PCR Amplification

PCR amplification or Molecular photocopying is a popular method used to amplify the short DNA fragments. PCR is an acronym used for Polymerase chain reaction. It provides a modern, inexpensive, and rapid method of amplifying specific DNA sequences, while the traditional method was quite time-consuming (requires several days or a week).

Polymerase chain reaction can yield million to billion copies of the desired DNA sequence. Kerry Mullis was the first scientist who introduced PCR with its remarkable applicability in genetic and molecular biology. It is a selective method amplifying the specific or target segment of DNA or RNA into specific fragments.

PCR completely relies on thermal cycling and involves 20-40 thermal cycles. It primarily uses Taq polymerases and primers to amplify a single strand of DNA or RNA. PCR generally amplifies the target strand of 0.1-10 kbp in length. This post mainly discusses the definition, set-up, steps, advantages and limitations of PCR amplification.

Content: PCR Amplification

1. Definition
2. PCR Setup
3. Steps
   • Denaturation
   • Annealing
   • Extension
4. Advantages
5. Limitations
6. Conclusion

PCR Amplification Definition

PCR or polymerase chain reaction refers to the in vitro technique of preparing around million copies of the target DNA sequence through consecutive stages of heat denaturation, primer annealing, and primer extension. It is an advanced molecular technology that revolutionized DNA study more in medical and biological research like DNA mapping, DNA fingerprinting, forensics, detection of microorganisms, etc.

PCR aims to yield more copies of desired DNA sequence or provides different scopes in studying the DNA. For instance, DNA amplified by PCR can be used in DNA sequencing and DNA cloning, which substantially require DNA samples.

PCR Amplification Setup

The polymerase chain reaction is a molecular method, which set-up requires the following reactants:

• DNA template
• Primers (short stretches of DNA)
• DNA precursors
• Magnesium ion
• Taq polymerase enzyme
• Buffer solution

DNA Template

It is the most important component of PCR, whose concentration and quality determines the success of amplification. The concentration of the DNA template should be within 30 ng-50 ng, i.e. not too little or too high. Other than this, the DNA templates should be free of chemical contaminants to avoid misleading results of the gene amplification.

DNA Precursors

Deoxynucleoside triphosphates (dNTPs), including dATP, dCTP, dGTP, and dTTP serve as molecular precursors that construct new DNA strands. They also function as the building blocks of DNA. Thus, dNTPs are added equivalently (generally 0.2 mM) for optimal base incorporation.

Magnesium Ion

Mg2+ acts as a cofactor of DNA polymerases that facilitate the incorporation of DNA precursors during polymerization. The magnesium ions associate with an enzyme Taq polymerase to bring about the formation of a phosphodiester bond between the primer terminus and the phosphate group of dNTPs.

It is utilized through MgCl2 solution added to the PCR mixture, which concentration is optimized within a range of 1–4 mM. Its high and low concentration can affect the productivity of PCR.

Taq Polymerase Enzyme

It is a DNA polymerase enzyme obtained from Thermus aquaticus (Heat-loving bacterial strain) that thrives in hot springs and hydrothermal vents. Taq DNA polymerase possesses high thermostability (remains stable above 80°C).

It is most active at a temperature of 70°C, at which it can add on approximately 60 bases per second. The heat-stability of Taq polymerase is ideal for PCR, as the template DNA is subjected to thermal cycling to get new DNA strands.

PCR Primers

They are the short pieces of single-stranded DNA (15-30 bp long) that aid in new DNA strands synthesis. PCR uses two primers. The designing of two primers require some information prior to the desired DNA sequence from the target DNA.

Thus, the PCR primers are selective relative to a desired DNA sequence in the template DNA. Primers attach oppositely towards the edges of the intended DNA sequence via complementary base pairing.

Once they bind to the desired DNA sequence, the sequence can be lengthened by the DNA polymerase, and the target sequence gets copied. Ideal properties of primers include:

• Primer’s length should not be too short or long, as it might hybridize the non-target sites or target DNA, respectively.
• The base composition should be even, and the GC content must be between 40-60%.
• Any self-complementary sequences must be avoided to minimize mispriming or non-selective priming.
• The melting temperature or Tm value for the two primers must be within 5°C.

PCR Buffers

They maintain the pH alternations in the sample to the optimal pH (8.0 and 9.5), necessary for the enzymatic action of Taq DNA polymerase. Tris-HCl optimizes the Taq DNA polymerase’s activity. One should use the buffer provided with the DNA polymerase for optimal PCR yields and robust enzyme activity of  Taq DNA polymerases.

• KCl buffer: Here, the potassium or K+ ion promotes primer annealing.
• (NH4)2SO4 buffer: It is an alternative to the KCl buffer. Here, the ammonium or NH4+ ion enhances specificity by destabilising the weak H-bonds formed between the mismatched primer and template DNA during DNA elongation.

Polymerase Chain Reaction Steps

The PCR reactants are assembled in a tube and subjected to repetitive thermocycling to yield new DNA strands. PCR cycle involves three sequential stages:

Denaturation

• It an initial stage where you need to subject the solution to a high temperature of 96°C.
• This reaction cause separation or denaturation of target ds-DNA into template ss-DNA.
• Denaturation of the target DNA occurs via the disintegration of H-bonds between the bases.
• Then, maintain the temperature for a certain time to establish complete separation of the DNA.
• Later, you could use the single-stranded or separated DNA as the template to synthesize new DNA.

Annealing

• It is a second stage when you need to cool the sample up to 55-65°C of temperature.
• This reaction facilitates the binding of the two primers to their complementary sequences of the template DNA via H-bond.
• Primers are the short DNA sequences that act as a starting point to polymerize or extend the DNA.
• During this step, the two primers attach towards the template DNA’s edges, opposite each other.

Extension

• This is the last stage when you again need to expose the sample to a high temperature (72°C).
• At this temperature, an enzyme DNA Taq-polymerase stretches out short sequenced primers by incorporating DNA bases complementary to the template strand.
• Taq polymerase adds DNA precursors or nucleotide bases from the 5’ to 3’ direction.
• This step results in forming new DNA strands, which synthesis is determined by the length of the DNA sequence that has to be amplified.

Finally, cool the reaction chamber up to 4–15 °C for short-term storage of the PCR products. The reaction cycle of PCR repeats 25-35 times to produce millions to billions of copies. Generally, the reaction cycle takes 22-44 hours to complete but depends on the DNA length, the concentration of PCR ingredients, the working efficiency of the instrument, and more other factors.

Advantages

1. It is a simple molecular technique that requires easy handling.
2. PCR amplification gives a large number of PCR products rapidly.
3. It is a selective amplification technique in which forward and reverse primers selectively bind to the template DNA’s complementary sequence.
4. The process uses a closed system that minimizes the chances of contamination.
5. It becomes an important tool to get better yield, which can be used for many research purposes.

Limitations

1. Selective amplification requires information about the target DNA to design primers.
2. Optimization is quite difficult in the PCR set-up that can give misleading results. Thus, it requires a sterile room for the reagent preparation.
3. DNA polymerases are prone to error (about 40%) and may yield mutated DNA after 20 reaction cycles. Also, multiple primers may cause cross-hybridization, thereby giving misleading results.

Conclusion

Therefore, we can conclude that PCR gene amplification aims to double the PCR product achieved by 100% reaction efficiency in each cycle. PCR produces exponential copies of DNA throughout the reaction cycle. A single copy of DNA can yield up to one billion copies after 30 rounds of amplification. Its working efficiency primarily depends upon the reagent’s optimization during the setup.