Optimization of PCR

Optimization of PCR 4.1 Introduction Depending on the success of your PCR amplification it may be necessary to optimize the conditions. This Chapter deals with various aspects of PCR optimization including reagents, temperatures, enhancers and preventing contamination. There is also a troubleshooting guide that will hopefully help you identify the source of any problems. Control reactions It is important to perform control reactions in parallel with the test samples to indicate whether any specificity (Section 4.2) or contamination (Section 4.5) problems exist. At least two controls are essential, a reaction contain- ing no DNA and one containing no primers. You should think of the control reactions as being just as important as your test samples and there are times when you may wish to include more controls. For example, if you are beginning to work with a new pair of primers, it is a good idea to include controls containing single primers. In this way you can see whether any products are generated from either of the primers alone, rather than by the two working in combination. 4.2 Improving specificity of PCR Primer pairs do not all work under the same reaction conditions. In some cases under ‘standard’ conditions one pair of primers will work very efficiently and give rise to a unique product in large amounts. At the same time, and under identical reaction conditions with the same template DNA, another primer pair will give rise to either no product, or to a complex pattern of extraneous products. Even more perplexing, some- times you can take one primer (primer A) that you know works well with primer B, but when you use it in combination with a new primer C, the PCR fails. The rules governing the operating characteristics of a primer pair are not defined. In essence the strategy that is usually followed for a new primer pair is to start with ‘standard’ amplification conditions (such as Protocol 2.1). If the PCR is not optimal then one of the reaction parameters should be changed to increase or decrease the stringency of the reaction conditions appropriately. If no bands are detected then the stringency may be too high whilst if several bands are seen then the stringency should be increased. How do you set about this optimization task? Are there standard rules or is it an empirical ‘hit-and-miss’ process? The answer lies somewhere between these two extremes. The most important parameters that will 4 influence reaction specificity are the annealing temperature, the cycling regime and the buffer composition. High specificity in PCR is favored by: ● optimal concentration of Mg 2+ , other ions, primers, dNTPs and DNA polymerase; ● efficient denaturation, high annealing temperatures and fast ramping rates; ● touchdown PCR; ● hot-start PCR; ● booster PCR; ● limiting the number of cycles and their length; ● thermal cycler efficiency; ● PCR additives; ● template quality (Section 4.3); ● nested PCR (Section 4.4). Magnesium ions As you already know (Chapter 3) the concentration of magnesium ions (Mg 2+ ) is critical. It exists as dNTP-Mg 2+ complexes that interact with the sugar-phosphate backbone of nucleic acids and influence the activity of the DNA polymerase. So, altering the concentration of MgCl 2 can lead to one primer/template pair behaving significantly differently from another under identical conditions. The usual strategy for assaying the effect of Mg 2+ ion concentration is to adjust the standard buffer so that the MgCl 2 concen- tration varies between 0.5 and 5 mM, usually in steps of 0.5 or 1 mM. Often one concentration will show a significantly improved PCR product pattern. Remember also that you will change the Mg 2+ concentration if you alter the concentration of dNTPs in the reaction (Chapter 3). If the Mg 2+ concentra- tion is too low then yields are likely to be poor while excess Mg 2+ can reduce the fidelity of Taq DNA polymerase and lead to amplification of nonspecific products. Other ions A study by Blanchard et al. (1) has gone some way towards standardizing buffer conditions and variations that may lead to rapid optimization of PCRs. They used a set of buffers called TNK that contain Tris-HCl (pH 8.3), ammonium chloride (NH 4 Cl), potassium chloride (KCl) and magnesium chloride (MgCl 2 ) and analyzed the effects of varying the concentrations of these buffer components. Interestingly, they found that potassium and ammonium ions, which in many biological systems behave interchange- ably, gave opposite effects in the PCR. Increasing KCl leads to a reduced stringency by affecting the melting characteristics of DNA by neutralizing the negative charge of the phosphate groups of the backbone so that the hydrogen bonding between bases becomes more important. Indeed at very high KCl concentrations (>0.2 M) this stabilizing effect becomes so pronounced that the DNA strands will not denature at 94°C and therefore no PCR can occur. 66 PCR DNA polymerase concentration If you have no product band(s) or weak band(s) you may have too little DNA polymerase. Different versions of thermostable DNA polymerase can be supplied at a variety of specific activities and concentrations. To check whether this is the problem you can perform a titration with varying amounts of DNA polymerase. Thermostable proofreading DNA polymerases can have lower processivity than Taq DNA polymerase, affecting yield, and so more enzyme may be needed for successful amplification. It is also important to remember that thermostable polymerases will become inacti- vated at high temperatures and this can lead to reduced levels of product. So, try to limit the time the enzyme spends above 90°C by using a short denaturation time at 94°C, say 15 s, or a lower denaturation temperature of 92°C rather than the 94°C recommended in many protocols. Temperatures Denaturation It is important that the template is efficiently denatured in order to provide single-stranded templates for PCR. This is achieved during the initial, usually 5 min denaturation phase when the sample is heated to around 94°C. If this step is inefficient then partially denatured duplex molecules will rapidly reassociate to prevent efficient primer annealing and DNA extension. For GC-rich templates it may be necessary to increase the temperature of this step to, for example, 96°C. However, it is not clear that such an extended time is required for many applications. With the exception of GC-rich templates, or where you are using a hot-start enzyme, the time could probably be reduced to 1 or 2 min. This would have the additional benefit of extending the useful life of the thermostable DNA polymerase. At the start of each cycle there is a shorter denaturation step that should denature the PCR products for subsequent reaction. While many protocols use a 94°C step here, for many templates it may be sufficient to use a temperature of 90–92°C, although GC-rich ones may require a higher temperature. It is useful to try to use the lowest effective temperature for the shortest effective time in order to retain the highest DNA polymerase activity in the reaction. Annealing The success of a PCR relies heavily on the specificity with which a primer anneals only to its target (and not nontarget) sequence so it is important to optimize this molecular interaction. Whether a primer can anneal only to its perfect complement, or also to sequences that have one or more mismatches to the primer, depends critically upon the annealing temperature. In general the higher the annealing temperature the more specific the annealing of the primer to its perfect matched template and so the greater the likelihood of only target sequence amplification. The lower the temperature, the more mismatches between template and primer can be tolerated leading to increased amplification of nontarget sequences. In practice it is often feasible to start at a temperature such as 55°C and assess Optimization of PCR 67 the success of your PCR. If there is poor recovery of product and a high background of nonspecific products then empirical determination of an optimal annealing temperature may be necessary, coupled with optimiza- tion of the MgCl 2 concentration (see above). It is also worth checking that the time for annealing is not too long. Generally about 30–60 s is reported in methods and the shorter the better. Since the polymerase will have some activity at the annealing temperature, the longer you hold the reaction at this temperature the increased risk there is of amplification of nonspecific products. Adjusting the annealing temperature step can alter the specificity of pair- ing between template and primer. If there is no product, the temperature may be too high and can be reduced, for example from 55°C to 50°C in the first instance. At the new temperature the primers may be more efficient. If there are products in control lanes where only one primer is present this indicates that the single primer is annealing to more than one region of the template and generating products. In this case you should increase the annealing temperature. As described in Chapter 3 thermal cyclers are now widely available that have a gradient block, allowing the simultaneous determination of optimal annealing temperature profiles in one reaction. By aliquoting a reaction premix into a series of tubes, the only variable should be the annealing temperature applied by the gradient block. If you do not have access to such an instrument an appropriate way to optimize primer/template annealing is to test by setting up PCR reactions and carrying out a series of experiments with 2–5°C adjustments of the annealing temperature. There are examples of two-step PCR where the primers can anneal to the template at 72°C thereby allowing cycling between the denaturation temperature and the extension temperature. Two-step PCRs are often performed for difficult PCRs such as amplification of large fragments from genomic DNA. Several approaches that rely upon temperature-based control of primer annealing have proven useful in improving the specificity of primer anneal- ing and therefore of amplification of the desired product. These are considered in the next two Sections. Touchdown PCR Touchdown PCR starts initially with an annealing temperature higher than the T m of the primers and then at each of the earlier cycles of the PCR the annealing temperature is lowered gradually to below the T m . This ensures that only specific annealing of the primers to their correct target sequence takes place before any nonspecific annealing events. A good rule of thumb, described by Don et al. (2), when using primers about 20 nucleotides in length, is to reduce the annealing temperature by 1°C every 2 cycles moving from 65°C to 55°C over the first 20 cycles. The reaction should then be completed by another 10 cycles at a 55°C annealing temperature. Since the first products to be made are specific products, this increases the concen- tration of true target sequences in the early stages of the PCR thereby enhancing the accumulation of true product as the amplification continues at a less specific annealing temperature. 68 PCR Hot-start PCR Even if you take great care in designing primers and in determining the most appropriate annealing conditions, specificity problems can arise even before the first cycle of PCR. How is this possible? Consider what happens when the various reagents and template are added to the PCR tube at room temperature or on ice and then placed in a thermal cycler to start the reaction. The tube may be left for some time before being placed in the thermal cycler. It is then heated up to 95°C in order to denature the template. However, during the time it is standing at or below room temperature, until it reaches a temperature of around 65 to 70°C, non- specific primer/template and primer/primer annealing events may occur to provide substrates for the DNA polymerase. Any products formed in this manner will be templates for subsequent amplification resulting in non- specific products and/or primer-dimers. The simplest way of avoiding such spurious priming events by enhancing correct primer annealing is by the use of a ‘hot-start’ procedure (3–5), which relies upon the physical separation of reagents until a high temperature has been reached. One or more reactant is omitted until the temperature of the reaction is above 70°C. The final reactant(s) can then be added to allow the reaction to proceed. There are various strategies for performing hot-start PCR; the cheapest procedure is to set up the complete reactions without the DNA polymerase and incubate the tubes in the thermal cycler to complete the initial denaturation step at >90°C. Then, while holding the tubes at a tempera- ture above 70°C, the appropriate amount of DNA polymerase can be pipetted into the reaction. But remember if you are using mineral oil you must put the pipette tip through the mineral oil layer first, so that the polymerase is introduced into the reaction rather than floating around on top of the oil. This approach can be used in a research laboratory where relatively small numbers of reactions are being performed. However, it is not suitable for processing large numbers of samples due to: ● the time involved in making additions of enzyme to individual tubes; ● the ‘loss-of-concentration’ phenomenon leading to failure to add enzyme to one or more tubes; and ● the opportunity for contamination due to the need to open the tubes (Section 5). Various commercial reagents are now available to facilitate hot starts and such products are recommended for routine hot-start applications. Some examples are given below. Inactive DNA polymerase Probably the most common approach used for hot start is DNA polymerase whose polymerase and in some cases 3′→5′ exonuclease activity has been inhibited by the physical binding of inactivating monoclonal antibodies that prevent it reacting with substrates (Figure 4.1). This allows all the reaction components to be mixed together in the absence of any polymerization. When the reaction reaches a high temperature the anti- Optimization of PCR 69 body(s) denatures thereby releasing the thermostable DNA polymerase in an active form, allowing polymerization (Figure 4.1). There are many DNA polymerases of this type sold by a range of companies. These require sufficient time during the initial denaturing step to inactivate the anti- bodies, but usually this is achieved by a 5 min soak at 94°C. Even if it is not fully activated during this step, it will activate during thermal cycling at each denaturation step during the early cycles of a PCR. Hot-start procedures are most useful when low concentrations of a complex template, such as genomic DNA, are being used. However, artefactual amplifications can occur in any reaction and it is generally recommended that all PCRs should be performed under a hot-start procedure. Wax beads The principle of wax beads, such as Ampliwax (Applied Biosystems) or DyNAwax (Finnzymes), is to physically separate some reaction components until the entire reaction has reached a high temperature, where mispriming events will not occur. As illustrated in Figure 4.2, some reactants, such as buffer, dNTPs, primers, template DNA and Mg 2+ , are placed in the reaction tube. A wax bead is added and the reaction incubated in the thermal cycler at 75–80°C for 5–10 min to melt the wax. The tube is then cooled to below 35°C to allow the wax to solidify and form a barrier layer above the initial reactants. The thermostable DNA polymerase can then be pipetted on to the wax layer and the PCR cycling started. As the temperature rises the wax melts and the enzyme becomes mixed with the other reactants to initiate the PCR while the wax rises to the surface. The wax layer has the added 70 PCR Add DNA polymerase/MAb complex As temperature reaches >70°C antibody denatures and activates polymerase DNA polymerase remains inactive due to antibody inhibition Active DNA polymerase released when antibody denatures so PCR is initiated Figure 4.1 Hot-start PCR using a thermostable DNA polymerase-inactivating antibody complex. The antibody sterically blocks the enzyme active site preventing the DNA polymerase from functioning until the antibody is denatured at high temperature. benefit of providing a barrier against evaporation during thermal cycling, in place of mineral oil. It also serves as a physical barrier to protect samples from contamination during subsequent storage and processing. After PCR when the tubes cool the wax solidifies and samples can be taken by insert- ing a pipette tip through the wax layer. It may be possible to use an alternative source of paraffin wax such as that from Sigma-Aldrich which melts at 53–56°C. Taq Bead™ hot-start polymerase These small spherical beads supplied by Promega comprise wax encapsu- lating Taq polymerase that is released when the reaction reaches 60°C. Unlike the wax beads above the volume of wax is small and does not form a physical barrier above the reaction solution. The beads are suitable for use in either standard or heated-lid thermal cyclers, but for the former addition of a mineral oil overlay is necessary. Optimization of PCR 71 Add wax bead Add dNTPs, buffer, DNA, MgCl 2 Wax layer 75–80°C 5–10 min to melt wax Cool below 35°C to reset wax Add DNA polymerase Cool below 35°C, wax resets Thermal cycling Wax layerReagents mix during first cycle Figure 4.2 Hot-start procedure using wax beads. Some reagents are added to the tube before a wax bead is added and melted. Once the wax has solidified to form a barrier over the reactants, the missing reagents are pipetted onto the wax layer. When the wax layer melts during the first heating step of the PCR all the reagents become mixed and the reaction is initiated. Magnesium wax beads The presence of magnesium is essential for DNA polymerase activity. PCR reaction mixes set up in magnesium-free buffer can be activated at around 70°C when a StartaSphere™ wax bead (Stratagene) melts, releasing the correct amount of magnesium. These beads are small and nonbarrier- forming and so are compatible with heated-lid thermal cyclers. Mineral oil should be added for a non-heated lid thermocycler. Booster PCR The appropriate choice of annealing conditions allows primers to efficiently identify their complementary sequences when reasonable concentrations of DNA are being used, for example 1 µg of human genomic DNA (around 3 × 10 5 template molecules). However, at very low template concentrations, perhaps less than 100 molecules, the interactions between primers and template become less frequent. Instead there are more significant inter- actions between primers themselves, which can lead to primer-generated artifacts such as primer-dimers (Chapter 3). To enhance the specificity of template priming at low DNA concentrations a procedure called booster PCR can be employed (6). This involves performing the first few cycles of PCR at low primer concentration, so that the molar ratio of primer:template is around 10 7 –10 8 , the level normally found in a PCR (see Table 2.2). This enhances specific priming events and subsequently the concentration of primers can be ‘boosted’ during the amplification phase to maintain the 10 8 ratio of these reactants. Cycle number and length In general the number of cycles of PCR should be kept to the minimum required to generate sufficient product for further analysis or manipulation. This reduces the likelihood of errors arising and of nonspecific products accumulating. If the basic protocol (Protocol 2.1) does not yield sufficient product you could try to increase the amount of template in the first instance. Alternatively the number of cycles of PCR could be increased. It is possible to sample PCRs by removing an aliquot such as 0.1 vol (5 µl of a 50 µl vol) every 5 cycles at 25, 30 and 35 cycles during a 40-cycle reaction. The samples can then be analyzed by agarose gel electrophoresis to allow the appropriate number of cycles to be determined. This number will be the minimum number that gives good yield of a single product. Another consideration that can influence PCR specificity is the time taken to move between temperatures during PCR cycling (7). Generally the faster the ramping rates the higher the specificity and the faster the reactions are completed. Some instruments can now achieve ramp rates of up to 2.5°C per s –1 . Thermal cycler efficiency It is easy to forget that instruments may malfunction. If your PCRs begin to fail then you should ask whether the thermal cycler is reaching the 72 PCR correct temperatures. Newer instruments have self-diagnosis features that can identify problems. As thermal cyclers get old they can tend to become less accurate in terms of their temperature profiles and often need adjust- ments. It is therefore worth using an independent temperature monitoring system occasionally, and certainly if variability in standard PCR results occurs. Temperature verification systems are available but are often very expensive. A simple and inexpensive temperature monitoring system can be made from a thermocouple, placed in a reaction tube containing water, and a digital thermometer. Even less expensive is to use a thin digital thermometer that fits inside a reaction tube filled with water. The temperature reached by the thermal cycler during PCR cycling can easily be monitored. In addition the system allows any temperature variation across the block to be assessed. PCR optimization and additives It is possible to purchase optimization kits that comprise a variety of buffers and additives to optimize conditions for PCR. For example, Stratagene produce an Opti-Prime™ PCR optimization kit comprising 12 different buffers and 6 additives, allowing a range of buffer conditions to be tested. Once optimized conditions have been determined the appropriate buffer can be purchased separately. Epigene also produce a Failsafe PCR optimization kit comprising a range of buffers. Various ‘enhancer’ compounds have also been reported to improve the specificity or efficiency of PCR. These include chemicals that increase the effective annealing temperature of the reaction, DNA binding proteins and commercially available reagents. Such additives can be added to PCRs to enhance primer annealing specificity, reduce mismatch primer annealing and improve product yield and length. Additives that lead to a destabiliza- tion of base pairing can improve PCR particularly from difficult templates such as GC-rich sequences and may also increase specificity by their relatively greater destabilization of mismatched primer–template complexes. Although these compounds can be useful in some circumstances to improve suboptimal PCR conditions, some are not applicable to a wide range of templates and primer combinations. There is no ‘magic’ additive that will ensure success in every PCR and it may be necessary to test different additives under different conditions, such as annealing temperature. Such testing has been made easier with the advent of gradient thermal cyclers that allow the automatic testing of different annealing temperatures (Chapter 3). Compounds that have been added to PCR reactions include: ● dimethyl sulfoxide (DMSO), up to 10% (8); ● formamide at 5% (9); ● trimethylammonium chloride 10–100 µM (10); ● betaine (N,N,N-trimethylglycine) 1–1.3 M. A useful study and primary references are provided in Promega Notes http://www.promega.com/ pnotes/65/6921_27/6921_27_core.pdf; ● nonionic detergents (11) such as Tween® 20 at 0.1–2.5%; ● polyethylene glycol 6000 (PEG) 5–15%; ● glycerol 10–15%; Optimization of PCR 73 ● single-stranded DNA binding proteins such as Gene 32 protein (Amersham Pharmacia Biotech) added to 1 nM or E. coli single-stranded DNA binding protein at 5µM; ● 7 deaza-dGTP to reduce the strength of G–C base pairs; it is used at 150 µM with 50 µM dGTP as the G nucleotide mix; ● Taq Extender™ (Stratagene) increases Taq DNA polymerase DNA extension capacity leading to a greater proportion being fully extended. This is due to a reduction in the mismatch pausing when Taq DNA polymerase is dissociated from the template; ● Perfect Match® PCR Enhancer (Stratagene) apparently destabilizes mis- match primer template complexes where there are several mismatches close to the 3′-end. Perfect or near-perfect matched primer–template complexes including those with nonhomologous 5′-ends or tails are not destabilized and therefore generate good yields of product; ● Q-solution (Qiagen) modifies the melting behavior of template DNA, is used at a defined concentration for any template–primer combination and is not toxic. The two additives that are probably most useful are DMSO, which disrupts base pairing and is usually added to 5–10% (v/v), and betaine (~1 M), which equalizes contributions of GC and AT base pairs towards duplex stability. It is advisable to adjust the denaturation, annealing and extensions temperatures down by perhaps 2°C when using betaine to adjust conditions for the weakening of the duplex bonding interactions and enzyme stability. In an interesting study undertaken by Promega they used NMR to analyse the constitutents of two commercially available PCR enhancer solutions and discovered that they were solutions of betaine (see http://www.promega.com/ pnotes/65/6921_27/6921_27_core.pdf). An example showing the effect of DMSO addition is shown in Figure 4.3. 74 PCR M1 2 3 4M –DMSO + DMSO 7 kb Figure 4.3 Agarose gel showing the effect of DMSO on PCR amplification. Lanes 1 and 2 show PCR amplification without DMSO, while lanes 3 and 4 show PCR amplification with 5% DMSO (final concentration). Lanes 1 and 3 are performed at 60°C annealing temperature and lanes 2 and 4 are performed at 58°C annealing temperature. (Provided by Dr Luis Lopez-Molina, Laboratory of Plant Molecular Biology, Rockefeller University.) M12 3 4M −DMSO +DMSO 7 kb [...]... for PCR optimization BioTechniques 21: 134–140 Dieffenbach CW, Dveksler GS (1993) Setting up a PCR laboratory PCR Methods Appl 3: S2–7 Optimization of PCR 85 Dragon E (1993) Handling reagents in the PCR laboratory PCR Methods Appl 3: S8–9 Roux KH (1995) Optimization and troubleshooting in PCR PCR Methods Appl 4: S185–194 References 1 Blanchard MM, Taillon-Miller P, Nowotny P, Nowotny V (1993) PCR. .. effect of dilution of the DNA sample to enhance specificity of PCR DNA concentrations are shown in ng If the DNA sample contains a contaminant then diluting the sample can lead to dilution of the contaminant and successful amplification of the product In this case the product can be seen only in the lowest dilution of the sample containing 50 ng DNA 76 PCR The variety of DNA sources demands a variety of. .. product The second round of amplification uses a pair of nested primers that lie within the target region, leading to the amplification of only the correct product M; molecular size markers PCR1 and PCR2 ; amplified product(s) from the first PCR and the nested PCR respectively PCR is prone to contamination from DNA molecules present in the laboratory environment There are some major sources of DNA contamination... pretreatment of the reactant mix prior to PCR Such a practice is particularly critical in diagnostic laboratories where routine amplification of identical products is being performed on a daily basis and the possibility of cross-contamination is omnipresent, but must be avoided to prevent false positives and misdiagnosis Optimization of PCR 81 The resulting PCR products contain dU in place of dT and will... and failure to identify true product M PCR1 Smear of products prevent identification of true product Nested PCR with internal primers only amplifies from the true product and not from nonspecific products M PCR2 Single nested product band obvious Figure 4.5 Schematic illustration of nested PCR from genomic DNA The first-round PCR amplification results in a smear of nonspecific amplification products masking... It has also been reported that incorporating dUTP in place of dTTP during primer synthesis can prevent carryover contamination Treatment of the PCR products with UNG will destroy the original primer regions of the PCR product thereby preventing amplification of the full-length product during a subsequent PCR (13) UV irradiation UV treatment of reaction components by illumination at 254 nm has been used... template in a subsequent PCR The treatment involves addition of the isopsoralin at the start of the PCR and a post -PCR irradiation to modify the products to ensure they are unable to contaminate further PCRs 4.7 Troubleshooting guide This Section is designed to help identify possible causes for common PCR problems and to suggest solutions that may overcome the problem Generally if PCR does not work then... Increase the number of cycles Reactions can be sampled by removing an aliquot (0.1 vol i.e 5 µl of a 50 µl vol) every 5 cycles (at 25, 30 and 35 cycles) during a 40-cycle reaction to allow the appropriate number to be determined Optimization of PCR 83 Table 4.1 continued Problem Possible reasons Things to try No PCR product Poor primer design No PCR product Template quality or quantity No PCR product Thermal.. .Optimization of PCR 75 4.3 Template DNA preparation and inhibitors of PCR PCR may be inhibited by a wide range of compounds derived from the biological specimens or method and reagents used to extract the DNA Typical biological samples used for PCR are animal tissues and bodily fluids, including peripheral blood cells, urine,... face with your PCRs Table 4.1 PCR troubleshooting guide Problem Possible reasons Things to try No PCR product One or more component missing or faulty No PCR product Insufficient number of cycles Use a checklist to ensure all components are added to tubes Check the concentrations of all reagents, including primers, template, dNTPs, MgCl2 and buffer If possible use a premix containing most of the reagents . preparation. Optimization of PCR 75 2 kbp M 50 ng 100 ng 200 ng Figure 4.4 The effect of dilution of the DNA sample to enhance specificity of PCR. DNA concentrations. Touchdown PCR Touchdown PCR starts initially with an annealing temperature higher than the T m of the primers and then at each of the earlier cycles of the PCR