Please see Current PCR Methods for a discussion on various PCR methods; PCR Machines for a survey on PCR machines based on literature; and Software Programs in Biomedical Research for a discussion and survey results on molecular biology software programs including those for PCR primer design.

Multiple PCR polymerases are available from commercial suppliers. The polymerases are from different sources and with varying degrees of replication fidelity and with different extension speed. Table 1 lists major PCR polymerases and some of their important features. Taq polymerase was initially purified from T. aquaticus cells, and later the gene was cloned and expressed in E. coli cells. The recombinant Taq polymerase, named AmpliTaq DNA polymerase, was commonly used [2]. Taq polymerase can be chemically inactivated at low temperatures, and this modification can be reversed at a high temperature; this temperature-dependent reversible modification of the Taq protein, AmpliTaq Gold, present in mixtures such as Thermo Power SYBR™ Green PCR Master Mix, can be used as the hot start PCR enzyme [2]. Phusion DNA polymerase fuses Pfu DNA polymerase with the DNA binding protein, Sso7d, from S. solfataricus [3] to increase its processivity performance whiling retaining the high fidelity of Pfu polymerase [2]. Specialized DNA polymerases have also been designed [4]. In the case of random mutagenesis, such as Agilent Random Mutagenesis Kits [5], error-prone polymerases are preferred.

Labome systematically curates information on reagents, instruments, and software programs from randomly selected formal articles. Table 2 lists the most commonly cited PCR polymerases and their major suppliers from a survey of formal articles citing DNA polymerases for PCR. For example, Liu Y et al measured RNA levels of multiple genes from mouse peritoneal macrophages in qPCR with SYBR Green Realtime PCR Master Mix from TOYOBO (QPK-201) by QuantStudio 7 Flex from Thermo Fisher Scientific after total RNA extraction with TRIzol reagent and reverse transcription using ReverTra Ace qPCR RT Master Mix with gDNA Remover from TOYOBO (FSQ301) [36]. Chopra S et al used PerfeCTa SYBR green fastmix from Quantabio and TaqMan Universal PCR master mix from Thermo Fisher for quantitative RT-PCR [17]. Flaherty SE et al evaluated the expression of multiple genes in bone-marrow-derived macrophages with quantitative PCR using QIAGEN PCR SYBR Green I QuantiTect Master Mix [22].

The error rates of DNA polymerases are dependent on the PCR conditions. For example, when the reaction pH changed from pH 8 to 9, the error rate of Pfu decreased ∼2-fold, and the error rate of exo−deficient Pfu increased ∼9-fold [37]. Dr. Peter McInerney and colleagues compared six commonly used DNA polymerases in PCR applications in vendor-recommended buffers in 2014 [1]. Figure 1 lists the tables from their article, indicating that Pfu (Agilent), Phusion (Finnzymes) and Pwo (Roche) produced 10 X fewer errors than the regular Taq polymerase and that most errors are transition mutations. Q5 High-fidelity polymerase from NEB, based on a novel DNA polymerase, appears to be one of popular low error rate polymerase [38, 39]. According to its supplier, NEB, it has an error rate ~280-fold lower than that of Taq DNA Polymerase. Boettcher S et al used Q5 High-Fidelity DNA polymerase for New England Biolab (M0491L) for TP53 MITE-seq screen [40]. de Goffau MC et al aimed to detect bacterial 16S rRNA gene exhaustively from human placental tissues with the same Q5 polymerase [41]. D Cervettini et al also used it for their work on evolving new aminoacyl-tRNA synthetase-tRNA pairs [42]. Invitrogen Platinum SuperFi DNA Polymerase with 100× higher fidelity compared to native Taq, was used in high annealing stringency PCR to detect APP gene somatic mutations in Alzheimer disease and normal neurons [23]. Lee J et al used PrimeSTAR GXL DNA polymerase from Clontech to detect any off-target mutation in TALEN-mediated LMNA genomic editing [43].

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Figure 1. Direct comparison of six commonly used DNA polymerases by Dr. Peter McInerney and colleagues [1].

PCR reactions can be directly performed on biological samples without any prior purification of DNA molecules. Dr. Miura and colleagues compared six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer and concluded that KOD FX was the best for blood-based direct PCR due to its resistance against blood component inhibition and against mild detergents [44].

DNA templates, primers, ddH₂O, DNA polymerase from commercial providers with specific reaction buffers, dNTPs, MgSO₄ (optional), and DMSO (optional).

Mix the reagents and set up the PCR cycles according to the manufacturer’s manual. Briefly, the PCR cycle is as follows:

1. Initialization step. This is only essential for Hot-start PCR. This step heats the solutions to 94-98°C for DNA polymerase activation. The time of this step depends on the polymerase used.
2. Denaturation step. DNA is double-stranded molecules, and DNA amplification needs primers interacting with single-stranded DNA template. During this step, reaction mixtures are heated to 94-98°C for 20-30 seconds to disrupt the hydrogen bonds between two strands and to generate single-stranded DNA molecules. This is the first step of PCR cycle.
3. Annealing step. After denaturation, DNA templates in reaction mixtures are single-stranded. As primers are complementary to the DNA template, when the reaction temperature is reduced to 50-65°C, the primers will match with the template sequence and form hydrogen bonds between complementary bases. The annealing temperature depends on the Tm of primers used, generally about 3-5°C lower than the primer Tm. This step will sustain for about 20-40 seconds for fully annealing and then polymerase will locate to primer-template hybrid to start DNA assembly.
4. Elongation step. At this step, DNA polymerase begins DNA synthesis, and so the temperature should be the DNA polymerase’s optimum temperature. Generally 72°C is chosen, but some enzymes work better at 68°C. This step is quite similar to the DNA replication in vivo and DNA polymerase adds dNTPs to the primers in 5’ to 3’ direction complementary to the template and finally result in a new double-stranded DNA fragment. The elongation time depends on the length of the target DNA fragment and the ability of DNA polymerase. In general DNA polymerase generate a thousand base per 60 seconds.
5. 2 to 4 step is called a cycle, and the amount of target fragment is doubled after every cycle. 30-35 cycles are used in one PCR process. During the early PCR cycles, the PCR products accumulate at an exponential rate, while during the late PCR cycles, with the reduction of dNTPs, primers and the inactivation of DNA polymerase at the denaturation temperature, the reaction slows down and the rate of PCR is limiting.
6. Final elongation. When 30-35 cycles are finished, the final elongation is at the temperature of 68-74°C for about 5-10 minutes, in order to fully extend the remaining single-strand DNA.
7. Storage. The final products can be stored at 4-10°C in PCR machines.

Before starting PCR experiments, DNA templates (either from genomic DNA or from cDNA prepared by reverse transcription), specific primers (designed by yourself or with software) must be prepared, and appropriate commercial enzymes (choose enzymes meeting your experiment aims) should be selected.

• DNA polymerase. There are many commercial DNA polymerases, and they have different properties. Generally, there are two categories: high-fidelity polymerase, and common Taq polymerase. If you want to PCR a specific DNA fragment without any mutations, choose the high-fidelity polymerase. If you just want to detect the existence of a specific sequence, just choose the common Taq polymerase. If you want to PCR a fragment for T-vector ligation, be careful because products of most high-fidelity polymerases have no A-tailing, so you should choose common Taq polymerase or add A-tailing after PCR experiments.
• The specificity of primers affect the probability of PCR success, and good primer design is critical for successful PCR amplification. When you start to design primers, several principles described below should be considered carefully:
  • The length of a primer. Generally, PCR primers are about 18-22 bp. This is long enough for primer specificity and binding at the annealing temperature.
  • Melting temperature(Tm). Tm is defined as at this temperature half of the DNA duplex will dissociate to become single strand DNA. Primer Tm in the range of 52-58C is optimal.
  • GC content. The optimal GC content should be 40-60%.
  Many software can be used for primer design, such as primer5 and Oligo. The primers suggested by these software usually satisfy our needs.

• No band
  • Wrong set up. Repeat the PCR to make sure that all reagents are added in a correct amount.
  • Program for PCR is wrong. Check the program in PCR machine is right.
  • Something wrong with DNA gel. Load positive control such as plasmids to make sure the DNA gel is OK.
  • Annealing temperature is not suitable. Run a temperature gradient in 2°C increments
  • Low template. Increase the amount of template.
  • Bad primers. Primer blast or redesign primers.
  • Inhibitors in DNA template. Check the DNA template to make sure it is clean enough.
  • Complex structure in the template. Add DMSO, BSA (or acetylated BSA to inactivate any trace of nucleases, although it may affect PCR under certain conditions [45] ) or betaine. Use touchdown PCR.
• The amount of PCR products is quite low.
  • Annealing temperature is not suitable. Run a temperature gradient in 2°C increments
  • Low template. Increase the amount of template.
  • Insufficient cycles. Increase the cycles to 30-40.
  • Low primer amount. Increase the amount of primers.
  • Complex structure in the template. Add DMSO, BSA (or acetylated BSA) or betaine. Use touchdown PCR.
  • Extension time is not long enough. Extend the elongation time according to 1kb/min.
  • Denaturation time is too long. Long denaturation time will inactivate the DNA polymerase.
  • Inhibitors in DNA template. Check the DNA template to make sure it is clean enough.
• Multiple bands
  • Premature replication. Often occurs when using non-hot start polymerase. Prepare the mixture on ice or use hot-start polymerase.
  • Low primer annealing temperature. Run a temperature gradient in 2°C increments.
  • Reaction buffer not melt thoroughly or not mixed completely. Just melting the buffer thoroughly and mixing completely.
  • Non-specificity of primers. Verify the primers by blast or redesign the primers.
  • Primer amount is quite high. Just lower the amount of primers.
  • Too much template. Use <50ng plasmid DNA and <200ng genomic DNA.
  • Exogenous DNA contamination. Just be careful.
• Smeared products
  • Needing hot start. Using hot start DNA polymerase.
  • Too much template. Use <50ng plasmid DNA and <200ng genomic DNA.
  • Too many enzymes. Lower the DNA polymerase amount.
  • Too many cycles. Reduce the cycle number to 30.
  • Primer concentration is not optimal. Repeat the reactions with the gradient amount of primers.
  • Bad primers. Primer blast or redesign primers.
• Band in the negative control

  Reagents, pipettes, work area may be contaminated. Use new reagents and pipettes. Change or clean the work area.

• Just a band of wrong size
  • Contamination. Use new reagents and pipettes. Change or clean the work area.
  • Using wrong templates or primers. Check primers and templates.
  • Gene isoforms. Sequencing and blast.