Polymerase chain reaction (PCR) is an efficient and cost-effective molecular tool to copy or amplify small segments of DNA or RNA. PCR combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are applied repeatedly through numerous cycles. It results in the exponential production of the specific target DNA/RNA sequences by a factor of 10^7 within a relatively short period.
This in vitro amplification technique can amplify a single copy of nucleic acid target by using two synthetic oligonucleotide “primers” that bind to the target genomic sequence, which are extended by a Taq polymerase (a thermostable DNA polymerase) . An automated process of repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94°C), annealing of primers to their complementary sequences (50°C), and primer extension (70°C) is employed for the amplification of target sequence.
PCR was originally developed in 1983 by the American biochemist and Nobel Laureate Kary Mullis.
Primer: A short segment of nucleotides, which is complementary to a section of the DNA or RNA, which is to be amplified in the PCR. Two short DNA sequences designed to bind to the start (forward primer) and end (reverse primer) of the target sequence is used in PCR.
Taq polymerase: A thermally stable DNA polymerase originally isolated from the thermophilic bacterium Thermus aquaticus, which resist inactivation during denaturation temperatures and allows primer extension at high temperature.
Components of Polymerase Chain Reactions (PCR)
I was too excited when our lab got the Real-Time PCR first time (File photo).
- DNA template (the sample DNA that contains the target sequence)
- Deoxyribonucleoside triphosphates (dNTPs)
- PCR buffer
- Primers (forward and reverse)
- Taq polymerase
Steps of polymerase chain reaction-PCR
To perform PCR, extracted sample (which contains target DNA template) is added to a tube containing primers, free nucleotides (dNTPs), and Taq polymerase. The PCR mixture is placed in a PCR machine. PCR machine increases and decreases the temperature of the PCR mixture in automatic, programmed steps which generates copies of the target sequence exponentially.
Polymerase Chain Reaction (PCR) has three major steps.
- Denaturation (strand separation) : The separation of the two hydrogen-bonded complementary chains of DNA into a pair of single stranded polynucleotide molecules by a process of heating (94°C to 96°C)
- Annealing (primer binding): The temperature is lowered (45-60 °C) so the primers can attach themselves to the single stranded DNA strands.
- Extension (synthesis of new DNA): It starts at the annealed primer and works its way along the DNA strand (72°C).
Once the first round is completed, the process is repeated by cycling back to the first reaction temperature and next round of denaturation, annealing and extension is started(an automatic process in thermocycler). This 3 steps temperature cycle is repeated approximately 30 times which results exponential amplification of target gene sequence.
Detection of PCR products
Labeled probe that is specific for the target gene sequence is used to detect PCR amplified gene product (also known as amplicon). Based on the nature of the reporter molecule used, probe generates radioactive, colorimetric, fluorometric, or chemiluminescent signals. Probe based detection of amplicons serves two purposes
- It allows visualization of the PCR product
- It provides specificity by ensuring that the amplicon is the target sequence of interest and not the result of non-specific amplification.
Apart from DNA based hybridization method, sometimes simple gel electrophoresis method is sufficient to confirm the presence of specific amplicons.
Types of polymerase chain reaction-PCR
Several modification of PCR methods have been developed to enhance the utility of this method in diagnostic settings based on their applications. Some of the common types of PCR are;
- Real-Time PCR
- Nested PCR
- Multiplex PCR
- Quantitative PCR
- Arbitrary Primed PCR
Applications of PCR
- Identification and characterization of infectious agents
- Direct detection of microorganisms in patient specimens
- Identification of microorganisms grown in culture
- Detection of antimicrobial resistance
- Investigation of strain relatedness of pathogen of interest
- Genetic fingerprinting (forensic application/paternity testing)
- Detection of mutation ( investigation of genetic diseases)
- Cloning genes
- PCR sequencing
References & further readings:
- Animation source: dnalc.org
- Image source: Wikipedia.org
- Bailey& Scott’s Diagnostic Microbiology- 12th Edition