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 oC to 65 o C. Reaction worked more efficiently with the lower extension temperature (pink arrow show missing products, yellow arros show unspecific products).
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DNA template
All multiplex reactions performed in this laboratory used human genomic DNA as a template. From both multiplex and single-locus PCR reactions, results showed that the amount of DNA template strongly influences the outcome of the reaction. In conditions in which the amount of DNA available is very low, reaction or cycling conditions can be adapted and modified to allow reaction to work efficiently.
The following five images provide examples illustrating the importance of the DNA template concentration.
Fig. 27. PCR amplification of very low amounts of genomic DNA using a degenerate primer. Amount of PCR product decreases with the decreasing amount of template.
Fig. 28. Multiplex PCR using primer mixture A in 1x PCR buffer. As the amount of template drops, most products become gradually weaker. Cycling conditions were identical. Arrow indicates the presence of an unspecific product.
Fig. 29. Multiplex PCR with mixture C* and single-locus PCR with one of the primer pairs form the same mixture. As the DNA template decreases, some bands become weaker in the multiplex reaction. Over the same range of concentrations, this effect is not so visible when only one primer pair is used.
Fig. 30. Multiplex PCR with mixture C* and PCR amplification using only one of the primer pairs from the same mixture. Very low template DNA concentrations were used (0.045 is the amount of DNA from 6 diploid cells). Again, the amount of PCR product decreases with the reduction in template DNA but less so when only one primer pair is used. PCR program used has a lower annealing temperature (about 5o C lower) than the program used for the reactions in Fig. 29.
Fig. 31. Multiplex PCR with mixture C* on two genomic DNA temlpates, one (yellow) carrying a polymorphism for one primer binding site and another one (green) with perfect match. As in Fig. 30 above, to amplify such reduced amounts of DNA template, the same program with low annealing temperature had to be used. Arrow indicates that the polymorphism at locus 4 is detected with the decrease in DNA template amount.
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Taq polymerase
Different concentrations of a Taq polymerase were tested using primer mixture C (Fig. 32). The most efficient enzyme concentration seemed to be around 0.4μl or 2 Units/25μl reaction volume. Too much enzyme, possibly because of the high glycerol concentration in the stock solution, resulted in an unbalanced amplification of various loci and a slight increase in the background. too little enzyme resulted in the lack of some of the amplification products
Fig. 32. Amplification products of mixture C, using 0.5 Units/25μl, 1 Unit/25μl, 2Units/25μl, 4 Units/25μl and 8 Units/25μl reaction volume are shown. Arrows indicate the expected positions of the amplification products. The most appropriate enzyme concentration was between 1-2 Units/25μl.
Five native Taq polymerases, from five different sources, were used to amplify multiplex mixture D in 1.6x PCR buffer using 2Units enzyme/25μl reaction (Fig. 33). In the same buffering conditions, all these enzyme performed similarly.
Fig. 33. Multiplex PCR of mixture D in 1.6x PCR buffer using Taq polymerases from five sources. Lanes 1 to 5 indicate that all enzymes work similarly at the same concentration. Lane 4* (green) shows the products obtained when the enzyme from lane 4 was used in the buffer provided by the vendor. An unspecific product appeared, indicating that buffer composition influences the results.
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Nucleotides (dNTP)
dNTP "instability"
One important observation, coming from experiments with multiplex PCR, is that dNTP stocks are very sensitive to cycles of thawing/freezing. After 3-5 such cycles, multiplex PCR reactions usually did not work well. To avoid such problems, small aliquots (2-5 μl) of dNTP (25 mM each), lasting for only a couple of reactions, can be made and kept frozen at -20o C. However, during long-term freezing, small amounts of water evaporate on the walls of the vial changing the concentration of the dNTP solution. Before using, it is essential to centrifuge these vials at high speed in a microfuge.
This low stability of the dNTP is not so obvious when single loci are amplified.
Another important observation is that, anytime nucleotides are diluted in water, the solution should be buffered (for example with 10mM Tris pH 7.7-8.0, final concentration).Otherwise, an acid pH will promote hydrolysis of dNTP into dNDP and dNMP and will render them useless for enzymatic DNA polymerizing reactions.
dNTP concentrations of about 200μM each are usually recommended for the Taq polymerase, at 1.5mM MgCl2 (Perkin Elmer Cetus). In a 25 μl reaction volume, theoretically these nucleotides should allow synthesis of about 6-6.5 μg of DNA. This amount should be sufficient for multiplex reactions in which 5 to 8 or more primer pairs are used at the same time. To work properly (besides the magnesium bound by the dNTP and the DNA), Taq polymerase requires free magnesium. This is probably the reason why small increases in the dNTP concentrations can rapidly inhibit the PCR reaction (Mg gets "trapped")whereas increases in magnesium concentration often have positive effects.
The relationship between the concentration of magnesium and that of the dNTPs was investigated by performing PCR with a degenerate primer in reactions that contained 200, 400, 600 and 800 μM each dNTP, combined with 1.5, 2, 3, 4 or 5 mM MgCl2 (Fig. 34). This test confirmed that any increase in dNTP concentration requires an increase in the concentration of magnesium ions in order for the reaction to work. At 200 μM each dNTP, reaction worked at all magmesium concentrations, but for this primer it worked better at 3 mM (which is about double the recommended magnesium concentration for the amount of dNTP). At 800 μM each dNTP, reaction worked only aboove 3 mM magnesium.
Fig. 34. PCR with a degenerate primer at different Mg and dNTP concentrations. Each of the Mg concentrations (1.5, 2, 3, 4, 5 mM) were combined with each of the following dNTP concentrations (each): 200μM, 400μM, 600μM and 800μM. Results indicate that increasing dNTP concentrations require increasing Mg concentrations for the PCR reactions to work.
Common dNTP use in PCR and multiplex PCR
In another test aimed at examining the proper dNTP concentration, a multiplex PCR using primer mixture D was performed. The MgCl2 concentration was kept constant (3mM) while the dNTP concentration was increased stepwise from 50 to 100, 200, 400, 600 and 1200 μM each deoxynucleotide (Fig. 35). The best results were achieved at 200 and 400 μm dNTP; reaction was rapidly inhibited after these values. Lower than usual dNTP concentrations still allowed PCR amplification, but with somewhat less efficiency (lane "50").
Fig. 35. Multiplex PCR amplification of mixture D in 2x PCR buffer (3 mM Mg) using increasing concentrations of dNTP (50mM, 100mM, 200mM, 400mM, 600mM and 1200mM each). Most efficient amplification is seen at concentrations of 200-400μM each dNTP. Further increase in the dNTP concentration inhibits the reaction when MgCl2 is kept constant.
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