Polymerase Chain Reaction: 9 Important Explanations

Contents

What is Polymerase Chain Reaction?

Polymerase chain reaction (PCR) is another widely used molecular biology technique for enzymatically producing DNA without utilizing cellular machinery, for example, Yeast or E. coli. The method permits a limited quantity of the DNA to be amplified several folds dramatically. PCR is regularly utilized in clinical, forensic, and biological labs for various purposes, such as determining genetic fingerprints, disease diagnosis, gene cloning, and paternity testing. 

This technique was created in 1983 by Kary Mullis. PCR is currently a crucial and significant procedure used in applied biology. These incorporate DNA cloning for sequencing, DNA-based phylogeny, or active investigation of gene expression and the determination of genetically transmitted diseases. In 1993, Mullis was granted with the Nobel prize in Chemistry for his work on PCR. 

The PCR is generally carried in a reaction mixture of a total volume of 0.1-0.2 ml in reaction tubes (0.2-0.5 ml volumes) in an instrument known as a thermal cycler. A Thermal cycler heats and cools down the DNA to reach the temperatures required in each step of the reaction. The thin-walled reaction tubes provide desired thermal conductivity allowing faster thermal equilibration. Usually, the thermal cyclers have heated tops or lid that prevents condensation of reaction components at the top of the tube. 

Requirements of Polymerase Chain Reaction

• DNA fragment to be amplified (template DNA or cDNA)
• Primers (forward primer and reverse primer) capable of recognizing ends of the template DNA. Primers are commonly 18-25 base pairs long.
• Thermo-stable DNA polymerase (Taq Polymerase) catalyzes the DNA synthesis. Taq polymerase is obtained from Thermus aquaticus.
• Nucleotides
• Buffer (provides an optimum medium for the activity of thermostable DNA polymerase)

Working principle of Polymerase Chain Reaction

PCR is called a chain reaction because the newly synthesized strands will act as the template for further synthesis of DNA in the subsequent cycles. 

PCR consists of 3 successive reactions:

• Denaturation of DNA: The process of denaturation separates the strands of double-stranded DNA. For human DNA, the denaturation temperature is about 93-95oC. The denaturation of DNA is done by breaking the hydrogen bonds between the two polynucleotide strands of the DNA. The denaturation process is often performed for 5 minutes or extended time to ensure that both the strands of the template DNA are fully separated from each other. The denaturation process also activates thermo-stable DNA polymerase.
• Primer annealing: After denaturation, forward and reverse primers are introduced in the reaction mixture. The solution is then cooled for the primer annealing. Usually, primer annealing occurs between 50-70^oC depending upon the Tm (melting temperature) of the DNA. Ideally, the annealing temperature is 5^oC below the Tm. The annealing temperature should be low to form a hybrid between the primer and the template DNA and high enough to prevent mismatched hybrids. The Tm of the DNA solely depends upon the G-C and A-T content of the DNA. The primer annealing step usually takes 1-2 minutes. Tm can be determined experimentally or calculated theoretically by the formula mentioned below:

Tm = [4 x (G+C)] + [2 x (A+T)]^oC

• DNA synthesis: The thermostable DNA polymerase (Taq polymerase) binds near the RNA binding region and incorporates the free nucleotides to elongate and synthesize a new strand of DNA adjacent to the template strand. The optimum temperature of the extension step depends on the type of polymerase used; for Taq polymerase, the optimum temperature is about 72^oC. This extension step’s duration depends upon the length of the template DNA and the efficiency of the thermostable DNA polymerase; usually, it is one kilobase pairs per minute.

All the reactions mentioned above are repeated 30 times to get approximately 1 billion copies of the template DNA.

Stages of Polymerase Chain Reaction

The process of PCR is divided into three stages:

• Exponential amplification: DNA polymerase performs the optimal function, and after completion of every cycle, the amount of product is doubled. Only a little quantity of the DNA need to be present at the starting point of reaction since the reaction is very sensitive.
• Leveling-off stage: DNA polymerase shows lesser activity because of the less availability of the reagents such as nucleotides.
• Plateau: no more product is formed due to exhaustion of reagent.

Uses of Polymerase Chain Reaction

PCR can be utilized to identify numerous genome linked abnormalities, a wide range of analyses, and experimentations. A few examples of the use of PCR are discussed below:

• Identification of infectious agents like CMV, Mycoplasma, Pneumonia, contagious and Protozoan related diseases, hepatitis, etc. in the sample.
• Determination of malignancy, specifically in the case of lymphoma and leukemia. 
• Paternity testing and genetic fingerprinting. 

PCR allows early identification of abnormalities which are presently the most developed in cancer research. PCR measures can be performed directly on the genomic DNA samples to recognize translocation specific malignant cells with a sensitivity of more than 10,000-folds compared to any other method. 

PCR also identifies non-cultivatable and slow-growing microorganisms, such as mycobacteria, anaerobic microorganisms, and viruses, from tissue culture techniques and animal-based models. PCR demonstrative applications in microbiology are the recognition of pathogens and the ability to differentiate between non-pathogenic from pathogenic strains by identifying the specific genes. 

PCR is utilized to intensify a short piece of a DNA strand. This DNA strand can be a complete gene or merely a part of a gene. Unlike living systems, the PCR cycle can amplify just short DNA fragments up to 10 kilobase pairs. Various other techniques can amplify DNA fragments up to 40 kb in size, which is still less than the size of chromosomal DNA of a eukaryotic cell – for instance, a human cell contains approximately three billion nucleotide base pairs. 

Paternal testing can be performed using PCR by taking the DNA of the individuals having the dispute. The DNA of the test individuals are amplified and digested through restriction enzymes and analyzed with gel electrophoresis. The pattern of DNA fragments on the agrose gel is known as the DNA fingerprint of the individual. Closely or blood-related individuals will have a similar DNA fingerprinting pattern compared to the distantly related or unrelated individuals. The fingerprints obtained can be further analyzed for the parent-child or siblings with two or more genetic (DNA) fingerprints. The DNA fingerprints obtained can be used to know the evolutionary relationships among the organisms. 

Its size can easily recognize the final product of the PCR on agarose gel electrophoresis. The agarose gel electrophoresis is performed by injecting the DNA samples into the agarose gel. The electric current is passed through the agarose gel to know the mobility of the DNA samples. Smaller DNA fragments move faster in the gel and vice-versa. The PCR product and the sample DNA fragment are identified by introducing a DNA ladder in the agarose gel. The DNA ladder contains DNA molecules of known size. 

Previously, identification of the genetic disorder by observing the genome was a difficult job. Later, by the development of PCR, this process is shortened. Any gene of interest can be easily amplified using suitable primers and then sequenced to detect mutations in the DNA. Early identification of some lethal viral infections can also be made through PCR. 

Types of Polymerase Chain Reaction

Based on some suitable modifications in the technique, Polymerase chain reaction is classified into the following types:

• Nested PCR
• Inverse PCR
• Reverse Transcription (RT-PCR)
• Asymmetric PCR
• Quantitative (PCR-Q-PCR)
• Quantitative real-time PCR (QRT-PCR)
• Touchdown PCR
• Colony PCR
• Allele-specific PCR
• Assembly PCR or Polymerase Cycling Assembly (PCA)
• Asymmetric PCR
• Linear After The Exponential-PCR (LATE-PCR)
• Dial-out PCR
• Helicase-dependent amplification
• Hot start PCR
• Inter-sequence specific PCR
• Inverse PCR
• Ligation mediated PCR
• Methylation-specific PCR
• Mini-primer PCR

Advantages of Polymerase Chain Reaction

• Polymerase Chain Reaction can be used for the identification of a variety of genetic abnormalities.
• PCR is used in a wide range of laboratory experiments and procedures in molecular biology.
• PCR can detect-Malignancies like leukemia, lymphoma, etc., and various other conditions like Toxoplasma gondi, Staphylococcal bacteremia, Malarial infections, etc.
• Genetic fingerprinting and paternity testing involves PCR as their crucial step.
• The high sensitivity and specificity of PCR makes it an important tool for biotechnology and molecular biology based experiments.

Disadvantages of Polymerase Chain Reaction

• Polymerase chain reaction requires thermal cycler, DNA electrophoretic assembly, DNA isolation kit, and other costly chemicals, which make the process expensive.
• Requires trained, qualified, and experienced technicians for conducting experiments.
• Basic safety level labs with de-humidifiers and laminar flow facilities are required.
• It’s hard to afford PCR-based testing for the regular public.
• Not all diseases could be identified and diagnosed through PCR.
• Possibility of getting false-positive and false-negative results.

Polymerase Chain Reaction is a commonly used and widely accepted technique for identification of several lethal infectious agents with sensitivity and specificity. PCR is being used in various advanced facility medical centers, modern labs, and medical institutes as a routine diagnostic and research module. Polymerase chain reaction plays a crucial role in identification of abnormalities representing atypical clinical symptoms; PCR enables early detection of these types of impairments. Early detection can help manage the individual in a much better way, which may reduce the social and economic burden on the subject’s family.

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Concept checker MCQs

Question no. 1: A student is performing PCR to amplify a sample of template DNA. But, he forgot to add DNA primer in the experiment. Which of the following option represents the best possible result?

A- The reaction will not take place.

B- Reaction will work, but the process will amplify non-specific regions (not the desired DNA portion of DNA.

C- Reaction will work but at a prolonged rate

D- Reaction will work, and the product will show undesired mutations.

Option A: “The reaction will not take place” is the correct answer.

Explanation: Primers are essential for the functioning of PCR. Taq polymerase needs them for binding (in the annealing step) to initiate DNA replication. Thus the PCR will not operate in the absence of primers, and no amplification will take place.

Question no. 2: A student/researcher wants to insert the human hemoglobin gene in an expression vector to clone and express the gene in mouse cells for analyzing the resulting phenotype. Which of the following sequence of procedures will allow the student/researcher to clone the gene successfully?

Option A:

1. Amplify the gene through PCR

2. Use a restriction enzyme for cutting the expression vector

3. Ligate gene and the digested vector

Option B: 

1. Ligate gene

2. Use a restriction enzyme for cutting the expression vector 

3. Amplify gene and the digested expression vector via PCR

Option C:

1. Use a restriction enzyme for cutting the expression vector

2. Amplify the gene through PCR

3. Ligate gene and the digested vector

Option D:

1. Use a restriction enzyme for cutting the expression vector

2. Ligate gene and the digested vector

3. Amplify the gene via PCR

Option A: Is the correct option 

1. Amplify the gene through PCR

2. Use a restriction enzyme for cutting the expression vector

3. Ligate gene and the digested vector

Explanation: Firstly, we will use PCR to amplify the gene of interest from the human genome having restriction sites at the ends (this will help its ligation to a restriction enzyme digested vector). Later, the expression vector needs to be digested using the same restriction enzyme, and then the digested vector and amplified gene were ligated together. The final product will be consisting of two segments: the original vector and the gene of interest.

Question no. 3: Which instrument is used to complete DNA amplification through PCR?

A- Genetic analyzer

B- UV spectrophotometer

C- Thermocycler

D- Centrifuge

Option C: Thermocycler is the correct answer

Explanation: The thermo-cycler can be set to operate a cycle of the denaturation, annealing, and extension stages, respectively, in a small amount of the reaction volume. A thermal cycler is used to perform PCR amplification.

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Dr. Abdullah Arsalan

I am Abdullah Arsalan , Completed my PhD in Biotechnology. I have 7 years of research experience. I have published 6 papers so far in the journals of international repute with an average impact factor of 4.5 and few more are in consideration. I have presented research papers in various national and international conferences. My subject area of interest is biotechnology and biochemistry with special emphasis on Protein chemistry, enzymology, immunology, biophysical techniques and molecular biology.