Polymerase Chain Reaction (PCR) Steps, Principle, Application

Polymerase chain reaction (PCR)

Polymerase chain reaction or PCR is a laboratory technique that is used to make multiple copies (millions or billions!) of a specific region of DNA in vitro (in a test tube rather than an organism).

• It can make millions to billions of copies of a specific sequence of DNA which allows the laboratorians to amplify a very small sample of DNA.
• In 1983, an American biochemist Kary Mullis at Cetus Corporation first developed this PCR machine.
• This technique is used for characterizing, analyzing and synthesizing DNA from virtually any living organism (plant, animal, virus, bacteria).
• Now PCR is used in medical laboratories for the analysis of ancient samples of DNA and the identification of infectious agents. It is also used in biomedical research and criminal forensics.
• This method relies on a thermostable DNA polymerase, Taq polymerase, and also requires DNA primers created especially for the DNA region of interest.
• The reaction in PCR is repeatedly cycled through a series of temperature variations, it helps in the multiplication of the mark region to be produced.
• Finally, the main purpose of PCR is to produce enough of the target DNA region that it can be examined or used in some other way.
• PCR consists of three basic steps such as: Denaturation, Annealing and Extension at 72°C.
• During Denaturation the two strands melt open to form single-stranded DNA and all enzymatic reactions stop. This is generally carried out at 92°C – 96°C.
• In the second step or during Annealing, the Annealing of primers to each original strand for new strand synthesis is carried out between 45°C – 55°C.
• At the third step or during extension at 72°C, The polymerase adds dNTPs complementary to the template at the 3’ end of the primers. Since both strands are copied during PCR, there is an exponential increase in the number of copies of the gene.
• The previously discussed three steps are continuously repeated 20-30 times in an automated thermal cycler that can heat and cool the reaction mixture in tubes within a very short time. As a result, it leads to the exponential accumulation of specific DNA fragments, ends of which are defined by 5’ ends of the primers. 
• The doubling of the number of DNA strands corresponding to the target sequences allows us to estimate the amplification associated with each cycle using the formula: Amplification = 2^n , where n = No. of cycles.

Polymerase chain reaction Principle

The desired nucleic acid sequence is first denatured to single strands, specific primers of targeted strands are added and the DNA polymerase will extend the DNA strand by adding deoxynucleotides and will produce new strands complementary to each of the target sequence strands (cycle 1). Both the double-stranded products which are formed within cycle 1 are denatured in cycle 2 and subsequently serve as targets for more primer annealing and extension by DNA polymerase. After completion of 25 to 30 cycles, at least 107 copies of target DNA may be formed by means of this thermal cycling.

Components Required for PCR

For PCR the following components are required;

• DNA template that contains the region to be amplified.
• Two primers complementary to the 3’ ends of each of the sense and anti-sense strand of the DNA.
• Thermostable DNA polymerase like Taq, Vent, Pfu etc.
• Deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP), the building blocks from which the DNA polymerase synthesizes a new DNA strand.
• Buffer solution which provides a suitable chemical environment for optimal activity and stability of DNA polymerase.
• Bivalent magnesium/manganese ions, which are necessary for maximum Taq polymerase activity and influences the efficiency of primer to template annealing.

Preparation of PCR Reaction Mixture

Add these following ingredients to a PCR tube;

• Sterile Water
• 10X Assay Buffer
• 10 mM dNTP Mix
• Template DNA (100 ng/µl)
• Forward Primer (100 ng/µl)
• Reverse Primer (100 ng/µl) 
• Taq DNA Polymerase (3 U/µl)

Note: Tap the tube for 1–2 seconds to mix the contents thoroughly. Then, add 25 μl of mineral oil in the tube to avoid evaporation of the contents. Place the tube in the thermocycler block and set the program to get DNA amplification.

Polymerase chain reaction Steps

Initialization

• In this step, the reaction mixture (DNA) is heated to 94 –96oC for 1–9 minutes, it will trigger the breaking of the hydrogen bonds in DNA strands.

Denaturation

• In this step, the reaction mixture is heated to 94–98o C for 20–30 seconds. 
• At these temperature points, the template DNA denatures due to disruption of the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

Annealing

• During this step, the temperature is reduced to 50–65o C for 20–40 seconds,it will allow the annealing of the primers to the single-stranded DNA template.
• Typically the annealing temperature is 3-5o C below the Tm (melting temperature) of the primers used. 
• Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. 
• The polymerase binds to the primer-template hybrid and begins DNA synthesis.

Extension/Elongation 

• The temperature range in this step depends on the types of DNA polymerase used. Generally, the optimum temperature of Taq polymerase is 75–80o C, where it shows the highest activity. But commonly 68-72o C temperature range is used with this enzyme.
• The DNA polymerase started to synthesize a new complementary DNA strand of the DNA template strand by joining dNTPs that are complementary to the template in 5′ to 3′ direction, condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxyl group at the end of the nascent (extending) DNA strand.
• The time required for the extension depends on the type of DNA polymerase used and on the length of the DNA fragment to be amplified.
• At the optimum tem[areture the DNA polymerase can polymerize a thousand bases per minute.
• Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential amplification of the specific DNA fragment.

Final elongation

• This step is performed at a temperature range of 70–74oC for 5–15 minutes after the last PCR cycle to assure that any left single-stranded DNA is fully stretched.
• Denaturation, annealing and extension steps are repeated 20-30 times in an automated thermal cycler that can heat and cool the reaction mixture in tubes within a very short time.
• As a result of this step, it will lead to the exponential accumulation of specific DNA fragments, ends of which are defined by 5’ ends of the primers.
• The doubling of the number of DNA strands corresponding to the target sequences allows us to estimate the amplification associated with each cycle using the formula; Amplification = 2n, where n = No. of cycles.

Final hold

• This step involves the short-term storage of the reaction mixture at 4°C for an indefinite time.

Gel electrophoresis to visualize the results of PCR

After completion of the PCR, perform agarose gel electrophoresis. Compare the amplified product with the ladder and determine its size. 

PCR Advantages

• PCR has high sensitivity compared to culture and staining.
• It has the ability to test for anti-microbial resistance.
• PCR is Quickly performed in 4-8 hours
• Shown to be more cost-effective with selective use than culture and staining.
• Increased ability to detect less common organisms such as viruses.

PCR Disadvantages

• PCR is Potentially lower specificity compared to culture and staining.
• Need for a narrow list of causative agents to use specific primers.
• Possibility of amplifying normal flora from corneal scrapings.
• Becomes less cost-effective when performed with a multi-organism PCR approach.
• Supply costs, machinery fees, training expenses.

PCR Applications

• PCR helps in the isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA.
• Used for DNA sequencing to discover the unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing.
• PCR fingerprint is used to identify genetic relationships between individuals.
• Used to amplify the extremely small amounts of sample in forensic analysis.
• Used in the analysis of ancient DNA.
• Quantitative PCR or Real-Time PCR is used to determine the amount of a given sequence present in a sample—a method usually used to quantitatively define levels of gene expression. 
• Used to diagnose genetic disorders.
• It is a sensitive test for tissue typing, vital to organ transplantation. 
• It can be used for early diagnosis of malignant diseases such as leukemia and lymphomas.
• PCR is used to diagnose different infectious diseases, mainly those caused by bacteria or viruses such as  HIV,  tuberculosis, etc.
• Used for genetic fingerprinting.
• Used in forensic DNA typing which helps in identifying or exonerating criminal suspects.
• Used for  DNA paternity testing.
• Used to generate the hybridization probes for Southern or northern blot.
• PCR assisted the DNA sequencing.
• It is also used in DNA cloning.
• Used for Sequence-tagged sites.
• Used for phylogenetic analysis of DNA from ancient sources.
• Used to study patterns of gene expression.
• Helps to analyze the genetic mapping.
• PCR can be used to create mutant genes with mutations chosen by scientists at will (Site-directed mutagenesis).

Factors affecting Amplification

Sample Volume

• Generally, the amplification is performed at 20, 50, or 100 µl volume in 0.2 or 0.5 ml microfuge tubes.
• Larger volumes do not allow adequate thermal equilibrium of the reaction mixture.

Template DNA

• During PCR, generally, a nanogram amount of plasmid DNA, or microgram amount of genomic DNA is used.
• The higher amounts of template DNA can lead to the inhibition or result in non-specific amplification.

Primers

• Primers are synthetic oligonucleotides which are ranging from 15 to 30 bases.
• For PCR required a forward and a reverse primer. The melting temperature (Tm) of both primers is the same.
• The 3’ ends of Primers do not have more than two bases complementary to each other, as this helps in the formation of PRIMER-DIMER.
• The G+C content of Primers ranges from 40-60%.
• Low concentrations of primers result in poor yield of the specific product and high concentrations of primers may result in non-specific amplification. The optimal concentration of primers is between 0.1-1 µM.

Deoxynucleotide-tri-phosphates

• The final concentration of each dNTP (dATP, dGTP, dCTP & dTTP) in a standard amplification reaction is 200µM.
• It is important to keep the dNTP concentrations above the estimated Km of each dNTP (10 to 15 µM) for best base incorporation.

Taq DNA polymerase buffer

• 10X assay buffer is consist of 100 mM Tris- Cl (pH 9.0), 500 mM Potassium chloride, 15 mM MgCl 2 and 0.1% w/v gelatin.
• Mg2+ is an important cofactor needed for the activity of Taq DNA Polymerase. A low concentration of Magnesium will result in no amplification and high amounts may lead to the production of unwanted products.

Taq DNA Polymerase

• It is a 94 kD (Kilodaltons) thermostable DNA polymerase. The optimal temperature for Taq DNA Polymerase is 72°C.
• 3’ to 5’ exonuclease activity is absent but has 5’ to 3’ exonuclease activity and 5’ to 3’ polymerase activity.
• For most amplification reactions 1.5 to 2 units of the enzyme is recommended as higher enzyme concentration leads to non-specific amplification.

References

• Liu, H. Y., Hopping, G. C., Vaidyanathan, U., Ronquillo, Y. C., Hoopes, P. C., & Moshirfar, M. (2019). Polymerase Chain Reaction and Its Application in the Diagnosis of Infectious Keratitis. Medical hypothesis, discovery & innovation ophthalmology journal, 8(3), 152–155.
• https://www.thermofisher.com/in/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-applications.html
• https://himedialabs.com/TD/HTBM016.pdf
• http://www.ispybio.com/search/protocols/StudentPCR.pdf
• https://en.wikipedia.org/wiki/Polymerase_chain_reaction
• https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotechnology/a/polymerase-chain-reaction-pcr
• https://microbenotes.com/polymerase-chain-reaction-pcr-principle-steps-applications/