• Polymerase
  • Overview of PCR polymerases

    Principle and application of Polymerase Chain Reaction (PCR)

    A revolutionary method of DNA amplication called PCR (Polymerase Chain Reaction) was first developed in 1985 by Dr. Kary Banks Mullis, a biochemist in Cetus Biotechnology Company. The  PCR method requires repeated cycles of a 3-step procedure to amplify a specific DNA region from a very low amount of template DNA: (i) denaturation of the template DNA, (2) primer annealing, and (3) DNA synthesis by polymerase. PCR was difficult in early days because it needs to add polymerase after each PCR cycle. PCR machines were developed after the discovery of Taq polymerase which maintains its activity over 90. Patents for the PCR method using thermostable polymerases were sold for $300,000,000 to Heffman-LaLoche Company. The PCR method has opened a new avenue in most areas of biology and medicine including molecular biology, microbiology, medical science, and forensic science. The PCR method is widely used to analyze hereditary diseases and for diagnosis of bacteria or virus infection, It also allows us to find criminals from a drop of blood or a single hair.

     

    Parameters that affect PCR amplification efficiency

    An optimum condition of PCR can vary depending on each PCR reaction. Each parameter for PCR should be adjusted for most efficient amplification of the desired DNA fragments. Common parameters that affect efficiency of PCR reactions are as follows: reaction time, incubation temperature, amount and type of polymerases, amount of substrate DNA, dNTP, and Mg2+.

     

    PCR polymerases

    The optimal amount of PCR polymerase ranges from 1 to 5 units in a 100-μl reaction volume. In general, two and half units of polymerase give rise to satisfactory results. Amounts of non-specific amplified products usually increase with higher amounts of enzyme used. On the other hand, use of too small amount of enzyme results in insufficient amount of amplified PCR products. nTaq (Cat.# P025, P050) synthesizes 1 kb per min. Half-life of nTaq DNA Polymerase at 95 is 50 min. nPfu-Forte is more heat stable and retains over 95% activity after 60 min at 95.

     

    Mg2+

    The optimal concentration of Mg2+ is between 1.5 to 5 mM. Generally, the use of 1.5 mM Mg2+ gives rise to satisfactory results in the presence of 0.2 mM dNTP. The concentrations of Mg2+ used affect the activity of PCR polymerase, fidelity of DNA synthesized, and primer annealing. Since Mg2+ is required to activate dNTP by chelating dNTP in a stoichiometric manner, increased concentration of Mg2+ is required if elevated levels of dNTP are used or a high level of EDTA are present in a reaction mixture.

     

    dNTP

    Concentrations of dNTP (deoxyribonucleotides triphosphates) are optimal at 50-500 μM for PCR, but 200 μM dNTP is most commonly used. Equimolar concentrations of four different deoxyribonucleotides triphosphates are required for a high efficiency of PCR. Low concentrations of dNTP often result in increased specificity and high fidelity of PCR reactions. Mg2+ concentrations should be increased with increased concentrations of dNTP. The dNTPs in the reaction that is not incorporated into DNA are damaged at a certain rate during repeated cycles of PCR reaction. After 50 cycles of PCR reaction, approximately 50% of dNTP will remain intact in the reaction mixture.

     

    Template DNA

    Recommended amounts of template DNA are 0.1-30 ng of plasmid DNA or a DNA fragment, or 50-500 ng of genome DNA in a 100-μl reaction volume. Amplification of DNA can be easily obtained with 105-106 molecules of DNA and as few as 10-100 DNA molecules can be readily amplified by multiple rounds of PCR or nested PCR. Cautions should be taken with inhibitory substances which are often present in impure DNA and interfere with PCR reaction.

     

    Reaction temperatures

    Initial denaturation

    Heat template DNA to 94-95 for 2-3 min, which convert duplex template DNA into singlestranded DNA in the first step.

     

    Denaturation during PCR cycles

    To convert amplified duplex DNA into single-strand DNA during the PCR cycles, denaturation for 20-30 sec at 94-95 is usually sufficient. Longer denaturation period than this reduces the efficiency of PCR reaction due to gradual inactivation of the PCR polymerases.

     

    Annealing

    This step allows primers to anneal to the template single-stranded DNA. Annealing temperature depends on the Tm of a primer sequence. In general, annealing is carried out at the temperature 5 lower than the Tm of the primer. Annealing at either higher or lower temperature than this often results in failure of PCR or amplification of nonspecific PCR products.

     

    Elongation

    The Taq polymerase requires 1 min of elongation time for amplification of 1-kb DNA fragment., whereas the Pfu polymerase requires 2 min/bk DNA. High PCR efficiency can be obtained with Enzynomics nPfu-Forte (elongation time, 1 min/kb, Cat.# P410, P425) which contains a PCR enhancing factor.

     

    Number of PCR cycles

    For most purposes, 25-35 cycles are appropriate. 40-45 cycles are often used when the amount of template DNA is limiting. Increasing the number of PCR cycles may result in amplification of nonspecific DNA.

    In theory, each cycle of PCR reaction doubles the amount of amplified DNA product (2n, n=number of PCR cycle). However, the actual amount of DNA amplified is lower than calculated due to many reasons such as inefficient annealing, gradual decrease in dNTP available for amplification, and loss of polymerase activity, which collectively render the reaction to reach a plateau. This is called a “plateau effect."

     

    Primers

    Selection or design of primer sequences is a key factor to successful amplication of desired DNA fragment in PCR reaction. It is imperative to design a pair of primers so that they anneal to the specific sequence in the template DNA. If they have homology to other regions than the sequence of interest, it is likely that nonspecific DNA is amplified as well. Appropriate concentrations of primers range from 0.1 to 0.6 μM. Use of excess amount of primers frequently gives rise to either nonspecific DNA amplification or primer dimers that result from the two primers (see below). The recommended length of primers is 15-30 nucleotides. The primers of 30 nucleotides or longer can be used to amplify longer DNA or to solve specificity problem. For optimal PCR efficiency, G+C content should be within 40-60% and Tm of the two primers should be similar. The 3’-end of primers should not contain more than 3 consecutive G or C in order to avoid non-specific amplification and the 3’-end of the two primers should not be complementary to each other in order to prevent the formation of primer-dimers. The 3’ end of the primers should not contain ATrich sequence, which hampers stable annealing to template DNA.

  • Table. PCR Troubleshooting

    Trouble : No PCR products

    Cause

    Solution

    Template DNA that could not be amplified

    (e.g., Template DNA with a high GC content)

    Add DMSO (2-5%) and reduce the enzyme concentration to 0.5 unit.

    Use other organic solvents that reduce Tm.

    Problems with template DNA.

    Examine concentration, quality and purity of template DNA.

    Confirm that template DNA is not degraded by agarose gel analysis.

    Carry out test PCR with a pair of other primers that were successful.

    Prepare a new template DNA.

    Low concentrations of polymerase

    Increase the amount of enzyme concentration by 0.5 unit.

    If necessary, increase the amount of enzyme up to 5 units in 100-μl reaction.

    Low concentrations of MgCl2

    Increase MgCl2 concentration by 0.25 mM.

    Inadequate PCR cycles

    Reduce annealing temperature.

    Increase the number of PCR cycles.

    Check whether the final elongation step (72, 5 min) is carried out.

    Unsuitable primers

    Prepare a new pair of primers.

    Inadequate primer concentration.

    Use of equal concentration of two primers.

    Titrate primers for their optimal concentrations (from 0.1 to 0.6 μM).

    Primer quality and storage

    Confirm that the primers are not degraded.

    Store primers at -15 to -30.

    Formation of primer dimers

    Carry out hot-start PCR

    Divide the PCR mixture into two submixtures, each of which is inactive, and

    combine them together just before PCR reaction.

     

    Trouble : Multiple or smeared PCR products

    Cause

    Solution

    Low annealing temperature.

    Increase the annealing temperature based upon the length of primers and their

    nucleotide sequences.

    Low concentration of primers or

    inappropriate primers

    Check whether primers are optimally designed.

    Determine the optimal primer concentration by titration between 0.1 to 0.6 mM.

    Use the same concentrations of the two primers

    Carry out nested PCR*

    Template DNA that could not be amplified

    (e.g., Template DNA with a high GC content)

    Add DMSO (2-5%) and reduce the enzyme concentration to 0.5 unit.

    Use other organic solvents that reduce Tm.

    Problem with DNA template

    Use template DNA after serial dilution.

    * Nested PCR : A PCR that uses the amplified products obtained from primary PCR as template and

    another pair of primers that are designed to anneal within the amplified product.

     

  • Selection guide of Enzynomics PCR polymerases 


     

    Characteristics of Enzynomics PCR polymerases 


     

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