What is the role of the 72 degree phase in a PCR reaction?

PCR (Polymerase Chain Reaction) is a biochemical technique developed by Kary Mullis in 1983 that is used to create large quantities of a sequence of DNA. Since this method of mass-producing DNA was first introduced, it has become significantly less labour intensive, more economical, and more routine. The technique relies on a few key players that team together to generate thousands of even millions of duplicates from only a few of even single of the DNA sequence of interest. These copies or duplicates are called amplicons.

There are 4 players involved in this process: a DNA template, DNA polymerase, primers, and nucleotides.  Each player has a unique and critical role to play. The DNA template is the sequence of interest to be amplified, for example a gene coding for a protein. The DNA polymerase used is a heat stabile enzyme (biological catalyst) that synthesises fresh strands of DNA complementary to the target sequence. The primers are short pieces of single-stranded DNA that bind specifically to the complementary 3’ end of the target sequence that allow the polymerase to synthesis new DNA. Nucleotide (dNTPs or deoxynucleotide triphosphates) are single units of the bases A, T, C, and G that act as ingredients to make new strands of DNA as DNA is composed of these 4 bases. If RNA is used rather than DNA, the RNA must be converted to cDNA (complementary DNA or DNA that is the complementary sequence to the RNA) using reverse transcriptase enzyme giving it the name reverse transcription PCR (RT-PCR).

The reaction can be divided into 4 steps:

Step 1: Separation- the two strands of the DNA double helix are “melted” apart to create single strands. This occurs at very high temperature of 94-96’C, hence why heat tolerate polymerases are used, such as Taq and Pfu).

Step 2: Annealing- the temperature is lowered to allow the primers to bind the DNA. Optimal primer binding temperatures vary but are usually around 60’C

Step 3: Synthesis- the temperature is raised to 72’C to allow the DNA polymerase enzyme to synthesis of new strands of complementary DNA from free nucleotides in solution.

Each step is extremely important and ensuring the correct temperature is programmed into the PCR thermocycler is vital. Different primers anneal at different temperatures and certain primer oligonucleotides are also better than other and so normally multiple primers are designed in silico that are of various lengths and each of these tested at various annealing temperature.

Each time the steps are fully completed, a cycle is said to be complete. The reaction may be cycled 20-40 times depending on the number of copies of the DNA target sequence required as well as the purity of the sample and the number of target sequences present in the original solution. At the end of cycle 1, if just one copy of the DNA sequence is present as a double-stranded molecule, the strands will have been separated, copied and will reanneal. This will create two partially double stranded DNA molecules. These partially double stranded DNA sequences contain the target sequence but also some adjacent DNA. In cycle 2, four partially double stranded molecules have been synthesised and again these contain both the sequence of interest and adjacent DNA. In cycle 3, 2 double stranded sequences are made that contain no contaminating adjacent DNA, alongside 6 partially double stranded target sequence-adjacent DNA molecules. The number of target sequence-only containing strands of DNA increases exponentially to create approximately a million copies by cycle 20, a billion by cycle 30 and a trillion copies by cycle 40 in a background of minute numbers of genes sequences not of interest.

What is PCR?

• The polymerase chain reaction (PCR) was originally developed in 1983 by the American biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work.
• PCR is used in molecular biology to make many copies of (amplify) small sections of DNA or a gene.
• Using PCR it is possible to generate thousands to millions of copies of a particular section of DNA from a very small amount of DNA.
• PCR is a common tool used in medical and biological research labs. It is used in the early stages of processing DNA for sequencing, for detecting the presence or absence of a gene to help identify pathogens during infection, and when generating forensic DNA profiles from tiny samples of DNA.

How does PCR work?

• The principles behind every PCR, whatever the sample of DNA, are the same.
• Five core ‘ingredients’ are required to set up a PCR. We will explain exactly what each of these do as we go along. These are:
  • the DNA template to be copied
  • primers, short stretches of DNA that initiate the PCR reaction, designed to bind to either side of the section of DNA you want to copy
  • DNA nucleotide bases (also known as dNTPs). DNA bases (A, C, G and T) are the building blocks of DNA and are needed to construct the new strand of DNA
  • Taq polymerase enzyme to add in the new DNA bases
  • buffer to ensure the right conditions for the reaction.
• PCR involves a process of heating and cooling called thermal cycling which is carried out by machine.
• There are three main stages:
  1. Denaturing – when the double-stranded template DNA is heated to separate it into two single strands.
  2. Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
  3. Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.
• These three stages are repeated 20-40 times, doubling the number of DNA copies each time.
• A complete PCR reaction can be performed in a few hours, or even less than an hour with certain high-speed machines.
• After PCR has been completed, a method called electrophoresis can be used to check the quantity and size of the DNA fragments produced.

Illustration showing the main steps in the polymerase chain reaction (PCR).
Image credit: Genome Research Limited

What happens at each stage of PCR?

Denaturing stage

• During this stage the cocktail containing the template DNA and all the other core ingredients is heated to 94-95⁰C.
• The high temperature causes the hydrogen bonds between the bases in two strands of template DNA to break and the two strands to separate.
• This results in two single strands of DNA, which will act as templates for the production of the new strands of DNA.
• It is important that the temperature is maintained at this stage for long enough to ensure that the DNA strands have separated completely.
• This usually takes between 15-30 seconds.

Annealing stage

• During this stage the reaction is cooled to 50-65⁰C. This enables the primers to attach to a specific location on the single-stranded template DNA by way of hydrogen bonding (the exact temperature depends on the melting temperature of the primers you are using).
• Primers are single strands of DNA or RNA sequence that are around 20 to 30 bases in length.
• The primers are designed to be complementary in sequence to short sections of DNA on each end of the sequence to be copied.
• Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a double strand of DNA. Only once the primer has bound can the polymerase enzyme attach and start making the new complementary strand of DNA from the loose DNA bases.
• The two separated strands of DNA are complementary and run in opposite directions (from one end – the 5’ end – to the other – the 3’ end); as a result, there are two primers – a forward primer and a reverse primer.
• This step usually takes about 10-30 seconds.

Extending stage

• During this final step, the heat is increased to 72⁰C to enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases.
• Taq DNA polymerase is an enzyme taken from the heat-loving bacteria Thermus aquaticus.
  • This bacteria normally lives in hot springs so can tolerate temperatures above 80⁰C.
  • The bacteria’s DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.
  • DNA polymerase from most other organisms would not be able to withstand these high temperatures, for example, human polymerase works ideally at 37˚C (body temperature).
• 72⁰C is the optimum temperature for the Taq polymerase to build the complementary strand. It attaches to the primer and then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction.
• The result is a brand new strand of DNA and a double-stranded molecule of DNA.
• The duration of this step depends on the length of DNA sequence being amplified but usually takes around one minute to copy 1,000 DNA bases (1Kb).

• These three processes of thermal cycling are repeated 20-40 times to produce lots of copies of the DNA sequence of interest.
• The new fragments of DNA that are made during PCR also serve as templates to which the DNA polymerase enzyme can attach and start making DNA.
• The result is a huge number of copies of the specific DNA segment produced in a relatively short period of time.

Illustration showing how the polymerase chain reaction (PCR) produces lots of copies of DNA.
Image credit: Genome Research Limited

This page was last updated on 2021-07-21

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