The optimization of DIAPOPS is closely related to the optimization of the PCR reaction itself (
). 1. Introduction
The optimization of DIAPOPS is closely related to the optimization of the PCR reaction itself ( ).
Generally, a well-performing standard PCR will also perform well with the same parameters in DIAPOPS.
Normally, all optimization performed to a obtain a standard liquid phase PCR which is both sensitive and specific will have the same effect on the DIAPOPS results, e.g. hot-start ( ), addition of substances ( ), such as glycerol or Tween, etc.. However, examples have been encountered where additional optimization was necessary in order to achieve optimal DIAPOPS results. At the Nunc A/S Research Laboratory, optimization of various parameters in the DIAPOPS procedure has been found to improve DIAPOPS results from systems which initially gave problems. The optimization either targets an improvement of the general amplification; both the liquid and the solid phase, or only targets the solid phase amplification and the hybridization detection of the solid phase product.
2. The importance of using a known template during the optimization
In the optimization of both standard PCR ( ) and DIAPOPS ( ), it is an advantage to use purified DNA in the same concentration as template for the amplification. If the experiments performed during the optimization are carried out using DNA of unknown purity and with different concentrations, results from individual experiments can not be compared. This is particularly important when the analyzed DNA contains residues that may be inhibitory to the heat-stable polymerase. Due to this, variable results can originate from variable concentrations of the inhibitory substances, and not because the parameters in the procedure are changed. To avoid variable results in the optimization period caused by different template concentrations or by inhibitory substances in the sample, a purified PCR product can be used as template in the initial optimization.
After the first optimization, a new optimization may be necessary for the analysis of crude and unpurified samples. This optimization focuses on removal of inhibitory substances or on the addition of other reagents in order to eliminate the inhibitory effect ( ). The advantage of having separate optimization steps is that two different parts of the procedure are optimized separately. In the first optimization step, optimal conditions for the DNA amplification are selected. In the second optimization, the fundamental optimal conditions are known, and the small changes necessary for optimal amplification of the crude sample preparation can be tested and selected. If the initial target DNA is not known and purified, both optimizations must be carried out simultaneously. This is not recommended as it is difficult to predict and interpret in which steps the altered parameters take effect during optimization.
3. Liquid phase PCR product analysis
As described in the Trouble-Shooting part of this Applications Guide ( ), the first thing to do if poor results with DIAPOPS are obtained is to analyze the liquid phase PCR product in an agarose gel ( ). The analysis should be of the liquid phase from the NucleoLink Strips giving the poor signal. If the product concentration on the agarose gel is normal and as expected, the problem is not to be found in the general amplification ( ). In this case, the optimization should focus only on the solid phase amplification or the detection by hybridization. If the liquid phase product concentration correlates with the hybridization detection signal and also yields poor results in the agarose gel, the problem lies with the general amplification.
3 a). DIAPOPS liquid phase PCR versus standard symmetric PCR
The DNA amplification in DIAPOPS is asymmetric ( ), in contrast to the symmetric DNA amplification in standard liquid phase PCR ( ). For this reason, the liquid phase DNA amplification in DIAPOPS may perform differently from the standard PCR liquid phase amplification. Normally, the liquid phase product concentration in DIAPOPS is not as high as the liquid phase product concentration in standard PCR. This is both due to the asymmetric amplification in DIAPOPS, and to the fact that some of the products formed in the liquid phase are removed from the liquid phase because they hybridize to the solid phase product during elongation.
In some systems using a long cycle time at each temperature (e.g. 1-2 min.) ( ), it has been observed that the liquid phase PCR product concentration from the NucleoLink Strips was lower than the concentration from the corresponding standard symmetric PCR. In other systems, multiple bands have been seen on the agarose gel from the asymmetric PCR in DIAPOPS, in contrast to the symmetric PCR where only the correct band was seen. The detection limit on the agarose gel from the asymmetric PCR with these systems was inferior to the standard symmetric PCR. Furthermore, although the samples with high initial template concentrations yielded high signals in DIAPOPS, the DIAPOPS signal decreased rapidly yielding a low or non-existent signal from samples with a lower initial template concentration. The correlation between the liquid phase PCR results and the DIAPOPS signals indicates that the problems were not in the solid phase amplification but rather with the general amplification. However, in all systems tested so far by the Nunc A/S Research Laboratory, it has been possible to optimize the DIAPOPS DNA amplification to give the same detection limit on agarose gel as standard PCR with 1:1 primer ratio in uncoated wells.
4. Cycle time of the PCR program
In the optimization of both standard PCR and DIAPOPS, the annealing temperature of the cycling program is a very important factor ( ). In standard symmetrical liquid phase PCR, the time the program stays at each temperature is normally not very critical. However, with the asymmetric DNA amplification used in DIAPOPS an optimization of these times may be necessary. A careful optimization of the cycle times may exclude the difference in liquid phase product concentration observed between the standard liquid phase PCR and DIAPOPS.
 Figure 1: DIAPOPS signals from a dilution series as a function of the cycle time of the PCR program. With long cycle times annealing and denaturation times were 2 and 1 min. respectively, while synthesis time was 1.5 min. In the program using short times in each cycle the denaturation and annealing time were 20 sec, and synthesis time was only 10 sec. |
Figure 1 shows an example of the change in DIAPOPS signals as a function of the change in cycle times in the PCR program. It is clear that the signal both improves the dynamic range and the limit of detection. In most systems tested, the asymmetric PCR in coated NucleoLink Strips still yielded a lower concentration of liquid phase PCR products compared with symmetric PCR, but there was no difference in the limit of detection. The detection limit and the dynamic range in the DIAPOPS detection of the solid phase products are generally better than for the corresponding standard PCR where the PCR products are detected on an agarose gel.
4 a). Different optimal cycle times in separate systems
As the DIAPOPS results were improved when the cycle times were reduced in the system used in Figure 1, this approach was also tested in other systems. The very short cycle times of 10-20 seconds at each temperature, ideal for the system presented in Figure 1, were not ideal in all systems. Normally, 20-40 seconds yielded the highest performance, but most systems could be improved by individual optimization of these cycle times. The difference in optimal cycle time at each temperature is probably caused by the difference in annealing temperature and product length. In the system presented in Figure 1, an annealing temperature of 40ºC was used. As it takes longer time for the thermal cycler to reach this low annealing temperature of 40ºC, the NucleoLink Strips are kept for a longer time at lower temperatures. If a system utilizing a higher annealing temperature is only allowed 10-20 seconds at this temperature, the primers may not have sufficient time for an efficient hybridization. Therefore, longer cycle times are required for higher annealing temperatures. Furthermore, the length of the product is important. A very short product only needs a short elongation time to be completely synthesized. If the product is longer, longer time for elongation is necessary.
5. BSA blocking of coated NucleoLink Strips before addition of PCR mix
The NucleoLink surface is designed to covalently bind DNA with a high efficiency ( ). However, other substances such as proteins can also adsorb to the surface. After covalent binding of solid phase primers, the wells are washed with NaOH containing Tween ( ). It is expected that Tween will block the surface and prevent further adsorption of any substances. However, when the wells are further blocked with BSA before PCR, there is an increased level of amplification ( ). The lower level of amplification seen in NucleoLink Strips not blocked with BSA is probably caused by an adsorption of PCR enzyme, e.g. Taq polymerase, to the surface of the wells. The most likely explanation is that the blocking effect by Tween is decreased by the heat treatment. When BSA is used as a complimentary blocking agent, less enzyme will adsorb to the surface, and the amplification rate increases. The pre-PCR blocking ( ) is now included in the standard DIAPOPS procedure ( ).
6. Primer ratio in the PCR mix for the asymmetric DIAPOPS amplification
DIAPOPS utilizes an asymmetric amplification ( ) with different concentrations of the two primers in the liquid phase of the PCR. A symmetric DNA amplification with a primer ratio of 1:1 will cause a low efficiency of the solid phase DNA amplification, and only yield low DIAPOPS signals.
 Figure 2: DIAPOPS signals from a dilution series as a function of the primer ratio. There is a raised background signal ( ) in one of the blank samples. but in this case a PCR product band was observed on the agarose gel. For this reason, it was concluded that the high background value was due to a carry-over contamination ( ) with either old PCR product or with plasmid template. |
In the system presented in Figure 1, optimization of the primer ratio was further tested. Normally, the optimal primer ratio in DIAPOPS 1:8 (primer 1 : primer 2) when primer 1 is selected as the solid phase primer. However, with this system a significant improvement was observed, when the ratio was altered to 1:16, as presented in Figure 2. This ratio is now used with this system. As illustrated with this example, the optimal primer ratio may differ from one system to the other. Therefore, an experiment where different primer ratios are tested should be carried out for each new system. This experiment should include a primer ratio of 1:8, which is most likely to be optimal.
 Figure 3: DIAPOPS signals as a function of primer ratio. The data are not from the system presented in Figures 1 and 2. Results from the test with a primer ratio of 1:16 are not included in the figure. The ratio of 1:16 was tested in another experiment, and showed the same tendency as 1:32 and 1:64, being less dynamic than the corresponding ratio of 1:8. |
Figure 3 illustrates the results from a primer ratio optimization experiment in a DIAPOPS system different from the one used in Figures 1 and 2. The decrease in DIAPOPS signals observed in Figure 3 with primer ratios of 1:32 and 1:64 is caused by a decrease in the efficiency in the liquid phase DNA amplification because the solid phase amplification is closely correlated to the concentration of liquid phase amplification ( ). This decrease in liquid phase PCR product concentration is caused by the suboptimal primer ratio. Therefore, the decrease in DIAPOPS signals is not observed because the solid phase amplification is less efficient, but because the liquid phase amplification is influenced by the difference in concentration between the two primers. In the system illustrated in Figure 3, the optimal primer ratio is 1:8.
7. Selection of the solid phase primer
The first step of the DIAPOPS procedure is to select the solid phase primer ( ). If the two primer sequences are optimally selected ( ) i.e. with minimal primer-dimer formation and minimal self-hybridization, both primers can equally well be selected as the solid phase primer. In that case the selection of the solid phase primer could be based on the probe. If a probe has previously been selected, the strand the probe recognizes is the obvious choice for the solid phase strand, and the primer initiating this strand should be selected as the solid phase primer.
7 a). Primer-dimer formation of the solid phase primer
Primer-dimers are formed when the two primers are able to hybridize to each other during the amplification ( ), and the hybrid forms a substrate for the polymerase ( ). Formation of primer-dimers will inactivate the primers, and the primers should be selected with a minimal possibility of primer-dimer formation ( ). However, it is not always possible to select optimal primers ( ). The solid phase amplification is more sensitive to primer-dimer formation compared to the liquid phase PCR due to the lower amount of primers on the surface ( ). For this reason, the solid phase primer should be selected as the primer with the least possibility of forming primer-dimers ( ).
Contrary to expectation, it has been observed that the primer most likely to form primer-dimers in some systems are the best selection for solid phase primer. This is presumably because the concentration of the selected solid phase primer in the liquid phase is the lowest of the two primers, and thereby reducing the primer-dimer formation. This effect has been seen in only two systems and therefore, the explanation is therefore only presumptive.
Since there are two theories pointing in opposite directions concerning the selection of the solid phase primer, both primers should be tested in new systems where primer-dimer formation is likely.
8. A linker in the 5'-end of the solid phase primer
In the amplification part of the DIAPOPS procedure, the solid phase primers are elongated ( ). For this to happen, the solid phase primer must hybridize to a PCR product synthesized in the liquid phase. To achieve the most efficient hybridization, the solid phase primer should be maximally extended into the liquid phase and be as far as possible from the solid support. This can be done by adding a linker to the 5'-end of the solid phase primer before covalent binding ( ). Linkers of thymidine (T) * have been successfully tested ( ). When the linker is of 10 T's or more, a good solid phase detection is achieved ( ). It is very strongly recommended that the solid phase primer should be made with a linker of at least 10 T's in the 5'-end of the primer sequence ( ). Furthermore, it is very important that the solid phase primer is phosphorylated in the 5'-end of the linker ( ).
9. Hybridization detection of the solid phase PCR product
The solid phase PCR products are detected with a labelled hybridization probe ( ). The ability of this probe to recognize the solid phase product depends, in addition to the sequence, upon the hybridization and washing conditions. These include temperature, salt concentration, pre-hybridization and blocking, and concentration of other added reagents. In general, the method described in the DIAPOPS procedure ( ) will be optimal for most probes with a length of 17-25 bases. However, it is not essential to use the described hybridization procedure. In general, every efficient hybridization procedure will also be efficient with DIAPOPS.
If false positive amplicons with a high degree of homology to the true positive probe sequence can be synthesized, there is a possibility that the probe can hybridize to the false product. If this is the case, the washing conditions after hybridization should be optimized so that only the true positives are recognized. This can be achieved by e.g. lowering the salt concentration from 0.5 x SSC to 0.1 x SSC or increasing the temperature by 5ºC.
In order to increase the DIAPOPS signals it was demonstrated with one system that the use of an additional probe not only doubled the signal, but enhanced it to an even greater extend. This synergetic effect can be used to optimize detection. The use of two probes will always give a better signal, and should be used if there is a possibility of finding more than one appropriate probe in the sequence. It has not been tested whether more than two probes will further enhance the signal. Theoretically, it should be feasible, and should yield a positive effect in both the level of DIAPOPS signals and in the limit of detection.
10. Selection of enzyme/substrate combination for detection of the solid phase product
A correct selection of the substrate/enzyme combination for the detection of the label on the probe hybridized to the solid phase products can greatly enhance the DIAPOPS results. The most important feature is the sensitivity of the substrate. The two enzymes tested at the research laboratory of Nunc A/S are horse radish peroxidase (HRP) ( ) and alkaline phosphatase (AP) ( ).
10 a). Substrates
One of the most sensitive substrates for AP is 4-Methylumbelliferyl phosphate (4-MUP) ( ). The product of the phosphatase reaction - 4-MU (4-methylumbelliferyl), is a fluorescent compound, consequently a fluorescence plate reader is necessary when using this compound. ParaNitroPhenyl Phosphate (pNPP) ( ), is another substrate for AP. An overnight incubation is necessary, when pNPP is used at a concentration of 1 mg/ml, in order to obtain results in DIAPOPS. However, it is possible to a higher concentrations of 10 mg/ml and get results after 30 min. of incubation at room temperature ( 
).
The compound ortho-PhenyleneDiamine (OPD) ( ) used with HRP is not sensitive, and is therefore not expected to perform well in DIAPOPS. A sensitive substrate for the peroxidase reaction is 3,3',5,5'-tetramethylbenzidine (TMB) ( ). This substrate forms a blue color during the reaction, but turns yellow when the enzymatic reaction is terminated by acid addition ( ). It often yields signals and signal to noise ratios equal to those obtained with 4-MUP ( ). The only problem with TMB is the requirement for addition of acid. After this addition, it is not possible to detect the products again by rehybridization ( ), as the acid cleaves the covalent bond.
11. Conclusion
Normally a good PCR will also perform well in DIAPOPS, but there are some differences primarily because the DIAPOPS utilizes an asymmetric PCR. In the first analysis with DIAPOPS, the experimental conditions may not be optimal. Optimization of these conditions may focus on two different factors: the general amplification and the specific solid phase amplification and detection. The optimization of the general amplification mainly focuses on the length of time at each temperature in the thermal cycling.
Optimization of the solid phase DNA amplification and detection of the solid phase PCR products focuses on the ratio of primers in the asymmetric amplification, the linker in the 5'-end of the solid phase amplification, the probe, and the selection of the correct enzyme/substrate combination.
*Use of a polythymidine linker in solid phase amplification is covered by patents owned by GenSet.