Optimization of Annealing Temperature and other PCR Parameters

Nowadays there are many different suppliers and systems for polymerase chain reaction (PCR) and of its derivative techniques, quantitative, or real-time, PCR (qPCR) and reverse transcription PCR (RT-PCR). Competition gives you the opportunity to simplify diagnostics and to make research more financially favorable.  Genaxxon constantly addresses these challenges.

Suppliers endeavor to provide kits that can be adapted to existing systems. However, it is never possible to achieve 100% compatibility.  The use of different buffer systems and buffer compositions (salts in the reaction buffer) which affect the net pH value, collectively influence the respective annealing temperatures in a given PCR reaction.  It is our experience that end-users underestimate the influence of the annealing temperature on the fidelity of PCR, real-time PCR and RT-PCR, and very often this is the last variable which is changed in the PCR protocol. It is incorrect to assume that a predicted Tm (melting temperature of primers) that is provided with each oligonucleotide/primer/probe, will remain constant, regardless of the kit used.

For these reasons, it is unrealistic to expect that a long-used PCR protocol will generate the desired product with the same efficiency when used with reagents from an alternative source.  Therefore, it is critical that the protocol is systematically tested and optimized with the chosen kit.

It is possible to save time and money with careful planning. Firstly, any new kit, regardless of cost or supplier should be tested using gradient PCR. This will identify the optimum annealing temperature in a given set of conditions to be identified, and it will reveal the parameters that will minimize the incidence of spurious artefacts. Usually the difference in the annealing temperature between different systems is only 2-3°C, but the effect is enormous and may lead to a completely negative result. Unexpected negative results may reflect a flawed experimental design and/or sub-optimum reagent concentrations. We hope that the following cost-effective suggestions will assist you in troubleshooting problems that may arise with your PCR protocols.

DNA Template

Amplification of templates with high GC content, strong secondary structure, low concentrations, or which produce products greater than 5 kb, may require adaptation of the following parameters:

  • Use high quality, purified DNA templates.
  • Approximately 10E4 copies of target DNA are required to detect product in 25-30 PCR cycles.
  • Use 1pg–1ng of plasmid or viral templates.
  • Use 1ng–1µg of genomic templates.
  • Higher DNA concentrations decrease amplicon specificity (i.e., extra bands are more likely), particularly when a large number of cycles are employed.
  • Use the higher DNA concentrations when fewer cycles are desired (e.g. to increase fidelity).


  • Generally 20-30 nucleotides in length.
  • Ideal GC content is 40-60%.
  • Space GC residues evenly within the primer.
  • Primer pairs should have Tms within 5°C of each other.
  • Avoid secondary structure (i.e., hairpins) within each primer and potential dimerization between the primers present.
  • When engineering sites into the end of primers, 4-6 extra bases should be added 5´ to the site.
  • Final concentration should be 0.05-1 µM, typically 0.1-0.5 µM of each primer.
  • Higher concentrations may increase secondary priming and create spurious amplification products.

Magnesium Concentration

  • 1.5-2.0 mM is optimal for Taq DNA Polymerase, but the ideal concentration depends on template, buffer, DNA and dNTPs (each has the potential to chelate magnesium).
  • If [Mg2+] is too low, no PCR product will be seen.
  • If [Mg2+] is too high, undesired PCR products may be seen.

Deoxyribonucleotide triphosphates (dNTPs) >

  • Typical concentration is 200 µM of each dNTP.
  • 50-100 µM enhances fidelity of polymerization, but reduces yields.
  • Higher concentrations increase yields particularly in long PCR, but can reduce fidelity.

DNA Polymerase

  • The choice of the correct polymerase depends among other things on the purpose as well as on the template used (standard PCR: Taq DNA Polymerase S > with high accuracy, Taq Polymerase E > with high yield; master mixes: Standard PCR master mix > or RedMasterMix > with red dye).
  • For multiplex PCR, there exist special multiplex master mixes >.
  • Hot start applications are recommended to increase specificity or if you use difficult templates. For this purpose there exist special Hot Start Polymerases >.
  • For PCRs for the purpose of cloning or other procedures requiring a low error rate, there are thermostable high fidelity proofing polymerases such as Pfunds >, ExactRun >, ReproFast >, or ReproHot (KOD) Proofreading Polymerase >. These enzymes make far fewer errors during amplification and increase the chances of an amplicon without mistakes.
  •  For genotyping and other applications where a high discrimination rate is required, there is a new highly selective DNA polymerase, SNP Pol DNA Polymerase >. It specifically distinguishes mismatched primer-template complexes and ony produces specific amplicons in case of perfectly matched primer pairs.
  • For the real-time quantitative PCR > there exist highly specialized qPCR master mixes. Hereby, the choice of the correct master mix depends on the qPCR device you are using. It is important to determine whether and how much ROX should be contained in the qPCR master mix and whether it should be a qPCR green master mix with green fluorescent dye > or a qPCR probe  master mix > without a fluorescent dye. A new lyophilized qPCR probe master mix in beads format, which is stable at room temperature, is available from Genaxxon (LyoBalls >).
  • The amount of DNA polymerase used in the PCR reaction can significantly influence the PCR result (use 1.25-1.5 units Taq Polymerase > for a 50μL volume).

General Guidelines

Annealing Temperature and Duration

  • Match the Tms within 5°C of each other.
  • Typical annealing temperatures are 5°C below the lowest primer's Tm and often fall in the range of 50-60°C.
  • Test higher annealing temperatures if spurious amplification products are observed.
  • Typical annealing times are 15-30 seconds.

Extension Time

  • Extensions are normally performed at 68°C.
  • In general, use extension times of one minute per 1000 base (1 kb) pairs (e.g. 3 minutes for a 3 kb product). 
  • For products less than 1 kb, use 45-60 seconds.
  • Products greater than 3 kb, or reactions using more than 30 cycles, may require longer extensions.

The amplification of templates with high GC content, strong secondary structures, low concentrations or amplicons more than 5 kb often require optimization of the PCR conditions. Typically, 15-30 seconds of denaturation should be performed at 95 ° C during PCR.

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