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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.
(examples of polymorphism are also shown on page 09)