PfuDNA聚合酶PCR实验方法介绍

General Advice

PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this technique means that the sample should not be contaminated with any other DNA or previously amplified products (amplicons) that may reside in the laboratory environment.

Guidance in Avoiding Contamination

• DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas.

• A Laminar Flow Cabinet equipped with a UV lamp is recommended for preparing the reaction mixture.

• Fresh gloves should be worn for each PCR step.

• The use of dedicated vessels and positive displacement pipettes or tips with aerosol filters for both DNA sample and reaction mixture preparation, is strongly recommended.

• The reagents for PCR should be prepared separately and used solely for this purpose. Autoclaving of all solutions, except dNTPs, primers and Pfu DNA Polymerase is recommended. Solutions should be aliquoted in small portions and stored in designated PCR areas. Aliquots should be stored separately from other DNA samples.

• A control reaction, omitting template DNA, should always be performed, to confirm the absence of contamination.

These are only rough guidelines. Detailed instructions about PCR laboratory setup and maintenance may be found in PCR Methods and Applications, 3, 2, S1-S14, 1993.

Preparation of Reaction Mixture

To perform several parallel reactions, we recommend the preparation of a master mix containing water, buffer, dNTPs, primers and template DNA in a single tube, which can then be aliquoted into individual tubes. Pfu DNA Polymerase should be added last. This method of setting reactions minimizes the possibility of pipetting errors and saves time by reducing the number of reagent transfers.

Reaction Mixture Set Up

• Gently vortex and briefly centrifuge all solutions after thawing.

• Keep solutions on ice.

• Add, in a thin-walled PCR tube, on ice:

Reagent	Final  concentration	Quantity, for 50µl of reaction mixture
Sterile deionized water	-	variable
10X PCR buffer with MgSO4*	1X	5µl
2mM dNTP mix	0.2mM of each	5µl
Primer I	0.1-1µM	variable
Primer II	0.1-1µM	variable
Template DNA	50pg-1µg	variable
Pfu DNA Polymerase	1.25u/50µl	0.5µl

* If using 10X PCR buffer without pre-added MgSO₄, refer to the table below to determine required volume of 25mM MgSO₄ solution (for 50µl total volume).

Final concentration of MgSO4 in 50µl reaction mix, mM	1.0	1.25	1.5	1.75	2.0	2.5	3.0	4.0
Volume of 25mM MgSO4, µl	2	2.5	3	3.5	4	5	6	8

• Gently vortex the sample and briefly centrifuge to collect all drops from walls of tube.

• If using a thermal cycler without a heated lid, overlay the sample with a half volume of mineral oil or add an appropriate amount of wax.

• Place samples in a thermal cycler preheated to 95°C and start PCR.

Recommended thermal cycling conditions:

Step	Temperature, °C	Time, min	Number of cycles
Initial denaturation	95	1-3	1
Denaturation	95	0.5-2
Annealing	37-70	0.5-2	25-35
Extension	70-75	2-4
Final extension	70-75	5	1

 Note 

Composition of the Reaction Mixture

1. Template DNA.
   Usually the template DNA amount is in the range of 50pg-1ng for plasmid or phage DNA and 0.1-1µg for genomic DNA, for a total reaction mixture of 50µl. Higher template DNA amounts usually increase the yield of nonspecific PCR products, but if the fidelity of synthesis is crucial, maximal allowable template DNA quantities in conjunction with limiting number of PCR cycles should be used to increase the percentage of "correct" PCR products. Nearly all routine methods are suitable for template DNA purification. Although even trace amounts of agents used in DNA purification procedures (phenol, EDTA, Proteinase K, etc.) strongly inhibit Pfu DNA Polymerase, ethanol precipitation of DNA and repetitive treatments of DNA pellets with 70% ethanol is usually effective in removing traces of contaminants from the DNA sample.

2. Primers.
   Guidelines for primer selection:

• PCR primers are usually 20-30 nucleotides in length. Longer primers provide sufficient specificity.

• The GC content should be 40-60%. The C and G nucleotides should be distributed uniformly within the full length of the primer. More than three G or C nucleotides at the 3'-end of the primer should be avoided, as nonspecific priming may occur.

• The primer should not be self-complementary or complementary to any other primer in the reaction mixture, in order to avoid primer-dimer and hairpin formation.

• The melting temperature of flanking primers should not differ by more than 5°C, so the GC content and length must be chosen accordingly.

• All possible sites of complementarity between primers and the template DNA should be noted.

• If primers are degenerate, at least 3 conservative nucleotides must be located at the primer's 3'-end.

• Estimation of the melting and annealing temperatures of primer: 
  If the primer is shorter than 25 nucleotides, the approx. melting temperature (T_m) is calculated using the following formula:
  T_m= 4 (G + C) + 2 (A + T)
  G, C, A, T - number of respective nucleotides in the primer. 
  If the primer is longer than 25 nucleotides, the melting temperature should be calculated using specialized computer programs where the interactions of adjacent bases, the influence of salt concentration, etc. are evaluated. Optimal annealing temperature is generally 5°C lower than the melting temperature of the primer-template DNA duplex.

• The 3’=>5’ exonuclease activity associated with Pfu DNA Polymerase may degrade the primers. It is therefore important that Pfu DNA Polymerase be added last to the reaction mixture. Degradation of primers can be efficiently prevented by the introduction of phosphorothioate bonds at their 3’-termini (1). The use of longer primers with maximized GC content can be advantageous.

MgSO₄ concentration.
Pfu DNA Polymerase prefers MgSO₄ to MgCl₂. Since Mg²⁺ ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgSO₄has to be selected for each experiment. Too few Mg²⁺ ions result in a low yield of PCR product, and too many increase the yield of non-specific products. Optimal MgSO₄ concentration is in the range of 2-4mM. Increasing MgSO₄concentration from 2 to 10mM did not vary significantly the error rate. If the DNA samples contain EDTA or other chelators, the MgSO₄ concentration should be raised proportionally. 

dNTPs.
The concentration of each dNTP in the reaction mixture is usually 200µM. It is very important to have equal concentrations of each dNTP (dATP, dCTP, dGTP, dTTP), as inaccuracy in the concentration of even a single dNTP dramatically increases the misincorporation level. dNTPs concentrations of 100-250µM of each dNTP result in the optimal balance of product yield (greater at higher dNTP concentration) versus specificity. In addition, the optimal concentration of dNTPs should be selected empirically.

Pfu DNA Polymerase.

• The concentration of the enzyme required for optimal PCR product yield and specificity depend on target to be amplified and the presence of inhibitors in the reaction mix (e.g., if the template DNA used is not highly purified). The optimal enzyme concentration is 1.25-2.5u/50µl.

• 3’=>5’ exonuclease activity associated with Pfu DNA Polymerase may degrade primers. It is important to add the enzyme to the reaction mixture at last and place PCR mixes and tubes on ice.

• Pfu DNA Polymerase has no detectable reverse transcriptase activity.

Reaction overlay. 
If necessary, the reaction mixture can be overlaid with mineral oil or paraffin (melting temperature 50-60°C) of special PCR grade. One-half of the total reaction volume is usually sufficient. 

Temperature Cycling

Amplification parameters depend greatly on the template, primers and amplification apparatus used.

1. Initial Denaturation Step.

• The complete denaturation of the DNA template at the start of the PCR reaction is of key importance. Incomplete denaturation of DNA results in the inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. The initial denaturation should be performed over an interval of 1-3min at 95°C if the GC content is 50% or less. 
  This interval should be extended up to 10min for GC-rich templates or denaturation temperature may be increased up to 97°C.

Denaturation Step.

• Usually 0.5-2min denaturation at 94-95°C is sufficient, since the PCR product synthesized in the first amplification cycle is significantly shorter than the template DNA and is completely denatured under these conditions. The GC content should be taken into consideration. Denaturation time can be optimized empirically.

Primer Annealing Step.

• Usually the optimal annealing temperature is 5°C lower than the melting temperature of primer-template DNA duplex. Incubation for 0.5-2min is usually sufficient. However, if non-specific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by increasing it stepwise by 1-2°C.

Extending Step.

• Usually the extending step is performed at 70-75°C. Pfu DNA Polymerase exhibits lower than that of Taq DNA Polymerase extention rate (0.5kb/min), so 2min extention time is recommended for every 1 kb to be amplified.

Number of Cycles.

• The number of PCR cycles depends on the amount of template DNA in the reaction mix and on the expected yield of the PCR product. For most amplification reactions, 25-35 cycles are usually sufficient. In general, we suggest using the fewest cycles possible to achieve acceptable yield of PCR product and ensure less amount of non-specific background product.

Final Extending Step.

• After the last cycle, the samples are usually incubated at 72°C for 5-15min to fill-in the protruding ends of newly synthesized PCR products.

• Pfu DNA Polymerase, unlike Taq DNA Polymerase, lacks terminal transferase activity and generates blunt-ended PCR products, which can be used directly for blunt end ligation without any pretreatment of the ends.

Reference

1.  Skerra, A., Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity, Nucleic Acids Res., 20, 3551-3554, 1992.