The polymerase chain reaction, or PCR is nothing short of a miracle in the field of biotechnologies: learn how to optimize this beautiful technique with our PCR annealing temperature calculator!
Here you will learn everything you need to know about PCR, the materials needed, and the process itself. We will answer the following questions:
- What is the annealing in PCR?
- How do I find the annealing temperature for the best results?
- How do I use our primer annealing temperature calculator? (don't worry, it's extremely simple!)
Keep reading to learn more!
A brief overview of DNA structure
🧬 DNA — deoxyribonucleic acid — is the manual of life on Earth. It's a simple molecule that contains the instructions to build a perfectly functional organism, from bacteria 🦠 to raccoons 🦝. Its structure is the famous double helix, where two strands of a biopolymer link and twist. The structure is similar to the single helix, one of the other nucleic acids fundamental to life, the RNA (ribonucleic acid).
You can picture a DNA molecule as a twisted ladder. Each step is made of a pair of molecules called nucleotides, each one in turn composed of a nucleoside and a phosphate. The steps are linked through the latter one. There is more: each nucleoside contains a nitrogenous base and a five-carbon sugar. Before the next step, let's check it out again:
- DNA = sequence of paired nucleotides;
- Nucleotide = nucleoside + phosphate; and
- Nucleoside = nitrogenous base + five-carbon sugar.
A really brief explanation of DNA replication
The cells in your body and every cell on this planet (apart from some exceptions) constantly follow a reproduction cycle (we have a tool to help you with counting them — the cell doubling time calculator). The process involves a copy of the genetic code (the DNA) to create two cells with a complete set of genes, the instructions needed to "build" an individual. The process itself is complicated, to say the least: we will be able to explain it only briefly to introduce the actors of the PCR.
How to use our PCR annealing temperature calculator
We will quickly teach you how to use our optimal primer annealing temperature calculator!
Let's go for an example. We chose a strand of DNA from a cat gene from an online repository. The target strand we looked for is ~ 2 kb, where kb means kilobases, or a thousand nucleotides. We are NOT going to write it here!
Ta = 0.3 · Tmp + 0.7 · Tmt - 14.9
= 66.77 °C
FAQs
What is the annealing in PCR?
The annealing step in PCR is the second stage of a thermal cycle where the target DNA has its terminations paired with the two 5' → 3' primers, from which the enzyme polymerase can start the replication.
What is the primer annealing temperature in PCR?
The annealing step must happen at a specific temperature to ensure a good matching between the two strands. The annealing temperature lies in the range between 55 and 60 degrees Celsius.