Fig. 11. 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).
Primer amount and buffer concentration. To improve the amplification of some of the DNA products from Fig. 11 above, the amount of primers was increased 2-5x for those loci. At the same time, the PCR buffer concentration was increased to 2x. These modifications allowed a much more efficient and reproducible amplification, with no unspecific products.
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Primer amount in PCR
Absolute value of primer concentration in multiplex PCR. The amount of DNA primer(s) available during the PCR reaction influences the results. Primer concentration taken in a common PCR reaction (for example when amplifying a single locus) is about 100-500 nM each primer. (Primers can be purchased from various sources at concentrations between 10-25 mM each. Usually, 0.5-1ml primer solution is sufficient for a 25-100 ml PCR reaction) In a multiplex PCR test using equimolar primer mixtures (Fig. 13), individual primer concentrations were varied between 500 and 15 nM each primer. Given that mixture A used 14 primers (7 loci) and mixture B 10 primers (5 loci), the final primer concentration varied between 7000 and 200 nM (mixture A) and between 5000 and 150 nM (mixture B). Although equimolar primer mixtures did not usually provide optimal amplification of all loci, this test allowed the observation that too high and too low primer amounts may need to be avoided.Too high primer concentrations may inhibit the multiplex reaction whereas too low amounts may not be sufficient.
Fig. 13. Multiplex PCR with mixtures A and B (see also Fig. 1). Numerical values indicate the concentration of each primer in the final reaction. Mixture A includes 14 primers and mixture B includes 10. Reactions work best at around 200 nM (each primer) in mixture A and 60 nM (each primer) in mixture B.
Primer and template concentrations. Within limits, increasing primer concentration may improve the outcome of the PCR reaction, and should be considered as a way to optimize PCR reactions.
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PCR buffers
A commonly used PCR buffer, includes only KCl, Tris and MgCl2 (for example, Perkin Elmer Cetus); a somewhat more complex buffer was previously proposed for multiplex reactions of the DMD gene exons (Chamberlain et al. (1988) in Nucleic Ac Res 16: 11141-11156). These buffers were compared in multiplex PCR reactions, for their efficiency in supporting the activity of the Taq polymerase. Figure 15 shows that, PCR reactions on four different genomic DNA templates were consistently more efficient (more PCR product) when performed in 1.6x PCR buffer than 1x DMD buffer. Same amount of template DNA and primer were taken in all reactions, which were run in the same conditions at the same time. The same amount of product was loaded in each lane on the gel.
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Fig 15. Comparison of two differnt PCR buffers. Multiplex mixture F was used in PCR amplification of 4 different genomic DNA templates. Reactions were performed in identical conditions with the exception of the buffers. Results indicate a higher yield of products in reactions performed in 1.6 x PCR buffer.
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Salt (KCl) concentration
For the successful PCR or multiplex PCR amplification of many loci (especially products between 100-1000 bp) raising the buffer concentration to 1.4x-2x (or only the KCl concentration to about 70-100mM) dramatically improves the efficiency of the reaction. In fact the effect of the KCl concentration was more important than any of the adjuvants tested (DMSO, glycerol or BSA). Generally, many primer pairs producing longer amplification products worked better at lower salt concentrations, whereas many primer pairs producing short amplification products worked better at higher salt concentrations. This is illustrated in the three figures below in which there is a realtive shift in the intensity of the products from the longer one towards the shorter ones as the ionic strength increases. An increase in salt concentration makes longer DNA denature slower than shorter DNA, so shorter molecules will be amplified preferrentially. Some primers, however, worked well over a wide range of buffer/salt concentrations. Examples of multiplex reactions at different buffer concentrations are shown in Fig. 16, 17 and 18.
Fig. 16. Multipex PCR amplification of mixture A at increasing buffer (salt) concentrations.
In Fig. 16, both primers for locus 6 have a melting point around 58° C whereas primers for locus 5 have a melting point around 52° C. At the same ionic strength (1x buffer) and annealing temperature (54° C), amplification of locus 6 will be favored over 5. To increase binding of primers for locus 5 while keeping the annealing temperature the same, stringency of the PCR buffer needs to be decreased. This can be easily done by increasing the KCl (or buffer) concentration. A different example is locus 7, where both primers have a similar melting point with primers for locus 6 (58° C). They are not as well amplified in 1x buffer, but respond well to increase in the salt concentration. In this case, the explanation may be that the entire product 7 has a lower GC content than product 6. This makes the DNA helix of product 7 less stable when exposed to the extension temperature. Some of the new strands may detach from the template, before the polymerase fully amplifies them. Decreasing the stringency of the buffer (1.6x-2x) might "stick" the newly synthesized strands better to the template, allowing the polymerase to finish its task.
PCR reactions in which only KCl or Tris-HCl concentrations were varied, showed that the described effect is due to the salt (KCl). Tris-HCl concentration did not influence the outcome of the reactions over a large range of concentrations (from 0.75x to 5x) whereas MgCl2concetrations have a somewhat different effect.
Fig. 17. Multipex PCR amplification of mixtures B and C* at increasing buffer (salt) concentrations.
Fig. 18. Multipex PCR amplification of mixtures D and E at increasing buffer (salt) concentrations.
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Designing PCR programs
The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. For any given primer pair, the PCR program can be selected based on the composition (GC content) of the primers and the length of the expected PCR product. In the majority of the cases, products expected to be amplified are relatively small (from 0.1 to 2-3 kb). (For long-range PCR (amplifying products of 10 to 20-30 kb) commercial kits are available). The activity of the Taq polymerase is about 2000 nucleotides/minute at optimal temperature (72-78o C) and the extension time in the reaction can be calculated accordingly.
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