The main purpose of gene cloning is to study genes: their structure, function and regulation. This information can be used to alter genes and modify properties of the organism.Technically speaking, gene cloning is used most frequently to amplify one particular gene. Having one particular gene in our "hands" we can study it using various biochemical techniques. PCR - polymerase chain reaction is a new method that provides an alternative way to amplify a specific gene from any DNA that contains it.
PCR was invented by Kary Mullis
Two underlying principles that make PCR possible are:
1. DNA hybridization - a short DNA oligonucleotide can form a duplex with the complementary sequence in a single-stranded DNA molecule.
2. DNA polymerase cannot initiation DNA replication, but can only "continue" it from a primer.
An oligonucleotide hybridized to a complementary sequence in the template DNA strand can be recognized as a primer by DNA polymerase, which can then synthesize a new DNA strand.
Thermostable DNA polymerase is used for the PCR. This enzyme, which is isolated from thermophilic bacteria, like for example Thermus aquaticus, does not denature at high temperature and remains active through the entire PCR reaction. Therefore, in order to perform PCR we just need to mix in the reaction tube:
1. DNA containing the gene to be amplified
2. Primers complementary to the flanking regions of the gene
3. Thermostable DNA polymerase
4. Nucleoside triphosphates (dATP, dGTP, dCTP, dTTP)
and then place the reaction mixture in the PCR machine that can rapidly change the temperature of the reaction mixture.
The newly synthesized fragments serve as templates for new rounds of DNA replication. After several rounds, the predominant DNA is identical to the sequence bracketed by and including the two primers. In each PCR cycle, the number of copies of DNA sequence bracketed by and including the two primers is duplicated.
PCR has multiple applications in Gene cloning, Biodiversity studies, Diagnostics, Forensics, etc.
A gene can be directly amplified from the chromosome if flanking sequences are known
BIODIVERSITY
Studying nucleotide sequences of DNA collected directly from Nature gives us an idea of diversity of microorganisms and their evolutionary relationships
DIAGNOSTICS
FORENSICS
