Molecular Biology

What is the Polymerase Chain Reaction



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What is the Polymerase Chain Reaction?

The Polymerase Chain Reaction (PCR) is a molecular biology technique use to amplify a single or a few copies of a piece of DNA. It has many uses in modern sciences, for example protein engineering, cloning, forensics (DNA Fingerprinting) and for analysis of environmental samples. The process was first developed in 1987 by a team led by Kary Mullis. The original DNA is replicated by DNA polymerase, the enzyme in our bodies that is used for DNA replication, doubling the number of DNA molecules. A second cycle is carried again doubling the number molecules. A ‘chain reaction’ starts exponentially increasing the amount of DNA molecules present. This makes it possible to gain sufficient DNA for a specific task from a single drop of blood or strand of hair.


The Process of the Polymerase Chain Reaction.

Requirements:

Thermal Cycler PCR amplification mix (usually consisting of :) Sample dsDNA with a target sequence Thermostable DNA polymerase 2 olignucleotide primers complimentary to bases on the target sequence Deoxynucleotide triphosphates (dNTP’s) Reaction buffer containing a mixture of magnesium ions and other components.

3 Stages:

Heat Denaturation

A DNA sequence is denatured by heat of about 90-95°C. The hydrogen bonds holding the strands together break so they unravel and separate.

Primer Annealing

In the presence of excess dNTP’s (the ‘building blocks’ of new DNA material), olignucleotide primers are added. The primers are complementary to the target strand but on opposite strands. As the mixture cools (50-65°C) each strand of DNA molecules becomes annealed with an olignucleotide primer complementary to either end of the target sequence.

Primer Extension

DNA polymerase is then added and complementary strands are synthesized at a temperature of 60-75°C. The polymerase causes synthesis of new material in the direction away from each of the primers.


This cycle of steps 1, 2 and 3 is then repeated as many times as needed to create a large enough amount of copied DNA material for the specific job that is needed doing.


The Uses of the Polymerase Chain Reaction.

The Polymerase Chain Reaction is most commonly used in the field of Forensic Sciences when needing to multiply the amount of DNA to create a large enough amount for processing. There are several variations on the PCR process for use in other fields though. For example in medicine, one method allows us to check for RNA viruses.

In forensics, the use of PCR can be done when regarding current or cold cases. Due to the ability to amplify degraded DNA to a fair amount, historical cases have been re-examined using the technology, such as the identification of victims of the Holocaust and the murder of Nicholas Romanov, the Tsar of Russia who was overthrown in the Russian Revolution. The Holocaust searches are for comparing DNA of current people to find if their relatives were murdered, whereas the Tsar case was to see if the remains found were who they thought it was.

In the case of identification using DNA is alright as you can take a sample from the other person. But in cold cases with no DNA to compare it to, you have to hope that the DNA is on the national database otherwise identification is nigh on impossible.

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