by David Briggs PhD and Greg Sjørgren
Introduction
 The MBS iStation Multi-block
thermal cycler. |
Multiplex PCR is a complex reaction that often results in low product yield, product drop out and nonspecific product amplification.
(1) This is due to the different optimal annealing temperatures and competition of the different targets and primers; the more complex the reaction, the more pronounced these effects can be. The results from a poorly optimized multiplex reaction can lead to false negatives or positives. This said, multiplex PCR procedures are used increasingly across a wide section of academia and industry for processes such as the amplification of short tandem repeat (STR) loci for identity and paternity testing, genetic linkage analysis and diagnostic assays. For these and other processes, the procedure provides a unique resource, which reduces protocol set-up time, provides significant savings in reagent consumption, and consequently improves cost efficiency.
Multi-block satellite thermal cycling
 Figure 1. Dynamic ramping ensures that all of the wells reach the set temperature at the same time, thereby ensuring that the annealing dwell time is the same for each temperature.
1 Denaturation
2 Extension
3 Dynamic ramping ensures each well arrives at the set temperature at the same time
4 Consistent dwell times across the gradient
5 Annealing. Click here to enlarge |
Unlike most standard thermal cyclers, multi-block satellite thermal cycling (such as the MBS iStation from Thermo Electron Corp., Milford, MA), can incorporate, as one of its multiple (up to 30) cycler units, a gradient that crosses four sets of paired heating elements (peltiers) and 12 wells. This creates the maximum number of temperature variables within a uniform temperature spread. In addition, the system uses dynamic ramping to ensure that all of the wells in a gradient PCR protocol reach the desired temperature simultaneously (Figure 1). This feature enables the production of uniform dwell times across all of the temperatures, removing a serious variable that could otherwise adversely affect the efficiency of the multiplex PCR optimization.
The above features are essential for rapid and precise protocol optimization for multiplex PCR development. In complex applications, such as in multiplex reactions, a delicate balance of primer sets, annealing temperature and dwell times needs to be achieved. Alteration in any of these parameters affects the result, with the loss or gain of the target products; the larger the size of the target products, the more delicate this balance. Altering the dwell times shifts the pattern of the bands, and clearly identifies the correct conditions for a given set of primers (Figures 3 and 4).
Accurate temperature control Reduced dwell times and improved performance are possible using the MBS iStation's method of temperature control. Accuracy is achieved by an algorithm - Simulated Plate/Tube Control that reduces the time required to reach target temperatures by using precise overshoots in block temperature. The overshoots occur at each set point and in the case of a gradient step all at the same time. This results in all the samples (not just the block) being at the set temperatures for the user-defined, programmed time period. These controls, in conjunction with precise gradient blocks, provide the temperature accuracy needed for annealing gradients at reduced cycling dwell times to enable robust multiplex PCR to be achieved.
 Figure 2. The block overshoot and temperature control ensures that the sample reaches the target temperature quickly, and that the sample remains at the set temperature for the specified dwell time. Click to enlarge. |
Efficient optimization of 10 different target sequences in one PCR reaction
Amplification. Multiple primer sets were used simultaneously to amplify ten different loci with product sizes of 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 bp, 50% GC (
4%) from a bacteriophage Lambda (48,502 bp) template. An MBS iStation 0.2 ml Gradient Block thermal cycler was used with an annealing gradient from 52 to 67 C with different MgCl2 concentrations and dwell times.
Reaction conditions. 20 l reactions in Omnifast 96-well skirted microplates with OmniTube strip domed caps, Lambda DNA (1,000 copiesl), primers (1 M), Optimized Buffer (1 X), MgCl2 (1.5, 2.0, 2.5, 3.0 and 3.5 mM), dNTP's (0.2 mM), Taq polymerase (0.05u/?l). 50 mM KCl, 10 mM Tris-HCl (pH 9.0 room temp.), 5%DMSO, 0.1%BSA.
Gel electrophoresis. PCR products were separated on a 4 mm Agarose Gel (0.5 X TBE/1.2% PCR Grade Agarose, and 0.5 g/ml Ethidium Bromide Solution). 5 l of each sample was loaded into the gel and the gels were run at 96V for 2 hours using a Thermo Classic Submarine Chamber and Electrophoresis Power Supply.
Results and discussion
Optimal annealing conditions for a 10 product multiplex reaction were quickly identified using the gradient function. By reducing dwell times, the effective optimal annealing range was increased, while overall cycle times and the production of non-specific product were both decreased. To perform a successful multiplex reaction, the best thermal conditions, i.e., temperature and time, need to be identified for any particular product target mix.
 Figure 3. Annealing gradient. Standard PCR Protocol 1: Total dwell time to hold step: 56 minutes {94 C/1min., (94 C/30sec., 52 to 67 C/30sec., 72 C/30sec.), 30 cycles, 72 C/10min., 4 C/Hold} END.A) Primer dimer. B) Second level of primer dimer and/or unincorporated primers. C) Optimum annealing range from 64.6 C to 66 C. D) The slower reaction is less robust with significant band dropout over the majority of the annealing temperature range. | |  Figure 4. Annealing Gradient. PCR Protocol 2 (Optimized): Total dwell time to hold step: 27.8 minutes {98 C/20sec., (94 C/5sec., 52 to 67 C/10sec., 72 C/20sec.), 30 cycles, 72 C/10min., 4 C/Hold} END. A) Primer dimer. B) Second level primer dimer and/or unincorporated primers are eliminated. C) Optimum annealing range from 54.8 C to 56.5 C. Nonspecific product above 1000 bp was not observed. D) The faster reaction is more robust, with less band dropout over the annealing temperature range. | |
Altering the annealing temperature alone will not result in identifying the most optimal conditions for a multiplex reaction, especially if the products are of differing size. The larger the product, the longer the time required for products to replicate. However, by increasing the dwell times, the chances of amplifying both primer, dimer and non-specific products also increases. This can all clearly be seen when comparing Figures 3 and 4. In both instances, the gradient temperature range used was identical. The only differences were in the dwell times.
Both figures show changing banding patterns as the annealing temperatures rise from 52 C to 67 C. In Figure 4 however, where the dwell times were reduced, a reduction in the primer/dimer formation (highlighted by B) and nonspecific product amplification above 1000 bp can be observed. In addition, the robustness of the amplification can be seen to increase with the reduced dwell times, as the temperature range where all products can be observed is broader in Figure 4 than Figure 3. Conversely, in Figure 3 there is greater product dropout over the majority of the temperature range. In conclusion, complex multiplex reactions can be optimized on the MBS iStation gradient thermal cycler. Optimization, however, requires the alteration of both annealing temperature and the dwell times. This is possible on the MBS, as the annealing temperature can have user-defined dwell times, even when using the gradient feature, due to the dynamic ramping.
About the author
All enquiries should go through David Briggs PhD, Product Manger, Molecular Biology Thermo Electron Corp. David.Briggs@thermo.com.
Thermo Electron Corp.
www.thermo.com
References
1. Chamberlain, J.S., Gibbs, R.A., Rainier, J.E., Nguyen, P.N., & Caskey, C.T. Nucleic Acids Res. 16:11141 (1988).