Polymerase chain reaction (PCR )

Polymerase Chain Reaction (PCR)- Principle, Steps, Applications

• PCR is an enzymatic process in which a specific region of DNA is replicated over and over again to yield many copies of a particular sequence.
• The most widely used target nucleic acid amplification method is the polymerase chain reaction (PCR).
• This method combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication applied repeatedly through numerous cycles.
• This method is able to amplify a single copy of a nucleic acid target, often undetectable by standard hybridization methods, and multiply to 10⁷ or more copies in a relatively short period.
• This thus provides ample target that can be readily detected by numerous methods.

Principle of PCR

The target sequence of nucleic acid is denatured to single strands, primers specific for each target strand sequence are added, and DNA polymerase catalyzes the addition of deoxynucleotides to extend and produce new strands complementary to each of the target sequence strands (cycle 1). In cycle 2, both double-stranded products of cycle 1 are denatured and subsequently serve as targets for more primer annealing and extension by DNA polymerase. After 25 to 30 cycles, at least 10⁷ copies of target DNA may be produced by means of this thermal cycling.

Requirements for PCR

• A PCR reaction contains the target double-stranded DNA, two primers that hybridize to flanking sequences on opposing strands of the target, all four deoxyribonucleoside triphosphates and a DNA polymerase along with buffer, co-factors of enzyme and water.
• Since the reaction periodically becomes heated to high temperature, PCR depends upon using a heat-stable DNA polymerase.
• Many such heat-stable enzymes from thermophilic bacteria (bacteria that live in high temperature surroundings) are now available commercially.
• The first one and the most commonly used is the Taq polymerase from the thermophilic bacterium Thermus aquaticus.

Steps Involved

A. Extraction and Denaturation of Target Nucleic Acid

• For PCR, nucleic acid is first extracted (released) from the organism or a clinical sample potentially containing the target organism by heat, chemical, or enzymatic methods.
• Once extracted, target nucleic acid is added to the reaction mix containing all the necessary components for PCR (primers, nucleotides, covalent ions, buffer, and enzyme) and placed into a thermal cycler to undergo amplification.

B. Steps in Amplification

• Conventional PCR involves 25 to 50 repetitive cycles, with each cycle comprising three sequential reactions:

1. Denaturation of target nucleic acid
2. Primer annealing to single-strand target nucleic acid extension of primer target duplex.
3. Extension of the primer-target duplex.

Denaturation

• The reaction mixture is heated to 95°C for a short time period (about 15–30 sec) to denature the target DNA into single strands that can act as templates for DNA synthesis.

Primer annealing

• The mixture is rapidly cooled to a defined temperature which allows the two primers to bind to the sequences on each of the two strands flanking the target DNA.
• Primers are short, single-stranded sequences of nucleic acid (i.e., oligonucleotides usually 20 to 30 nucleotides long) selected to specifically hybridize (anneal) to a particular nucleic acid target, essentially functioning like probes.
• This annealing temperature is calculated carefully to ensure that the primers bind only to the desired DNA sequences (usually around 55^oC).
• One primer binds to each strand. The two parental strands do not re-anneal with each other because the primers are in large excess over parental DNA.

Extension

• The temperature of the mixture is raised to 72°C (usually) and kept at this temperature for a pre-set period of time to allow DNA polymerase to elongate each primer by copying the single-stranded templates.
• Annealing of primers to target sequences provides the necessary template format that allows the DNA polymerase to add nucleotides to the 3’ terminus (end) of each primer and extend sequence complementary to the target template
• Taq polymerase is the enzyme commonly used for primer extension, which occurs at 72°C. This enzyme is used because of its ability to function efficiently at elevated temperatures and to withstand the denaturing temperature of 94°C through several cycles.
• The ability to allow primer annealing and extension to occur at elevated temperatures without detriment to the polymerase increases the stringency of the reaction, thus decreasing the chance for amplification of non-target nucleic acid (i.e., nonspecific amplification).

The three steps of the PCR cycle are repeated.

• Thus in the second cycle, the four strands denature, bind primers and are extended. No other reactants need to be added. The three steps are repeated for a third cycle and so on for a set of additional cycles.
• By the third cycle, some of the PCR products represent DNA sequence only between the two primer sites and the sequence does not extend beyond these sites.
• As more and more reaction cycles are carried out, the double-stranded DNA are synthesized more in number. After 20 cycles, the original DNA has been amplified a million-fold and this rises to a billion fold (1000) million after 30 cycles.

C. Product Analysis

• Gel electrophoresis of the amplified product is commonly employed after amplification.
• The amplified DNA is electrophoretically migrated according to their molecular size by performing agarose gel electrophoresis.
• The amplified DNA forms clear bands which can be visualized under ultra-raviolet (UV) light.

Advantages of PCR

• PCR (polymerase chain reaction) is an extremely simple yet immensely powerful technique.
• It allows enormous amplification of any specific sequence of DNA provided that short sequences either side of it are known.
• Allow faster diagnosis and identification while enhancing sensitivity and maintaining specificity.

Applications of PCR

PCR already has very widespread applications, and new uses are being devised on a regular basis.

• PCR can amplify a single DNA molecule from a complex mixture, largely avoiding the need to use DNA cloning to prepare that molecule. Variants of the technique can similarly amplify a specific single RNA molecule from a complex mixture.
• DNA sequencing has been greatly simplified using PCR, and this application is now common.
• By using suitable primers, it is possible to use PCR to create point mutations, deletions and insertions of target DNA which greatly facilitates the analysis of gene expression and function.
• PCR is exquisitely sensitive and can amplify vanishingly small amounts of DNA. Thus, using appropriate primers, very small amounts of specified bacteria and viruses can be detected in tissues, making PCR invaluable for medical diagnosis.
• PCR is now invaluable for characterizing medically important DNA samples. For example, in screening for human genetic diseases, it is rapidly replacing the use of RFLPs.
• Because of its extreme sensitivity, PCR is now fundamentally important to forensic medicine. It is even possible to use PCR to amplify the DNA from a single human hair or a microscopic drop of blood left at the scene of a crime to allow detailed characterization.