MgCl2 concentration
Relationship between MgCl2 and dNTP concentration
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.
Relationship between MgCl2 and buffer (or salt) concentration
Two of the most important ingredients influenceing the results of a PCR reaction are the buffer (especially salt) and the magnesium concentrations. To study their relationship, a multiplex PCr was performed using mixture C (Fig. 36, below). Two sets of reactions were performed at two "extreme" concentrations of salt (KCl), 1x (50mM) and 3x (150 mM), and various magnesium concentrations (yellow values). Two other sets of reactions were performed at two "extreme" magnesium concentrations, 1.5 and 10.8 mM and various salt (KCl) concentrations (blue values). The dNTP concentration was kept constant, at 200 mM each deoxynucleotide. The following observation can be drawn:
at 1x salt concentration and 200 mM each dNTP, reaction worked best at about 1.5 mM magnesium. At higher magnesium concentrations unspecific products appeared, but they gradually decreased in intensity towards 21.6 mM (probably because MgCl2 is a salt, decreasing the stringency of the buffer - same way KCl does).
at 3x salt concentration and 200 mM each dNTP, reaction worked best between 1.5 and 3.5 mM magnesium. As the stringency of the buffer was already lower than usual (due to the high KCl concnentration), further increase in MgCl2 increased the "combined" stringency of teh reaction even more. Thus, fewer long unspecific products were obtained and the reaction was almost completely inhibited towards 21.6 mM magnesium.
at 10.8 mM MgCl2 and 200 mM each dNTP, reaction worked best around 2x salt (KCl) concetration (mostly specific products amplified). However, it is obvious that overall amount of PCR product is reduced compared to the reactions taking place at 1.5 mM magnesium. In this respect, high magnesium concentrations seem to inhibit the reaction more than high KCl (3x) concentrations. Therefore, it is likely that this magnesium inhibition is more than just a reduction in stringency of the reaction mixture.
at 1.5 mM magnesium and 200 mM each dNTP, reaction worked best around 2x salt (KCl) concentration (all products amplified, few unspecific products visible). Overall product amount is higher than in the reactions taking place at 10.8 mM magnesium.
Fig. 36. Realtionship between magnesium and salt (KCl) concentration in PCR reactions. For a detailed description of the figure, please read text above.
Effects of variations in MgCl2 concentration only
A recommended MgCl2 concentration in a standard PCR reaction is 1.5mM, at dNTP concentrations of around 200µM each. To test the influence of MgCl2, a multiplex PCR with mixture C was performed, keeping dNTP concentration at 200µM each and gradually increasing MgCl2 from 1.8 to 10.8 mM (Fig. 37). The overall amplification became gradually more "specific" (unspecific bands disappeared) and the products acquired comparable intensities (at 10.8mM). However, higher concentrations of MgCl2 appeared to inhibit the polymerase activity, decreasing the amount of all products. Taking into consideration the amount of PCR products, the best magnesium concentration should be between 1.8 and 3.6 mM. The large unspecific product (arrow) appeared due to the lower annealing temperature at which the reaction took place.
Fig. 37. Multiplex PCR amplification with mixture C at 2x KCl and increasing magnesium concentrations. Overall reaction becomes more specific at 10.8 mM magnesium, but the products are reduced in intensity. The most optimal magnesium concentration is somewhere between 1.8 and 3.6 mM where the PCR product amount is higher. The unspecific product (arrow) appears due to a lower than usual annealing temperature used for this reaction.
[NextPage]
Gel electrophoresis
Comparison of agarose type (non-polymorphic loci)
Two types of agarose from the same manufacturer (both in use in this laboratory) were compared for their efficiency in separating the multiplex PCR products (Fig. 38). Multiplex PCR with primer mixtures A (one sample) and F (4 samples) was performed. Same amount of each reaction was loaded on a 3% agarose gel of each type. Electrophoresis time was about 1.6-1.7x longer for the regular (SeaKem LE) agarose gel.
In accordance to the manufacturer's specifications, the NuSieve agarose separates short products better than the regular agarose, and in a reduced amount of time. Although the gels had the same thickness, results also indicate that the "special" NuSieve agarose is more transparent than the regular agarose. Although NuSieve agarose is much more expensive, it provides some cost reduction by requiring less amount of agarose for the same separation power and by requiring less amount of separation time. These particular advantages can make such "specialized" agaroses useful for particular applications.
It is worth mentioning that other agaroses (from different manufacturers) used, perform similarly.
Fig. 38. Separation of the same multiplex products of mixtures A and C (four lanes) on two different agaroses. Arrow indicates a few unspecific products in lane 2 and circle indicates primers (or primer-dimers), both of these being stronger on the NuSieve gel. This shows tthat NuSieve gels have a higher transparency. Also, separation on NuSieve gels was achieved in les amount of time, over a shorter gel length. The unmarked lane(s) is the 1 kb ladder (GIBCO).
Agarose gel (running time)
Agarose gels can be run at various voltages, depending on the separation desired and the available time. It was noticed that, at least for PCR products smaller than 600 bp, separation is better and bands are sharper if gels are run very fast (3-4 hours for a 15-20 cm long 2-3% agarose gel). When the same gel runs at a low voltage overnight (14-16 hours) the products become less separable or "puffy" due to the diffusion in the gel (compare Fig. 39 below with lane C in Fig. 1).
Fig. 39. Multiplex PCR with mix C was performed on 9 DNA samples to screen for microdeletions (chromosome Y loci. Gel separation was performed overnight (14 hours). Products appear diffuse, less intense, and less separable (product 1 and 2 are "fused" together). Green and magenta arrows indicate lack of loci #1 and # 4 (microdeletions) in some of the DNA samples tested. he unmarked lane(s) is the 1 kb ladder (GIBCO).
Agarose gels and polymorphic loci
As depicted also in Fig. 7, agarose gels can be used to separate PCR products of plymorphic loci. In most cases, two or three bands appear, due to heteroduplex formation between the long and short alleles. However, separation of multiplex PCR reaction products of many polymorphic loci (for example mixture K) coud become a problem for nondenaturing agarose gels. In mixture K, products were chosen so they differ by no less than 5 bp and no more than 45 bp. As depicted in Fig. 7 and Fig. 40 (below), agarose gels do not have sufficient separation power. Bands become to overlap and it is difficult or impossible to find and label each band. Denaturing polyacrylamide gels are recommended in such cases (see below).
Fig. 40. Multiplex PCR amplification of mixture K, using four different DNA samples. A 2.5% agarose gel was used to separate the products. As depicted also in Fig. 7, 2-3 bands become visible for each product. When together, many of these bands start overlapping, making identification of individual products/alleles impossible. The unmarked lane(s) is the 1 kb ladder (GIBCO).