Applications of polymerase chain reaction (PCR)

July 12, 2018

1. Cloning genes

It is used to amplify the gene which can then be inserted into a vector.
The DNA can then be transferred into an organism (the GMO) where the gene and its product can be studied more closely.
Expressing the cloned gene can also be away of mass producing useful proteins e.g. medicines or enzymes.
A normal cloned gene can be compared with an un-cloned mutant form of gene.

Image source: igenelab

2. Detection of low abundance of nucleotide sequences

Viruses have long latency periods (e.g. HIV).
So they are difficult to detect at the early stage of infection.
But PCR offers rapid and sensitive method for detection when small proportion of cell is harboring the virus.

3. Adding novel DNA sequences to the ends of a PCR amplified sequence

It can be used to add specific sequences to the end of a DNA molecule.
These specific sequences can be restriction enzymes sites or any other sequence of interest.
In cycle 1, the added bases simply do not hybridize to anything.
In cycle 2, the bases are replicated, effectively making them a part of PCR products.
In subsequent cycles, the added sequence will be replicated as part of the DNA molecules.

4. Genetic fingerprinting

In forensic analysis of DNA samples, DNA from a single human hair is sufficient to determine whether the sample comes from specific individual.
Except for identical twins, fingerprints are unique.
In forensic technique, DNA (e.g. blood, semen, saliva or hair) etc. from a crime scene can be genetically compared to sample from a suspect.
Likewise genetic fingerprints are also used for paternity tests.

5. Detection of hereditary diseases

Each gene in question can be easily amplified through PCR by using the appropriate primers and then sequenced to detect mutations.
Viral diseases can also be detected months before the actual symptoms occur.

Image source: thebiologynotes

6. Mutagenesis

It is used to access the function of a gene or in vitro protein evolution.
Mutation can be introduced into copied DNA sequences in two fundamentally different ways in the PCR process:

a) Site directed mutagenesis

PCR can be used to introduce a specific mutation into a specific base pair in a cloned gene.
A primer is designed that contain the mutant base pair in place of the wild type base pair.
The mutant primer is used in PCR reaction with the plasmid containing the cloned gene.
PCR is used to synthesize the entire plasmid after several rounds of replication, the large majority of plasmid molecules contain mutation.
This mixture of mutant and wild type molecules is transformed into E. coli and the plasmids in individual colonies are tested for the presence of the mutation by determining the DNA sequence of the cloned gene.

b) Random mutagenesis

It is based on the use of error prone polymerases in the PCR processes.
In the case of random mutagenesis, the location and nature of mutation cannot be controlled.
One application of random mutagenesis is to analyze structure-function relationships of a protein.
By randomly altering a DNA sequence, one can compare the resulting protein with the original and determine the function of each part of protein.

7. Other uses