Annealing Temperature Calculator
Find out how to optimize the Polymerase Chain Reaction taking help of the PCR Annealing Temperature Calculator over here. All you have to do is simply enter the Primer Melting Temperature, Target Melting Temperature in input fields and click on the Calculate button to avail the output Appealing Temperature in no time.
Polymerase Chain Reaction - Definition
Polymerase Chain Reaction(PCR) is a technique that oermits multiplication of small strand of DNA into a huge amount of identical copies just by providing the right enzymes, DNA Building Blocks. This Process in general uses a DNA Replication Phenomenon which when controlled targets a single fragment of genetic code and amplifies its signal.
DNA Structure
DNA is a single molecule that builds a perfect organism and has a structure of double helix. In this two strands of a biopolymer link and twist. If the structure is a single helix and one among the nucleic acids is fundamental to life it is known as Ribo Nucleic Acid. Each step in the DNA Molecule is made of pair of molecules known as Nucleotides. In addition each turn is further made of nucleoside and phospate. Each nucleoside in turn made of nitrogenous base and a five-carbon sugar.
- DNA = sequence of paired nucleotides;
- Nucleotide = nucleoside + phosphate; and
- Nucleoside = nitrogenous base + five-carbon sugar.
Pairing happens at connection between bases. There are 4 kinds of bases and they pair in the following way
- Adenine, A + T, Thymine; and
- Guanine, G + C, Cytosine.
Now that you have got an idea on DNA Structure check our further modules to be aware of DNA Replication as well in detail.
DNA Replication
Cells in the body follow a certain reproduction cycle and the process involves DNA in the creation of two cells having complete genes needed to build an individual. In the initial stage of DNA Replication DNA double helix is firstly flattened and then divided into two strands.Each strand exposes its bases so as to allow a complex group of enzymes to recreate complementary strands.
After the double helix opens priming of the strand happens and enzymes can't directly copy on DNA. Short Segment of RNA is attached to the Target Strand and from then Enzyme called Polymerase starts getting new nucleotides which pair to the strand's ones and connects via Phosphate Bridge.
DNA Polymerase moves in the 5′→3′ direction and thus operates on one single strand of the seperated double helix. However, copy on wrong strand happens in small batches and joins afterwards. The replication continues further as long as the DNA's Double Helix Progresses and unravels entirely and we get two daughter filaments and both of them carries half of the parent molecule.
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Ingredients of PCR
PCR needs certain ingredients inorder to carry out successfully. They are as follows
- DNA Template is the ingredient that undergoes amplification.
- DNA Primers are single strands that come in two types and correspond to specific side of each end. Only After you know the genetic sequence of the target you can synthesize.
- Polymeraze Enzyme is the one that amplifies and attaches new nucleotides to the parent strands.
- Nucleotides are needed in the creation of copies and provide energy to enzymes.
- Buffer Solution is the environment in which the reaction takes place and get stablized by divalent cations such as Mg+2
Steps Involved in a Polymerase Chain Reaction
Let us look into the process of Polymerase Chain Reaction. Firstly, combine all your ingredients into vial and let the process happen.
- Denaturation: In this particular step temperature is increased to 94–98 °C for about 20–30 seconds. It is in this step DNA Seperates in two single strands since hydrogen bonds between bases break.
- Annealing: In this step temperature is reduced to 50–65 °C for another 20-40 seconds. Here Primers attach to target DNA and it is the temperature that matches between target and primer.
- Elongation Phase: Here temperature rises to 75–80 °C for polymerase enzyme. Free Nucleotides Pair up with target DNA and creates new copy of original fragment.
After the Nucleotides gets depleted the process ends itself. To find out the number of fragments in a solution we use the formula NDNA = 2n. The increment is completely exponential and it all begings from a single fragment and is a pure chain reaction.
How to find the PCR Annealing Temperature?
Annealing Temperature is the temperature at which annealing step in a Thermal Cycle occurs. The value is dependant on denaturation temperature of both the target DNA and Primer. The formula to determine the Annealing Temperature is given by the expression Ta* = 0.3.Tmp+0.7.Tmt-14.9
- Where, Tmp = denaturation melting temperature of unstable primer.
- Tmt = Melting Temperature of Target
FAQs on Annealing Temperature Calculator Online
1. Should Annealing Temperature be lower than TM?
Yes, the Annealing Temperature should be less than TM.
2. What is meant by Annealing Temperature?
Annealing Temperature is the Temperature that occurs in the annealing step of a thermal cycle.
3. How do you Calculate the Annealing Temperature?
Annealing Temperature can be found using the formula Ta* = 0.3.Tmp+0.7.Tmt-14.9
4. What is a Normal Annealing Temperature?
A Normal Annealing Temperature ranges between 48-72°C.