Prof. Dr. Sevgi ERTUĞRUL KARATAY
PCR (Polymerase Chain Reaction)
It is a method for enzymatically amplifying a specific region between two segments of known sequence within the genomic DNA of any organism. The device used in the PCR process is called thermal cycler.
It was first discovered by Kary Mullis in 1985.
Usage Area of PCR:
Sequence analysis of DNA and DNA mapping Diagnosis of genetic diseases
Determination of tissue type for tissue transplantation by demonstrating allele sequence variations
Genetic typing of forensic samples Agricultural research
Molecular biological studies (cloning, etc.)
PCR- Basic Components
Template DNA
DNA primers specific to the target region (forward and reverse primers) DNA polymerase enzyme (Taq Polymerase)
dNTPs (dATP, dTTP, dGTP, dCTP) Buffer
Mg ++ ion Ultra pure water
PCR- Basic Components
PCR consists of three main phases.
Stage 1: Denaturation
At this stage, double-strand DNA becomes single-strand at 95 C. ̊
PCR- Basic Components Stage 2: Anneling
Afer denaturation, the temperature is lowered to 55-60 C ̊ depending on the primer length and the primers specifically bind to the separated target DNA strands
The forward primer binds to the 5 ’end of the target DNA. The reverse primer is also connected to the 3 ’section.
The bonding temperature is generally selected as a value below the melting temperature of the primers.
PCR- Basic Components Stage 3: Extension
Starting from the region where the primers bind, DNA is amplified by the Taq polymerase enzyme.
The best operating temperature of Taq polymerase enzyme is 72 C.̊
The Taq polymerase enzyme introduces nucleotides into the 3
’end of the primers using nucleotides in the 5’-3’ direction. This results in a copy of the target DNA.
PCR- Basic Components
Primers are normally designed as at least 18-20 base pairs.
This allows the primers to bind to a single site in the target DNA.
Taq polymerase uses magnesium as a cofactor. Therefore, magnesium concentration is extremely important for PCR.
The Taq polymerase enzyme is derived from a thermophilic bacterium called Thermus aquaticus.
PCR-Types
Touchdown PCR Nested PCR Anchored PCR Asymmetric PCR
Multiplex PCR
Reverse transcription PCR Real time PCR
etc.
