Guidelines for Preventing Contamination of PCR
During PCR more than 10 million copies of a template DNA are generated. Therefore, care must be taken to avoid contamination with other templates and amplicons that may be present in the laboratory environment. General recommendations to lower the risk of contamination are the following:
· Prepare your DNA sample, set up the PCR mixture, perform thermal cycling and analyze PCR products in separate areas.
· Set up mixtures for PCR in a laminar flow cabinet equipped with an UV lamp.
· Wear fresh gloves for DNA purification and reaction set up.
· Use containers dedicated for PCR. Use positive displacement pipettes, or use pipette tips with aerosol filters to prepare DNA samples and set up PCR.
· Use certified reagents, including high quality water (e.g., Water, nuclease-free).
· Always perform No-Template-Control (NTC) reactions to check for the absence of contamination.
For detailed instructions for the set-up of a PCR laboratory and its maintenance, refer to PCR Methods and Applications, 3, 2, S1-S14, 1993.
PCR frequently is contaminated by amplicons from previous PCR held in the same room. One of the most popular and efficient methods for prevention of carryover contamination is a use of uracil DNA glycosylase*(UDG) (1). A part or all of the dTTP in the PCR reaction is substituted by dUTP and therefore all PCR products generated in your working environment contain dUTP. Prior to each PCR, short incubation with UDG eliminates such contaminating amplicons carried over from the previous PCR. Incorporation of dUTP does not affect the intensity of ethidium bromide staining or the electrophoretic mobility of the PCR product, therefore the reactions can be analyzed by standard agarose gel electrophoresis.
TaqDNA polymerase and all other non-proofreading polymerases will incorporate dUTP into a PCR product, but proofreading polymerases or enzyme mixes containing such proofreading polymerases (e.g, Fermentas DreamTaq™ DNA Polymerase, High Fidelity Enzyme Mix or the Long PCR Enzyme Mix), do not incorporate dUTP or may incorporate with much less efficiency.
*Use of such enzyme in certain territories may be covered by patents and may require a license.
Reference
1. Longo, M.C., et al., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions, Gene 93, 125-8, 1990.
Guidelines for Primer Design
Use theREviewer™primer design software or follow general recommendations for PCR primer design below:
· PCR primers are generally 15-30 nucleotides long.
· Optimal GC content of the primer is 40-60%. Ideally, C and G nucleotides should be distributed uniformly along the primer.
· Prefer one or two G or C at the 3'-end of the primer, but avoid placing more than three G or C nucleotides at the 3'-end to lower the risk of nonspecific priming.
· Avoid primer self-complementarities, complementarities between the primers and direct repeats in a primer to prevent hairpin formation and primer dimerization.
· Check for possible complementary sites between primers and template DNA.
· When designing degenerate primers, place at least 3 conservative nucleotides at the 3'-end.
· Differences in melting temperatures (Tm) of the two primers should not exceed 5°C for conventional PCR.
Estimation of Primer Melting Temperature
· For primers containing less than 25 nucleotides, the approx. melting temperature (Tm) can be calculated using the following equation:
Tm = 4 (G + C) + 2 (A + T), where G, C, A, T – number of respective nucleotides in the primer.
· If the primer contains more than 25 nucleotides specialized computer programs e.g,REviewer™are recommended to account for interactions of adjacent bases, effect of salt concentration, etc.
· For calculation of primer melting temperature only consider nucleotides homologous to the template.
Considerations for Subsequent Cloning of PCR Products
· When introducing restriction endonuclease sites into primers for subsequent digestion and cloning of the PCR product, refer to tables"Cleavage Efficiency Close to the Termini of PCR Fragments"or"Reaction Conditions for FastDigest® Restriction Enzymes"to determine the number of extra bases outside of the recognition sequence that are required for efficient cleavage.
Components of the Reaction Mixture
Template DNA
Optimal amounts of template DNA in the 50µl reaction volume are in the 0.01-1ng range for both plasmid and phage DNA, and in the 0.1-1µg range for genomic DNA. Higher amounts of template increase the risk of generation of nonspecific PCR products. Lower amounts of template reduce the accuracy of the amplification.
All routine DNA purification methods are suitable for template preparation e.g., Genomic DNA Purification Kit (#K0512), GeneJET™ Plasmid Miniprep Kit (#K0502). Trace amounts of certain agents used for DNA purification, such as phenol, EDTA and proteinase K, can inhibit thermostable DNA polymerases. Ethanol precipitation and repeated washes of the DNA pellet with 70% ethanol normally remove trace contaminants from DNA samples.
Primers
The recommended concentration range of primers is 0.1-1µM. Too high primer concentrations increase the probability of mispriming and thereby appearance of nonspecific PCR products.
For degenerate primers and primers used for long PCR higher primer concentrations in the range of 0.3-1µM are often favorable. Therefore start optimization from standard concentrations and increase if necessary.
Mg2+Concentration
Mg2+in general stabilizes primer-template complexes. PCR buffers forTaqDNA Polymerase are supplemented with Mg2+, while in PCR withPfuDNA Polymerase MgSO4is a preferable component. Due to the binding of Mg2+to dNTPs, primers and DNA templates, Mg2+concentration needs to be optimized for maximal PCR yield. The recommended concentration range is 1-4mM. If the Mg2+concentration is too low, the yield of PCR product could be reduced. On the contrary, non-specific PCR products may appear and the PCR fidelity may be reduced if the Mg2+concentration is too high. If DNA samples contain EDTA or other metal chelators, the Mg2+ion concentration in the PCR mixture should be increased accordingly (1 molecule of EDTA binds 1 molecule of Mg2+(1)).
Recommended Mg2+concentrations:
· TaqDNA Polymerase at Fermentas is supplied with two buffers:Taqbuffer with KCl andTaqbuffer with (NH4)2SO4. K+stabilizes primer annealing whereas NH4+has a destabilizing effect especially on weak hydrogen bonds between mismatched primer-template base pairs. Therefore for standard PCR withTaqDNA Polymerase and 0.2mM dNTPs the recommended MgCl2concentrations are in general lower 1.5±0.25mM when usingTaqbuffer with KCl compared to 2.0±0.5mM when usingTaqbuffer with (NH4)2SO4. Due to antagonistic effects of NH4+and Mg2+,Taqbuffer with (NH4)2SO4offers higher primer specificity in a broad range of magnesium concentrations at variety of annealing temperatures.
· For standard PCR withPfuDNA Polymerase, 2mM MgSO4is recommended.
Volumes of 25mM MgCl2or 25mM MgSO4solutions required to reach a specific concentration of magnesium ions in the 50µl reaction volume:
Volume of 25mM MgCl2or MgSO4, µl / 2 / 2.5 / 3 / 3.5 / 4 / 5 / 6 / 8
dNTPs
The recommended concentration of each dNTP is 0.2mM. In certain PCR applications higher dNTP concentrations are required. Due to the binding of Mg2+to dNTPs, Mg2+concentration needs to be adjusted accordingly. It is essential to have equal concentrations of all four nucleotides (dATP, dCTP, dGTP and dTTP). If the nucleotide concentrations are not balanced, the PCR error rate may dramatically increase. Fermentas PureExtreme® dNTP Mixes contain either 2mM or 10mM, or 25mM of each nucleotide. The concentrations of all four dNTPs are perfectly balanced to provide fidelity and to increase the yield of PCR products.
To achieve 0.2mM concentration of each dNTP in the PCR mixture, use the following volumes of dNTP Mixes:
50µl / 5µl / 1µl / 0.4µl
25µl / 2.5µl / 0.5µl / 0.2µl
20µl / 2µl / 0.4µl / 0.16µl
To prepare 1ml of working solutions of dNTPs (dNTP Mixes) from individual 100mM dNTPs or dNTP Set, use the following volumes of reagents:
Component / dNTP Mix, 2mM each (#R0241) / dNTP Mix, 10mM each (#R0191) / dNTP Mix, 25mM each (#R1121)dATP, 100mM / 20µl / 100µl / 250µl
dTTP, 100mM / 20µl / 100µl / 250µl
dGTP, 100mM / 20µl / 100µl / 250µl
dCTP, 100mM / 20µl / 100µl / 250µl
Water, nuclease-free / 920µl / 600µl / -
Total volume / 1ml / 1ml / 1ml
Thermostabile DNA Polymerases
TaqDNA Polymerase.TaqDNA polymerase is the most commonly used enzyme for PCR. It is suitable for most amplifcation reactions that do not require high fidelity enzyme or PCR products longer than 3kb.
Normally, 1-1.5u ofTaqDNA Polymerase are recommended for a 50µl volume of a PCR mixture. Nonspecific PCR products may appear at higher concentrations of the polymerase. However, it may be necessary to increase the amount ofTaqDNA Polymerase to 2-3 u, if the PCR mixture contains inhibitors, for instance, due to contamination of the template DNA.
TaqDNA polymerase, if PCR is assembled at room temperature, exhibits low but noticeable activity during the reaction set-up. As a result, non-specific priming events, such as mispriming or formation of primer dimers, which occur at ambient temperatures, will lead to generation of nonspecific amplification products during PCR. Therefore, PCR reaction set-up should always be performed on ice.
DreamTaq™ DNA Polymerase.DreamTaq™ DNA Polymerase is an enhancedTaqDNA polymerase optimized for all standard PCR applications. It ensures higher sensitivity, longer PCR products and higher yields compared to conventionalTaqDNA Polymerase. DreamTaq™ DNA Polymerase uses the same reaction set-up and cycling conditions as conventionalTaqDNA Polymerase. An optimization of reaction conditions is generally not required. It is supplied with optimized DreamTaq™ buffer, which includes 20 mM MgCl2. DreamTaq™ DNA Polymerase generates PCR products with 3’-dA overhangs. PCR with DreamTaq™ DNA Polymerase is inhibited by dUTP, but the enzyme can incorporate modified nucleotides.
Hot StartTaqDNA Polymerases.Hot start PCR uses enzymes, which have no activity at room temperature and are activated only at high temperatures during PCR cycling (e.g. TrueStart™ Hot StartTaqDNA Polymerase or Maxima® Hot StartTaqDNA Polymerase). In hot start PCR non-specific amplification is reduced and target yield is increased. Using hot start DNA polymerases, PCR can be set-up at room temperature. TrueStart™ Hot StartTaqDNA polymerase has very short activation time (1min) and can be used without changing of regular PCR cycling protocol. Maxima® Hot StartTaqDNA Polymerase is activated in 4min.
PfuDNA Polymerase.PfuDNA Polymerase is a thermostable DNA polymerase with proofreading activity. It is one of the highest fidelity enzymes among thermostable DNA polymerases and is widely used in applications which require high fidelity amplification, e.g. cloning and expression. Normally, 1.25-2.5u ofPfuDNA Polymerase are used in a 50µl volume of PCR mixture. The actual amount of enzyme required for optimal PCR yield and fidelity depends on the target to be amplified and on the presence of inhibitors in the PCR mixture.PfuDNA polymerase is a slower enzyme thanTaqDNA polymerase and it requires an elongation time of 2min/kb. Also,PfuDNA polymerase often requires more PCR cycles to produce sufficient amount of PCR product. Due to the intrinsic 3'=>5' exonuclease activityPfuDNA polymerase should always be the last component added to the reaction mixture to avoid degradation of primers. It is also recommended to use longer PCR primers. Alternatively, phosphorothioate primers (exo- resistant primers) can be used to avoid primer degradation byPfuDNA Polymerase (2).
PCR Enzyme Mixes.Long PCR Enzyme Mix and High Fidelity Enzyme Mix are blends ofTaqDNA Polymerase and a thermostable DNA polymerase with a proofreading activity. The two enzymes synergistically generate long PCR products in greater yields and higher fidelity thanTaqDNA Polymerase alone. The Long PCR Enzyme Mix is also used for efficient amplification of GC-rich DNA regions. Normally, 1.25-2.5u of Enzyme Mix are used in a 50µl volume of PCR mixture. Due to the 3'=>5' exonuclease activity of proofreading enzyme Enzyme Mixes should always be last components added to the reaction mixture to avoid degradation of primers. It is also recommended to use longer PCR primers. Alternatively, phosphorothioate primers (exo-resistant primers) can be used to avoid primer degradation by enzyme mixes.
PCR Master Mixes.Thermostable DNA polymerases can be provided in a Master Mix format, a ready to use 2X concentrated solution, which includes DNA polymerase together with a PCR buffer and nucleotides. The Master mix is the most convenient and cost effective product for routine or high throughput PCR, where time for setting up a reaction and reproducibility of results are most important factors.
Fermentas offers two PCR Master Mixes. The PCR Master Mix (2X) containsTaqDNA polymerase and is suitable for routine PCR. The PyroStart™ Fast PCR Master Mix (2X) contains a hot startTaqDNA polymerase and is formulated to work in fast thermal cycling conditions to reduce time not only dedicated to PCR set-up, but also to PCR cycling. PCR of less than 1kb target can be completed in 25min using this product.
References
1. David, H., Modern Analytical Chemistry, Mc Graw Hill, 315, 2000.
2. Skerra, A., Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity, Nucleic Acids Res., 20, 3551-3554, 1992.
Cycling Parameters
Amplification parameters greatly depend on the template, primers and parameters of the thermal cycler used. At Fermentas, functional PCR tests are performed on the GeneAmp® PCR System 9700 (Applied Biosystems) or Mastercycler® ep gradient S (Eppendorf).
Initial DNA Denaturation.It is essential to completely denature the template DNA at the beginning of PCR to ensure efficient utilization of the template during the first amplification cycle. If GC content of the template is 50% or less, an initial 1-3min denaturation at 95°C is sufficient. For GC-rich templates, this step has to be prolonged to 10min. If longer initial denaturation step is required, or DNA is denatured at a higher temperature, theTaqDNA Polymerase can be added after DNA is denatured to avoid a decrease in its activity.PfuDNA Polymerase can withstand a prolonged initial denaturation step due to its higher thermostability.
Hot Start PCR.Hot start PCR usesTaqDNA polymerases which are inactive at room temperature and are activated during the initial DNA denaturation/ enzyme activation step at 95°C. TrueStart™ Hot StartTaqDNA polymerase has a very short activation time (1min) and can be used without changing the regular PCR cyling protocol. Maxima® Hot StartTaqDNA Polymerase activates in 4min, therefore the initial denaturation step should be set to 4min.