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The different Phases of PCR and Why They Are Important

The different Phases of PCR and Why They Are Important

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.

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