Polymerase Chain Reaction (PCR) is a major technique which is used to analyze the DNA with high accuracy. It can be defined as “Fast, simple and inexpensive way to amplify (copy) small quantities of specific DNA fragments via different polymerase enzymes by using in-vitro methods”. The product of PCR is commonly known as “amplicon”.

Why may you want to amplify DNA?

1. To synthesize copies of the gene which is infrequent or rare.
2. To check the relative abundance of two DNA samples

How PCR revolutionized the molecular biology?

It was very difficult to analyze DNA in past but now it has become very easy due to polymerase chain reaction. PCR revolutionize the scientific approaches by its versatile applications in the research field. Karry Mullis developed PCR in 1983, who was the winner of the noble prize in chemistry in 1993. Polymerase chain reaction is an easy, reliable and inexpensive process to repetitively replicate a segment of DNA, in molecular biology, the most widely used technique is PCR. Now PCR is a necessary technique which is used in research labs and in diagnostic labs. In 30 cycles of PCR millions of copies of template DNA fragment {230 – (2 x 30)} can be produced.

Components of PCR

dNTPs

dNTPs (deoxynucleotide triphosphate) are the major component of PCR. Polymerase enzyme adds dNTPs to the growing strand in the reaction after removing two phosphates.

Polymerase enzyme

The polymerase enzyme that is used in PCR should be thermally stable like Taq polymerase which can perform their function easily at 95°C and do not denature.
For example, Taq polymerase, Pfu polymerase etc.

MgCl2

MgCl2 helps in the enzymatic activity of polymerases for the addition of dNTPs to the strand. It also promotes hybridization of the primer to the template (denatured strand) at the target region. DNA molecules have a negative charge on it so due to repulsion between template strands and primers hybridization become difficult. Mg2+ ions embrace these strands and minimize the repulsion effect by neutralizing the net charge on the strands.
Its concentration is important to get a high yield of the final product. Due to the high concentration of MgCl2, non-specific binding of primers will increase. Low concentration reduces the primer binding.

DNA Template

This is the target DNA and contains the gene of interest which is to be amplified. The sequence of the target region of the template strand must be known.
The fragment of DNA to be amplified should be less than 3 kb in size and for ideal condition less than 1kb. By standard PCR techniques, fragments of 10 kb can be amplified but by increasing length of the fragment amplification process will become less efficient. By using special methods amplification of larger fragments (up to 40 kb) is possible.

Primers

Primer is a short fragment of DNA that serves as an initiating point for DNA synthesis. Forward and reverse primers are used in polymerase chain reaction. Both of these primers hybridize with each strand of template DNA at 5′ end in such a way that both primers have 3′ end towards each other. The size of primers that can be used for the polymerase chain reaction typically 20-30 bases in length.
Size of primer should be normal otherwise non-specific binding increases by using too short primers. Short primers hybridize easily with the sites other than the specific complementary region on target DNA. By using too large primer hybridization time increases due to this final yield of the PCR in a specific time decreases.

Buffer

Buffer solution maintains pH of the reaction mixture which is important for proper functioning and stability of DNA polymerase.

How PCR works?

The machine which is used for the polymerase chain reaction is known as a thermal cycler, the programmed machine that can perform an automated function has become the basic need for the polymerase chain reaction in these days.

Initiation/ denaturation

This is the first step in polymerase chain reaction. In PCR reaction template strand has double-stranded structure so to amplify the gene of interest it is necessary to melt the double-stranded structure. 92 °C to 94 °C for 1 minute is required to break the hydrogen bonding between the nitrogenous bases of the target DNA and denature the double-stranded structure.

Annealing

The hybridization process of the primers to the target DNA is called annealing. Low temperature is required for annealing process for 1minute. At 50-60°C some single strands of the template rejoin with each other but also primers bind easily with template strand at their complementary region.
It is important to note that annealing temperature should be less than Tm (melting temperature) of the primers that are used, otherwise primers will not anneal. GC content of the primer plays a major role, primer with high GC content has high melting temperature and primer with low GC content usually has a low melting temperature. Tm for the primers must be calculated before the reaction.

Elongation

After the annealing of primer to the template elongation starts. Taq Polymerase adds nitrogenous bases to the 3′ end of the primer at high temperature 74°C (for 2 minutes) just below to the optimum temperature of the Taq polymerase.
A set of “long product” is synthesized in this first stage of the PCR from each strand of the template target DNA. The product of this first stage PCR reaction has identical 5′ end but different 3′ end because reaction starts from the same site where primer gets attached but ends at different sites where reaction stops by chance.

Final Elongation

Final elongation is optional step to make sure that any remaining single-stranded DNA is fully elongated. Final elongation needs 70-74°C for 5 to 15 minutes after the last cycle of PCR. Final elongation is also helpful in the addition of 3′ overhang by polymerase enzyme that is used in TA cloning.

Final Hold

At the end, the final hold is maintained at 4-15°C for an indeterminate time.

PCR optimization

How temperature affects the PCR?

primer should be very specific to the sequence of interest and the role of annealing temperature is important for specific hybridization because DNA-DNA hybridization is a temperature dependent process. Hybridization of primers to the target region will not occur at high temperature hence primer and template remain dissociated. At low-temperature mismatch hybridization will be there (not all the base pairs correctly formed). So, amplification will occur at a non-target site in the template DNA molecule.
The ideal temperature for the hybridization of primers is 1-2°C less than melting temperature (Tm) of the primer-template hybrid. This temperature allows hybridization of the primer to target region but mismatch pairs will not form. Hence melting temperature of a primer-template hybrid must be known for better results. It can be calculated by using following formula:

Tm= (4 × [G + C]) + (2 × [A + T]) °C

In this [A + T] is the number of Adenine, thiamine base pairs in the sequence of primer and [G + C] is the number of guanine, cytosine base pairs in the primer sequence. It is important to note that melting temperature should be identical for both reverse primer and forward primer, otherwise one of these primers will not accurately bind.

 

Primer design

During primer designing following points are important.

• GC content: Melting temperature directly depends upon the GC content in the primer-template hybrid because three hydrogen bonds form between G and C nitrogenous bases in DNA double stranded structure. Greater the GC content higher will be the Tm. So, primers should have 40-60% GC content normally.
• GC clamp: within the last five bases at 3′ ends of the primers should have G or C bases that promote specific binding at the 3′ ends due to strong bonding. More than three G or C repeats should be avoided.
• Primer self-secondary structures: Due to intramolecular or intermolecular interactions sometimes, self-secondary structures are made
  

  Fig.04: Primer self-secondary structures

  which can lead to poor yield because they greatly decrease the availability of the primers to the reaction like.(Fig.04)

  • Self-dimer: sometimes by intermolecular interactions same(sense) strand hybridize with each other and form self-dimers.
  • Cross dimes: by intermolecular interactions between sense and antisense primers cross dimers are formed.
  • Hairpins: due to intramolecular interaction in the primer hairpin is formed.

Buffers

Most of the time in different concentrations of KCl and tris are used as a buffering agent for the polymerase chain reaction. KCl helps in primer binding but its high concentration may inhibit the activity of the polymerase enzyme. DMSO, glycerol, and BSA also used.

After the PCR: Studying product

The product after polymerase chain reaction can be analyzed by three different techniques.

• Gel electrophoresis of PCR products
• Cloning of PCR products
• Sequencing of PCR products.

Types of PCR

•  Colony PCR
•  Nested PCR
• Multiplex PCR
• AFLP PCR
• Hot Start PCR
• Long PCR
• Reverse Transcriptase PCR
• Real-time PCR

Applications of Polymerase Chain Reaction

A. Molecular identification

• Molecular archeology
• Detection of pathogens
• Genetic matching
• Drug discovery
• Mutation screening
• Genotyping
• Classification of organisms
• DNA fingerprinting

B. Sequencing

• Bioinformatics
• Human genome project
• Genome cloning

C. Genetic engineering

• Site-directed mutagenesis
• Gene expression studies