About this calculator
The Annealing temperature calculator is a molecular biology tool, which aims to assist you in finding the optimum temperature (in PCR experiments) at which the primer will bind. The calculator uses the DNA sequence, length of primers and GC content to estimate the melting temperature (Tm) and subsequently propose an appropriate annealing temperature (Ta).
What is PCR?
One of the most significant methods in the contemporary biology and genetics is Polymerase Chain Reaction (PCR). PCR is a technique that enables scientists to obtain millions of copies or billions of precise copies of a given sequence of DNA using a very minimal sample of the DNA. In science and medicine, a lot of things that we have grown accustomed to today, including genetic testing, diagnosis of diseases, forensic examinations, and even testing COVID-19, would not be feasible without the use of PCR.
How PCR Works
PCR is a thermal cycling method, that is the sample is heated and cooled several times to induce various reactions. There are three primary steps in every cycle:
- Denaturation (≈ 94–98 °C) At this point, the DNA breaks down into two single strands by disrupting hydrogen bonds between bases of the two strands of a double-stranded DNA molecule. This leaves the DNA available in the next step.
- Annealing (≈ 50–65 °C) A reaction mixture of this is cooled such that primers, short, synthetic pieces of DNA built to complement the target sequence, can bind to (or anneal) to complementary regions on the single-stranded DNA. This is the crucial process in which the annealing temperature is important. Excessively low and primers might get stuck at the inappropriate locations. Excessively high and they do not bind.
- Extension (≈ 72 °C) In this case, the DNA polymerase enzyme (which is commonly the Taq polymerase of Thermus aquaticus bacterium) grows new strands of DNA by adding nucleotides in the primers.
One cycle multiplies the quantity of DNA by two. One copy of DNA may increase to more than a billion copies after approximately 30 cycles.
What is the PCR annealing temperature?
The PCR annealing temperature (Ta) is the temperature at which primers bind (hybridize) to their complementary sites on single-stranded DNA in the thermal cycle; this is set at a temperature slightly lower than the primer melting temperature (Tm), so it binds specifically and stably; most often Ta = Tm − 3–5 °C and typically 50–68 °C. Ta depends on the length of the primer and GC content (higher GC content causes higher Tm), the concentration of salt and Mg²⁺ ions (higher ionic strength stabilizes duplexes and raises Tm), the concentration of the primer, the mismatch at the binding site (which reduces effective Tm), and primer additives such as DMSO or formamide (which lower Tm). The selection of Ta too low gives non-specific binding and spurious products; the selection of Ta too high gives less binding of the primer and weak or no amplification. To get predictable values, you should calculate Tm by nearest-neighbor methods as much as possible, Ta ≈ Tm − 3–5 °C to start with, and test the best Ta empirically using gradient PCR under your real reaction conditions.