Question

Explain the three steps involved in each cycle of polymerase chain reaction.

Answer 1

Hint: Polymerase Chain Reaction (PCR) is a laboratory based technique that is widely used to create multiple copies of DNA from a particular DNA section. It was first developed in the year 1983 by Kary Mullis.

Complete answer:
PCR uses:
- DNA template (target DNA whose copies are to be made)
- DNA polymerase enzyme (Taq DNA polymerase enzyme which is heat stable)
- Primers (short sequences of DNA or RNA that copy template DNA)
- Free nucleotides

During PCR - DNA template, Taq DNA polymerase, primers and free nucleotides are placed in a test tube which is kept in a PCR machine. A PCR machine is an automatic machine that can increase or decrease the reaction temperature in a programmed manner. A PCR is a cyclic process and a single PCR cycle involves three main steps.

These steps are as follows:
Denaturing:
It is the first step in Polymerase Chain Reaction (PCR). During this step, the DNA fragments are heated to a high temperature of about 94ᵒC to 95ᵒC. As a result of heating, the hydrogen bonds present between the two strands of DNA are broken down. This separates the two strands of DNA into two single strands. This process of separation of DNA strands is called denaturation. Each strand of DNA can now be used independently as a template to create multiple copies of DNA. The temperature is maintained in the range of 94ᵒC to 95ᵒC during the entire process till the two strands of DNA get separated from each other completely. This generally happens in 15 to 30 seconds.

Annealing:
It is the second step. This step is meant to allow the binding of primers to DNA templates. Primers are the short strands of DNA or RNA. Primers consist of just 20 to 30 nitrogen bases. During this step, the temperature is lowered and the reaction is cooled at 50ᵒC to 65ᵒC. This cooling enables the primers to attach to specific regions over DNA templates by forming hydrogen bonds. This attachment of primers is called annealing. When this happens, the DNA polymerase enzyme binds and starts synthesizing new complementary strands of DNA starting from primers. DNA polymerase uses free nucleotides for this purpose. There are two types of primers that are used – a reverse primer and a forward primer. The two primers are required as the two separated DNA strands are complementary that run in opposite directions. Annealing usually completes in 10 to 30 seconds.

Extending:
This is the last and final step. During this step the temperature is again increased to 72ᵒC. This is the optimum temperature for DNA polymerase (Taq polymerase). The Taq polymerase now starts working optimally. It extends the primers and finally a single cycle of PCR gets completed. At the end of each PCR cycle, each double stranded DNA molecule contains one old and one new DNA strand.

Note: Polymerase Chain Reaction (PCR) is a cyclic process. Each cycle of PCR involves three steps – denaturing, annealing and extending. At the end of each PCR cycle, the number or pieces of double stranded DNA gets doubled. The number of DNA copies after n cycles of PCR can be calculated by using formula - 2^n. For example - after 10 PCR cycles, the number of DNA copies will be 2^10 = 1024. The number of PCR cycles to be conducted depends on the requirement i.e. how many DNA copies are required. Most of the requirements are fulfilled in about 25 to 35 PCR cycles.