CONTENT
PCR guide: a discussion of the main parameters influencing the outcome of the PCR and multiplex PCR reaction in 16 pages/sections and using over 45 pictures
PCR troubleshooting: some commonly asked questions and likely solutions
Standards: provides images of the standard multiplex reactions used during this work. It is useful to consult before reading other pages of the PCR guide
Applications: includes examples of some applications of PCR and multiplex PCR. Still under construction.
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1 | Generalities | 9 | |
2 | Choosing PCR primers | 10 | |
3 | Reaction volumes | 11 | |
4 | Number of PCR products | 12 | Taq polymerase(s) |
5 | Primer amount | 13 | dNTP concentration |
6 | PCR buffers | 14 | MgCl2 concentration |
7 | Salt (KCl) concentration | 15 | Gel electrophoresis |
8 | Designing PCR programs | 16 | Adjuvants in PCR |
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PCR generalities
| Standard PCR reaction mix Consider the standard PCR reaction mix (25 µL reaction) below. All reactions are run for 30 cycles. Table 1. PCR reaction componentsCOMPONENTVOLUMEFINAL CONCENTRATION1. autoclaved ultra-filtered water (pH 7.0)20.7µL-2. 10x PCR Buffer*2.5µL1x3. dNTPs mix (25 mM each nucleotide)0.2µL200 µM (each nucleotide)4. primer mix (25 pmoles/µL each primer)0.4µL0.4 µM (each primer)5. Taq DNA polymerase (native enzyme)0.2µL1 Unit/25 µL6. genomic DNA template (100 ng/µL)1.0µL100 ng/25 µL * The PCR buffer used was made after the recommendations of the manufacturer/vendor (Perkin Elmer Cetus). The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2(the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mM MgCl2). | |
| Pipetting and DNA template |
Fig. 5. Multiplex PCR test reaction for pipetting errors.
Two genomic DNA samples (each 100 ng/ml) were used in multiplex PCR reactions with mix J, simultaneously amplifying eleven different loci (between 165 and 85 bp long). Labeling was done by adding radioactive dCTP to the reaction mix and separation of products was done on a sequencing PAA gel.
One microliter each of DNA sample A was taken in vials 1-4, and of DNA sample B in vials 5-8. On the left side, the DNA was pipetted separately in each vial. On the right side, the DNA was mixed with all other PCR ingredients and the mixture was split in equal parts in the vials.
The uneven amplification on the left side indicates that, even after thourough mixing, 1 microliter of genomic DNA may contain variable amounts of DNA. This may negatively influence interpretation of the data, especially in quantitative PCR and multiplex PCR reactions. On the right side, amplifications are much more consistent (compare 1-4 and 5-8). Small differences may be due to slight temperature differences in various places in the metal block of the thermocycler.
| First PCR program 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. Number of cycles. In general, 30 cycles should be sufficient for a usual PCR reaction. An increased number of cycles will not dramatically change the amount of product (see below).Table 2. Designing a first PCR program. | |
| Thermocyclers and PCR vials A number of different types of thermocyclers and PCR vials were used and tested in time. Some potentially useful observations were made: |
Fig. 6. Variation in amplification due to lack of proper contact between the metal block and some vials. A PCR mixture containing all ingredients was split in nine equal parts in the same typ/brand of vials, and the tubes were placed in different wells of the metal block of a thermocycler. Reactions 2, 4, 5 and 9 were negative. The same aspect was not reproducible: in another experiment, reactions in other positions could become negative. This was explained by slight variations in vial construction (wall shape or thickness) but not by temperature variations in the metal block (when the aspect should have been reproducible).
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Choosing/designing PCR primers
In designing primers for PCR, the following steps/rules were tested and proven to be useful:
Fig. 7. Multiplex PCR using primers 18-24 bp long
When PCR reaction Eight individual loci are amplified with similar intensities when the primer pairs are used separately. When equimolar amount of these primers are mixed together for a multiplex reaction (Mix K), some of the products are much weaker (#1, #2, #5, #6) than other. In this case, primers had "usual" length, between 18-24bp.
(primers used in this case amplify polymorphic loci, explaining the "double" or "triple" bands as seen on a regular agarose gel)
Fig. 8. Multiplex PCR using primers 30-35 bp long
Compared to the figure above, in this case the primers used for multiplexing were longer than 30 bp (up to 37 bp). Equimolar amounts of primer were used and all loci were amplified with comparable intensities in each reaction.
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Reaction volume
Q: Does the PCR reaction volume (negatively) influence the outcome?
A: No, especially since the introduction of the small, thin walled, 0.2 ml plastic vials fitting the 96 well metal blocks of the thermocyclers.
A number of observations are worth mentioning:
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Multiplexing primer pairs
Single locus PCR. First step in designing a multiplex PCR is choosing the primer pairs which can be combined. One important requirement is to find a PCR program allowing optimal amplification of all loci when taken individually (Fig. 9). This is achieved by adjusting the annealing and extension time and temperature.
Fig. 9. Single-locus PCR with 34 different primer pairs using the same cycling conditions. Arrows indicate position of the specific products in lanes 25, 28 and 33, in which other unspecific products also appear. Such primer pairs are difficult to use both by themself and in multiplex PCR. However, even though some unspecific products still appeared, primer pair 28 was multiplexed in mixture 5 (Figure 1) and used in a microdeletion screening project. The unspecific products did not interfere with data interpretation. Examples of multiplex reactions using these primers are shown in Fig. 1.
Multiplexing equimolar primer mixtures. The next step is combining the desired primer pairs in multiplex mixture(s), using equimolar amounts of each primer. PCR amplification of the multiplex mixtures can be performed, first using exactly the same PCR program as with individual primer pairs. Very often, this will results in preferential amplification of some loci. Such a situation will require further adjustment in cycling conditions and primer concentration. Although, sometimes unspecific products can be seen in single-locus PCR (yellow arrow in PCR product # 2), these unspecific products usually become invisible when the multiplex reaction is performed. This is probably due to the concurrent ampification of many specific loci, which overwhelms the unspecific products (although they are probably still present in small quantities).
Fig. 7 (duplicate). Single locus PCR and multiplex PCR with equimolar amounts of primers from mixture K, performed in the same cycling conditions. In Some products of mixture K become weak or invisible, requiring further adjustment of primer amount(s) and of cycling conditions. Primers used in mixture K amplify polymorphic loci, explaining the appearence of multiple bands on a nondenaturing agarose gel.
Fig. 10. Equimolar amounts of the same primers used for mixture K (see also Fig. 7 above), where amplified in pairs. In lanes 1, 2 and 4, one locus was amplified less efficiently than the other one (arrows). As mentioned before, amplification of the "weaker" loci can be improved increasing the amount of primers or adjusting the reaction conditions.
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Adjustment of cycling conditions
For example, 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.
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