Like any other PCR, multiplex reactions should be done at a stringent enough temperature, allowing amplification of all loci of interest without "background" by-products. Although many individual loci can be specifically amplified at an annealing temperature of 56°-60° C, experiments showed that lowering the annealing temperature by 4-6° C was required for the same loci to be co-amplified in multiplex mixtures. This is demonstrated in Fig. 19 below, showing the same PCR reactions performed in conditions in which the only parameter changed was the annealing temperature. For the multiplex a PCR amplification of mixtures C and C*, an annealing temperature of 54° C seems the most appropriate, although the individual loci (for example "Y") could be amplified at 60° C. At 54° C, although some unspecific amplification probably still occurs in the multiplex reaction, it is overcome by the concurrent amplification of an increased number of specific loci and thus remains invisible. In PCR, due to differences in base composition, length of product or secondary structure some loci are more efficiently amplified than others When many loci are simultaneously amplified (multiplexed), the more efficiently amplified loci will negatively influence the yield of product from the less efficient loci. This phenomenon is due in part to the limited supply of enzyme and nucleotides in the PCR reaction. Therefore, in the multiplex procedure the more efficiently amplified loci compete better and take over the less efficiently amplified products, thus rendering them less visible or invisible. (Figure 19 below, depicts a complex situation in which annealing temperature, number of simultaneously amplified loci and buffer concentration were changed in parallel reactions). |
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Fig. 19. Multiplex amplification of mixture C* (first three lanes in each gel), primer pair "Y" (lanes 4 to 6, blue arrows) and mixture C (lanes 7 to 12 in 1x or 2x PCR buffer) on three different template DNAs using three PCR programs differing in annealing temperature (48° C, 54° C or 59° C). Lanes 1-9 on each gel show reactions in 1x PCR buffer. Lanes 10-12 on each gel show reactions in 2x PCR buffer. Lanes 7-12 on each gel (under "1x" and "2x" ) were with primer mixture C. The unmarked lanes are the marker (1 kb ladder). The five arrows to the left side of the first gel indicate the expected products of mix C* (five products). The longest specific product on each gel is marked by a red arrow. Magenta arrow indicates a strong unspecific product. Yellow arrows indicate the two extra products expected in mix C (total of seven products) compared with C*. Blue arrows indicate position of product Y (either by itself or in the multiplex mixture) in the first gel or the lack of product Y in some of the reactions from the last two gels. Multiplex amplification at 48° C shows many unspecific bands. In 1x PCR buffer, the Y product is stronger when amplified in mixture C* (5 primer pairs) than in mixture C (7 primer pairs) showing that, at least for some products, an increased number of simultaneously amplified loci can influence the yield of some individual loci. Raising the PCR buffer concentration from 1x to 2x allows a more even amplification of all specific products and helps in decrease the intensity of many longer unspecific products (compare lanes 7-9 vs. 10-12). The strong 470-480bp unspecific band (magenta arrow) seen with 2x buffer was eliminated by varying the proportion of different primers in the reaction (compare with C in Fig 1). At 59° C the Y product can be seen only when 2x buffer is used or when the locus is amplified alone.
Number of cycles
Primer mix C* was used to amplify two different genomic DNA templates, stopping the reaction after increasing numbers of cycles (Fig. 20). For the same DNA template, results were reproducible among all vials although one of the two genomic DNAs was better, probably due to the higher quality and/or amount of DNA. The most obvious variation in the amount of products was around 24 cycles (for ethidium bromide stained gels). 28-30 cycles are usually sufficient in a reaction. Little or no quantitative changes (i.e., relative amounts of PCR products) were observed with increasing cycle number up 45. Little quantitative gain was noticed when increasing the number of cycles up to 60 (Fig. 21)
Fig. 20. Multiplex amplification of mixture C* using two different DNA templates and increasing the numbers of cycles by units of three.
Fig. 21. Multiplex amplification of mixture C* using tthe same PCR program and increasing the number of cycles by units of ten (up to 60). No additional ingredients were added in the reactions.
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Annealing time and temperature
Annealing time
An annealing time of 30-45 seconds is commonly used in PCR reactions. Increase in annealing time up o 2-3 minutes did not appreciably influence the outcome of the PCR reactions. However, as the polymerase has some reduced activity between 45 and 65o C (interval in which most annealing temperature are chosen), longer annealing times may increase the likelihood of unspecific amplification products (data not shown)
Annealing temperature is one of the most important parameters that need adjustment in the PCR reaction. Moreover, the flexibility of this parameter allows optimization of the reaction in the presence of variable amounts of other ingredients (especially template DNA). For example, the PCR product depicted in Fig. 22 could be amplified easily at annealing temperatures of 55 o C in the presence of 1-100 ng genomic DNA template. Below this limit, there was no detectable PCR product on agarose gels (this primer pair amplifies a polymorphic locus, explaining the two bands seen on non-denaturing agarose gels). It was observed that the specific product can be detected again, even in the presence of very low DNA template concentrations, if the annealing temperature is also decreased. In the reactions depicted in figure 22, the DNA template amount was decreased to 3.1 pg (which is about half the DNA content of a diploid human cell). Remarkably, only one allele was preferentially amplified when the template DNA was approximately 6.6 pg. To achieve these results, reaction was performed at 45 o annealing temperature (a 10 degrees drop from usual). No unspecific products are seen. However, if the same reaction is performed in the presence of a higher amount of DNA template, the low annealing temperature results in the appearance of many unspecific secondary products. Thus, it appears that by decreasing the amount of DNA template, the number of potentially unspecific sites is also decreased, making possible the drop in annealing temperature.
Fig. 22. PCR amplification of a plymorphic locus in the presence of decreasing, low amounts of genomic template DNA and at an annealing temperature 10 o C lower than normal.
Lanes A-F show slight variation in the amount of product, when vials with identical reaction mixture were placed in different position in the metal block of a thermocycler. Amount of template was 800pg/reaction.
Polymorphisms and annealing temperature
Annealing temperature is important in finding and documenting polymorphisms. Slight mismatches, (even 1 base-pair mutations) in one of sequences bound by the two primers used to amplify a DNA locus, can be detected by slight variations in annealing temperature and/or by multiplex PCR. In Fig. 23 such a polymorphism on human Y chromosome is detected in a few DNA samples by amplifying that locus along with other ones using multiplex mixture C (see also Fig. 1). In Fig. 24, same polymorphism is detected by performing PCR reaction only with the specific primer pair, but increasing the stringency of the annealing temperature.
Fig. 23. Single-locus PCR on 7 different template DNAs with a primer pair amplifying a polymorphic locus (yelow). Multiplex PCR of the same templates when the primer pair is part of mixture C. Reactions were performed in the same cycling conditions (annealing at 54 o C). The slight mismatch in primer binding (polymorphism) is detected only in the multiplex reaction by the lack of the amplification product (magenta arrows).
Fig. 24. Same primer mismatch described above can be detected by single-locus PCR reactions after increasing the stringency of the annealing temperature. Samples 3 and 4 show a decrease of product at 61 o C annealing temperature but have a "normal" appearance at 59 o C annealing temperature (magenta arrows).
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Extension time and temperature
Extension time
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.
