Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is molecular biological technique used for amplifying (creating copies of) DNA without the use of living organisms such as E coli, yeast. It is used in medical and biological research laboratories for detection of hereditary diseases, diagnosis of infectious diseases, identification of genetic fingerprints, cloning of genes and paternity testing.

The concept of PCR was at first put forward by H Ghobind Khorana et al in 1971 but is seemed to be impractical before gene sequencing and viable thermostable DNA polymerase. Later, after 15 years in 1986 Kary Mills developed the PCR technique. PCR is a process by which DNA is artificially multiplied through repeated cycles of duplication in the presence of DNA polymerase.

The PCR process was patented by Cetus Corporation where Kary Mills worked where he developed the technique. Taq polymerase enzyme was also covered by the patent. The pharmaceutical company Hoffmann-La-Roche purchased the right to patent in 1992 and currently holds them.

DNA polymerase occurs naturally in living organisms and functions to create copies of DNA when cell divides. It functions by binding to single stranded DNA and creating complementary strand. The original concept of PCR technique developed by Mills uses the enzyme in vitro. Double stranded DNA was separated into two single strands by heating at 96 degree C. However, at this high temperature, DNA polymerase was destroyed and required to be replenished after heating stage of each cycle. Thus, it required great deal of time, large amount of DNA polymerase and continued attention throughout the PCR process.

Later, this PCR process was modified by using DNA polymerase obtained from thermophilic bacteria that grow in geysers at 110 degree C. This DNA polymerase was thermostable and do not break down when the reaction mixture was heated to separate strands.

The first thermostable DNA polymerase was obtained from Thermus aquaticus and called Taq polymerase. One of the disadvantages of this Taq polymerase was that it sometimes maked mistakes while making copies of DNA leading to mutation of DNA sequences since it lacked 3’-5’ proofreading exonuclease enzyme. The polymerase Pwo and Pfu obtained from Archaea contained exonuclease enzyme and reduced the mistakes while making copies of DNA. The combination of Taq and Pfu is available now a days that provides both fidelity and accurate amplification of DNA.

PCR amplifies short, well defined DNA fragment. It requires a single gene or just a par of gene. As opposed to living organism, PCR can make copies of only short DNA fragment upto 10 kb ie 1000 base pairs. DNA is double stranded and therefore it is measured as complementary DNA building block (nucleotides as base pairs).

PCR requires
DNA template containing the region of DNA fragment to be amplified
Two primers determining the beginning and end of DNA fragment to be amplified
DNA polymerese to make copies of DNA fragment to be amplified
Nucleotide from which DNA polymerase synthesize DNA strand
Buffer for creating optimum chemical environment for DNA polymerase to perform

PCR is carried out in thermal cycler. It is a machine that cools and heats the reactions tubes within it in precise temperature that is required for each step of the reaction. Evaporation of the reaction mixture is prevented by placing heated lid on reaction tube or by placing thin oil layer on the reaction mixture.

Primer
DNA fragment to be amplified is determined by selecting the primer. Primers are artificial, short DNA strands upto 50 nucleotides that exactly match the beginning and end of the DNA strand to be amplified. They anneal with DNA template at these beginning and end points and DNA polymerase binds and begins synthesis of DNA strand.

The choice of the length of the primers and their melting temperature depends on several considerations. Melting temperature of primer- not to be confused with the melting temperature of DNA at firs step of PCR- is the temperature at which half of the primer binding sites would be occupied. Melting temperature increases with the length of the primer. Short primers would anneal at several points on the long DNA template resulting non specific copies. On the other hand, length of the primer is limited by melting temperature at which it melts. High melting temperature above 80 degree C will cause problem since the DNA polymerase is less active at this high temperature. The optimum length of primer is 20 – 40 nucleotide wit melting temperature of 60 – 75 degree C.

PCR has a series of 20-30 cycles and each cycle consists of 3 steps-
1st step- Double stranded DNA is heated at about 94-96 degree C to separate the strands. This step is called denaturation and breaks apart the hydrogen bond that binds together two DNA strands. Prior to the first cycle, DNA is denatured for extended time period in order to ensure that both template DNA and primers are separated into single strand. The time of this step is usually 1-2 minute/s.

2nd step- After denaturation, temperature is lowered so that primer anneals with the single stranded DNA. This step is called annealation. Temperature of this step depends on the primers and is usually 5 degree C below their melting temperature. Wrong temperature at this step causes primer not to bind with DNA template or binding at random. Time of this step is 1-2 minute/s.

3rd step- After annealation, DNA polymerase has to fill the missing strands. DNA polymerase binds at annealed primer and works its way along the DNA fragment. This step is called elongation. Temperature of this step depends on DNA polymerase. However, time of this step depends both on the DNA polymerase itself and the length of DNA fragment to be elongated. Usually by the rule of thumb, the time of this step is 1 minute for every 1,000 bp.

The PCR product is identified by its size using Agarose gel electrophoresis. The sixe of PCR product is determined by comparing it with DNA ladder.

Uses of PCR
Genetic fingerprinting
Detection of hereditary disease
Cloning of genes
Analysis of ancient DNA
Paternity testing
Genotyping of specific mutation
Mutagenesis
Comparison of gene expression

Genetic fingerprinting is a forensic technique to identify a person by comparing his/her DNA with a sample e.g:- urine, semen, saliva, blood, hair from crime scene can be genetically compared to the blood of suspect.
Genetic fingerprint is unique except for identical twins
Genetic relationship can be determined by comparing two or more genetic fingerprints for paternity test
A slight variation of this technique can be used to determine evolutionary relationship between organisms.