The ability of the polymerase chain reaction to amplify a single molecule means that trace amounts of DNA contaminants could serve as templates, resulting in amplification of the wrong template (false positives). Consider the points mentioned below to avoid PCR contamination from sources such as:
Laboratory Facilities
Sample Handling
Type of Thermal Cycler
A thermal cycler must, at a minimum, accurately and reproducibly maintain the three PCR incubation temperatures (see Thermal Cycling Profile for Standard PCR), change from one temperature to another ("ramp") over a definable time, arrive at the selected temperatures without significant over- or undershoot, and cycle between the temperatures repeatedly and reproducibly.
Note: Cycling conditions have to be adjusted depending on the respective thermal cycler or primer/template combinations.
Type of Reaction Tubes
The reaction tubes affect the rate at which heat transfers from the thermal cycler to the reaction mixture. Therefore, preferably use thin-walled reaction tubes that are designed for PCR and that fit precisely into the wells of the particular brand of thermal cycler you are using.
Template
The quality of the template influences the outcome of the PCR. For instance, large amounts of RNA in a DNA template can chelate Mg2+ and reduce the yield of the PCR. Also, impure templates may contain polymerase inhibitors that decrease the efficiency of the reaction.
Note: To get the purest template, always use a purification product specifically designed to purify DNA, such as the High Pure PCR Template Preparation Kit.
The integrity of the template is also important. Template DNA should be of high molecular weight. To check the size and quality of the DNA, run an aliquot on an agarose gel. When testing a new template, always include a positive control with primers that amplify a product of known size and produce a good yield.
The amount of template in a reaction strongly influences performance in PCR. The recommended amount of template for standard PCR is:
Low amounts of template, for example, <10 ng human genomic DNA, will require specific reaction modifications, such as changes in cycle number, redesign of primers, use of Hot Start, etc.
Primers
In most PCR applications, it is the sequence and the concentration of the primers that determine the overall assay success. For convenience, several primer design software programs are available. These can be used to ensure that the primer sequences have the following general characteristics:
Note: Optimal annealing temperatures are often higher than the Tm of the primers (approximately +5 to +10 °C) and have to be determined empirically. For both primers, the Tm should be similar.
Bases that do not hybridize to the template may be added at the 5´ end of a primer (e.g., for introducing restriction sites into the amplification product). Primer concentrations between 0.1 and 0.6 µM are generally optimal. Higher primer concentrations may promote mispriming and accumulation of nonspecific product. Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yields of desired product.
Note: For some systems, a higher primer concentration (up to 1 µM) may improve results. When testing new primers, always include a positive control reaction with a template that has been tested for function in PCR. This control shows whether the primers are working. The Human Genomic DNA from Roche is a good control template for evaluation of human primer sequences.
Choice of DNA Polymerase
The choice of a DNA polymerase can profoundly affect the outcome of PCR. For most routine PCR, Taq DNA Polymerase has long been the standard PCR enzyme. However, Taq DNA Polymerase has its limitations. For most assays, the optimum amount of thermostable DNA polymerase (or a blend of polymerases) will be between 0.5 and 2.5 units / 50 µL reaction volume. Increased enzyme concentrations sometimes lead to decreased specificity.
MgCl2 Concentration
Mg2+ forms soluble complexes with dNTPs to produce the actual substrate that the polymerase recognizes. The concentration of free Mg2+ depends on the concentrations of compounds that bind the ion, including dNTP, free pyrophosphate (PPi) and EDTA. For best results, always determine the optimal Mg2+ concentration empirically. The optimal Mg2+ concentration may vary from approximately 1 - 5 mM. The most commonly used Mg2+ concentration is 1.5 mM (with dNTPs at a concentration of 200 µM each). Mg2+ influences enzyme activity and increases the Tm of double-stranded DNA. Excess Mg2+ in the reaction can increase nonspecific primer binding and increase the nonspecific background of the reaction.
Deoxynucleotide Triphosphate (dNTP) Concentration
Always use balanced solutions of all four dNTPs to minimize polymerase error rate. Imbalanced dNTP mixtures will reduce Taq DNA Polymerase fidelity.
Note: For maximum convenience, a premixed, balanced mixture of dNTPs such as the PCR Nucleotide Mix may be added to the reaction mixture as a single reagent. In addition, PCR grade dATP, dGTP, dCTP, dTTP and a Deoxynucleoside Triphosphate Set, PCR Grade are available.
If you increase the concentration of dNTPs, you must also increase the Mg2+ concentration. Increases in dNTP concentration reduce free Mg2+, thus interfering with polymerase activity and decreasing primer annealing. For prevention of carryover contamination, a higher concentration of dUTP is usually used in place of dTTP (for details, see Preventing carryover contamination with uracil-DNA glycosylase). The final dNTP concentration should be 50 – 500 µM (each dNTP). The most commonly used dNTP concentration is 200 µM.
pH
Generally, the pH of the reaction buffer supplied with the corresponding thermostable DNA polymerase (pH 8.3 – 9.0) will give optimal results. However, for some systems, raising the pH may stabilize the template and enhance results.
Reaction Additives
In some cases, adding the following compounds can enhance the efficiency or specificity of PCR:
Initial Denaturation
It is essential to denature the template DNA completely. Initial heating of the PCR mixture for 2 minutes at +94 to +95 °C is enough to completely denature complex genomic DNA so that the primers can anneal to the template as the reaction mix is cooled. If the template DNA is only partially denatured, it will tend to "snap-back" very quickly, preventing efficient primer annealing and extension, or leading to "self-priming", which can lead to false-positive results.
Denaturation Step During Cycling
Denaturation at +94 to +95 °C for 20 – 30 seconds is usually sufficient, but this must be adapted for the thermal cycler and tubes being used. For example, longer times are required for denaturation in 500 µL tubes than in 200 µL tubes. If the denaturation temperature is too low, the incompletely melted DNA "snap-backs" as described earlier, thus giving no access to the primers. Use a longer denaturation time or higher denaturing temperature for GC-rich template DNA.
Note: Never use a longer denaturation time than absolutely required for complete denaturation of template DNA. Unnecessarily long denaturation times decrease the activity of Taq DNA Polymerase.
Primer Annealing
For most purposes, annealing temperature has to be optimized empirically. The choice of the primer annealing temperature is probably the most critical factor in designing a high specificity PCR. If the temperature is too high, no annealing occurs, but if it is too low, non-specific annealing will increase dramatically. Primer-dimers will form if the primers have one or more complementary bases so that base pairing between the 3´ ends of the two primers can occur.
Primer Extension
For fragments up to 3 kb, primer extension is normally carried out at +72 °C. Taq DNA Polymerase can add approximately 60 bases per second at +72 °C. A 45-second extension is sufficient for fragments up to 1 kb. For extension of fragments up to 3 kb, allow about 45 seconds per kb. However, these times may need to be adjusted for specific templates. For improved yield, use the cycle extension feature of the thermal cycler. For instance, perform the first 10 cycles at a constant extension time (e.g., 45 seconds for a 1 kb product). Then, for the next 20 cycles, increase the extension time by 2–5 seconds per cycle (e.g., 50 seconds for cycle 11, 55 seconds for cycle 12, etc.). Cycle extension allows the enzyme more time to do its job, because as PCR progresses, there is more template to amplify and less enzyme (due to denaturation during the prolonged high PCR temperatures) to do the extension.
Cycle Number
In an optimal reaction, less than 10 template molecules can be amplified in less than 40 cycles to a product that is easily detectable on a gel stained with ethidium bromide. Most PCRs should, therefore, include only 25 to 35 cycles. As cycle number increases, nonspecific products can accumulate (Figure 1).
Figure 1.Effect of excessive cycling on impure and pure templates. A PCR product (245 bp amplicon from exon 6 of the dopamine 2 receptor gene) was reamplified in a series of reactions. In one set of experiments, the template was not purified before it was used. In the second set, the template was purified by agarose gel electrophoresis before reamplification. In both sets, the template was amplified for either 40, 60, or 72 cycles. Aliquots (8 µL) of the products were analyzed on a 3% agarose gel.
MWM: Molecular Weight Marker
40, 60, and 72: Number of amplification cycles.
Result: In both sets, the lowest number of cycles (40) produced the most specific product. In both the 60 and 72 cycle amplifications, a smear appeared which contained multimeric "specific" PCR products.
Photo courtesy of U. Finckh and A. Rolfs, Free University of Berlin, Germany.
Final extension
Usually, after the last cycle, the reaction tubes are held at +72 °C for 5 – 15 minutes to promote completion of partial extension products and annealing of single-stranded complementary products.
Table 1 shows the application profile of PCR enzymes provided by Roche.
Obviously, in RT-PCR, a major factor to consider is the choice of reverse transcriptase used to synthesize cDNA. Since each of the available enzymes has different enzymatic properties, one may be more suitable for a specific experiment than the others. The most important enzyme properties are discussed below.
Temperature Optima
Higher incubation temperatures can help eliminate problems of template secondary structure. In addition, high temperature improves the specificity of reverse transcription by decreasing false priming. Thus, thermoactive reverse transcriptases that can be incubated at high temperatures (+50 to 70 °C) are more likely to produce accurate copies of mRNA, especially if the template has a high GC content.
Note: At these high temperatures, use only specific primers; do not use oligo(dT) or random hexamer primers.
Divalent Ion Requirement
Most reverse transcriptases require a divalent ion for activity. Enzymes that use Mg2+ are likely to produce more accurate cDNA copies than those that use Mn2+, since Mn2+ adversely affects the fidelity of DNA synthesis.
Specificity and Sensitivity
Reverse transcriptases have differing ability to copy small amounts of template (sensitivity). They also differ in their ability to transcribe RNA secondary structures accurately (specificity).
Enzyme Profiles
Table 2 shows the application profiles of RT-PCR enzymes and kits provided by Roche.
Table 2.Application profiles of RT-PCR enzymes and kits provided by Roche.
Priming affects the size and specificity of the cDNA produced. There are three types of primers that may be used for reverse transcription:
Successful RT-PCR requires a high quality, intact RNA template. Use the following guidelines to help prepare this template:
RT-PCR amplification of a particular mRNA sequence requires two PCR primers that are specific for that mRNA sequence. The primer design should also allow differentiation between the amplified product of cDNA and an amplified product derived from contaminating genomic DNA. There are two approaches to designing the required primers (Figure 2).
Figure 2.Primer design approaches.
Panel 1: Make primers that anneal to sequences in exons on both sides of an intron. With these primers, any product amplified from genomic DNA will be much larger than a product amplified from intronless mRNA.
Panel 2: Make primers that span exon/exon boundaries on the mRNA. Such primers should not amplify genomic DNA.
RT-PCR can be performed as either a two-step or an one-step procedure. Each has certain advantages.
1. Two-step procedures
A. Two tube, two-step procedure
In the first tube, first-strand cDNA synthesis is performed under optimal conditions, using either random hexamers, oligo(dT) primers (generating a cDNA pool), or sequence-specific primers. An aliquot of the RT reaction is then transferred to another tube (containing thermostable DNA polymerase, DNA polymerase buffer, and PCR primers) for PCR.
Advantages of this approach: This method is useful for experiments where multiple transcripts have to be analyzed from the same RT reaction or for specific applications, such as Differential Display Reverse Transcription (DDRT) or Rapid Amplification of cDNA Ends (RACE). Also, since the RT reaction is performed under optimal conditions, this approach produces the longest RT-PCR products (up to 14 kb in length, if the appropriate enzymes are used).
B. One tube, two-step procedure
In the first step, reverse transcriptase produces first-strand cDNA in the presence of Mg2+ ions, high concentrations of dNTPs, and either specific or nonspecific [oligo(dT)] primers (reaction volume, 20 µL). Following the RT reaction, an optimized PCR buffer (without Mg2+ ions), a thermostable DNA polymerase, and specific primers are added to the tube and PCR is performed. This approach may be useful when template amounts are limited, since the entire RT reaction is used in the subsequent PCR.
A two-step procedure has the following advantages:
2. One tube, one-step procedure (coupled RT-PCR)
Both cDNA synthesis and PCR amplification are performed with the same buffer and site-specific primers, eliminating the need to open the reaction tube between the RT and PCR steps. In addition to the higher sensitivity of this approach (as in the one tube, two-step reaction above), the one-step approach minimizes the chance of contamination, since the entire reaction is performed with minimal pipetting steps and without opening the tube. In addition, this approach permits direct analysis of a specific transcript, since the primers used in both steps are sequence-specific. Finally, the thermoactive reverse transcriptase used in this procedure allow a high RT reaction temperature (+50 to +72 °C), which reduces false priming and increases the specificity of the reaction by eliminating secondary mRNA structure.
A one-step procedure has the following advantages:
The polymerase chain reaction (PCR) can amplify a single molecule over a billionfold. Thus, even minuscule amounts of a contaminant can be amplified and lead to a false-positive result. Such contaminants are often products from previous PCR amplifications (carryover contamination). Therefore, researchers have developed methods to avoid such contamination.
One common strategy is substituting dUTP for dTTP during PCR amplification, to produce uracil-containing DNA (U-DNA). Treating subsequent PCR reaction mixtures with Uracil-DNA Glycosylase (UNG) prior to PCR amplification and subsequent cleavage of apyrimidinic polynucleotides at elevated temperature (+95 °C) under alkaline conditions (during the initial denaturation step) will remove contaminating U-DNA from the sample (Figure 3). This method, of course, requires that all PCR reactions in the lab be carried out with dUTP instead of dTTP.
Figure 3.Removal of contaminating U-DNA with uracil-DNA glycosylase.
Note the following when using dU-containing PCR products in downstream applications:
There are two ways of preventing carryover contamination when amplifying RNA:
Here are some troubleshooting hints that we have gathered regarding PCR, based on the five most common symptoms observed. Before you begin, please make sure that you have reviewed the other application hints.
Here are some troubleshooting hints that we have gathered if your PCR yields no product.
1. Non-optimal Mg2+ concentration
Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.
2. The amount of template in the reaction is not optimal
The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human DNA (105 - 106 copies) per 100 µL reaction. The amount of enzyme should be optimized for each template.
Suggestion:
3. An enzyme inhibitor is present in the reaction
Known inhibitors of PCR include:
Suggestion: Reduce or remove the concentration of any inhibitor in the reaction mixture.
4. Primer annealing temperature is too high or too low
Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).
Suggestion: Determine Tm/annealing temperature based on the following equations:
If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5
If primers are 14 - 70 bases
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo
5. Primers are degraded or not optimal
Primers should have the same number A and T's versus G and C's, and they should be at least 14 bases for specificity.
Suggestions:
6. Incomplete template denaturation
Insufficient heating during the denaturation step is a common cause of failure in a PCR reaction. It is very important that the reaction reaches a temperature at which complete strand separation occurs. A temperature of about +94 °C for 2 minutes should be adequate in most cases.
As soon as the sample reaches +94 °C, it can be cooled to the annealing temperature. Extensive denaturation is probably unnecessary and limited exposure to elevated temperatures helps maintain maximum polymerase activity throughout the reaction.
DNA reaction buffers with higher Mg concentration (4 - 5 mM) may require a higher denaturation temperature to allow complete separation of the DNA template strands. It is recommended to use the supplied buffer without adding further magnesium.
Suggestions:
7. Machine-based error
Suggestions:
8. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends of primers
Suggestions:
9. NaCl concentration above 50 mM
Suggestions: Reduce NaCl concentration
10. KCl concentration above 50 mM
Suggestions: Reduce KCl concentration
Here are some troubleshooting hints that we have gathered regarding misincorporation or low fidelity of PCR reactions.
1. Non-optimal Mg2+ concentration
Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.
2. Nucleotide concentration is too high or unbalanced
The standard concentration is 20 - 200 µM of each nucleotide.
Suggestions:
3. The pH of the reaction buffer is too high
A pH of 8.3 is optimal.
Suggestions:
4. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends of primers
Suggestions:
5. Damaged template DNA
Suggestions:
Here are some troubleshooting hints that we have gathered regarding nonspecific bands after PCR reactions.
1. Non-optimal Mg2+ concentration
Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.
2. Nucleotide concentration is too high or unbalanced
The standard concentration is 20 - 200 µM of each nucleotide.
Suggestions:
3. DNA contamination/carryover
Suggestions:
To prevent carryover, use good laboratory practices:
To eliminate contamination/carryover:
4. Primer annealing temperature is too low
Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).
Suggestion: Determine Tm/annealing temperature based on the following equations:
If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5
If primers are 14 - 70 bases
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo
5. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends of primers
Suggestions:
6. Primers are degraded or not optimal
Primers should have the same number A and T's versus G and C's, and they should be at least 14 bases for specificity.
Suggestions:
7. Primer concentration is too high
Suggestion: Adjust the primer concentration (0.1 - 1.0 µM of each primer is optimal).
8. Cycle number is too high
Most templates require 25 - 30 cycles.
Suggestion: Cycle number should be based on starting concentration of template DNA.
If the number of target molecules in your sample is... | Then we recommend the following number of cycles... |
3 x 105 | 25 - 30 |
1.5 x 104 | 30 - 35 |
1.0 x 103 | 35 - 40 |
50 | 40 - 45 |
9. Incorrect template to enzyme ratio
The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human DNA (105 - 106 copies) per 100 µL reaction. The amount of enzyme should be optimized for each template.
Suggestions:
Here are some troubleshooting hints that we have gathered regarding smeared bands after PCR reactions.
1. Non-optimal Mg2+ concentration
Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.
2. Nucleotide concentration is too high or unbalanced
The standard concentration is 20 - 200 µM of each nucleotide.
Suggestions:
3. DNA contamination/carryover
Suggestions:
To prevent carryover, use good laboratory practices:
To eliminate contamination/carryover:
4. Primer annealing temperature is too low
Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).
Suggestion: Determine Tm/annealing temperature based on the following equations:
If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5
If primers are 14 - 70 bases
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo
5. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends of primers
Suggestions:
6. DNase activity (indicated by smears visible on gel below expected band size)
Suggestions:
7. Oil contamination of gel sample
Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).
Suggestion:Spin the reaction tube and carefully extract the oil layer from the surface.
8. Incorrect template to enzyme ratio
The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human DNA (105 - 106 copies) per 100 µL reaction. The amount of enzyme should be optimized for each template.
Suggestions:
Here are some troubleshooting hints that we have gathered regarding low yield after PCR reactions.
1. Primer annealing temperature is too high
Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).
Suggestion: Determine Tm/annealing temperature based on the following equations:
If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5
If primers are 14 - 70 bases
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo
2. Template not clean or degraded
For example, protease contamination can degrade the polymerase.
Suggestions:
3. An enzyme inhibitor is present in the reaction
Known inhibitors of PCR include:
Suggestion: Reduce or remove the concentration of any inhibitor in the reaction mixture.
4. Not enough template in the reaction
The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human DNA (105 - 106 copies) per 100 µL reaction. The amount of enzyme should be optimized for each template.
Suggestions:
5. Extension temperature too high
Optimal extension temperature and time depends on fragment size:
Suggestions: Longer times, not higher temperatures should be used when longer templates or suspected secondary structure is present.
6. Enzyme activity is low
For Roche polymerases, 100% activity is guaranteed through the control date.
Suggestions:
7. Cycle number is too high
Most templates require 25 - 30 cycles.
Suggestion: Cycle number should be based on starting concentration of template DNA.
If the number of target molecules in your sample is... | Then we recommend the following number of cycles... |
3 x 105 | 25 - 30 |
1.5 x 104 | 30 - 35 |
1.0 x 103 | 35 - 40 |
50 | 40 - 45 |
8. Nucleotides hydrolyzed
Always store nucleotide stock solutions at a concentration of at least 10 mM, 100 mM is best. Significant hydrolysis occurs after storing at 1 mM for 2 months.
Dissolve NTPs or dNTPs in water at an expected concentration of 10 mM. Using 0.05 M Tris-base and pH paper, adjust the pH to 7.0. Dilute an aliquot of the neutralized NTP or dNTP appropriately and read the optical density at the wavelengths given in the following table. Calculate the actual concentration using the values for the extinction coefficients. Freeze away in small aliquots at -20 °C.
Base | Wavelength | Extinction coefficients for bases e (M-1 cm-1) |
A | 259 | 1.54 x 104 |
T | 260 | 7.4 x 103 |
G | 253 | 1.37 x 104 |
C | 271 | 9.1 x 103 |
U | 262 | 1.0 x 104 |
The lithium and sodium salts have equivalent stability and work equally well in PCR, sequencing, and labeling applications. Lithium salts are more soluble in ethanol than sodium salts. Thus, removal of lithium salts by ethanol precipitation is more efficient than removal of sodium salts. Using lithium salt nucleotide preparations reduces salt-induced artifacts and increases the legibility of sequencing gels.
9. Nucleotide concentration is too high or unbalanced
The standard concentration is 20 - 200 µM of each nucleotide.
Suggestions:
10. Primer concentration is too low
Suggestion: Adjust the primer concentration (0.1 - 1.0 µM of each primer is optimal).
11. Machine-based error
Suggestions:
12. Plateau effect
Possible causes of the plateau effect/solutions:
13. Evaporation
Evaporation can lead to higher concentrations of components, which may inhibit enzyme activity. The change in volume also leads to changes in the thermal profile inside the reaction tubes.
Suggestion: Use 100 µL mineral oil overlay/reaction.
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