This article discusses the in vitro DNA cloning method, also known as the polymerase chain reaction (PCR). It explores the different steps involved in PCR and its advantages and disadvantages.
In vitro: In vitro is Latin for “in glass”, i.e., performed in a laboratory. The in vitro gene cloning method uses the polymerase chain reaction (PCR).
In vivo is the opposite of in vitro - work performed in a whole living organism.
Polymerase chain reaction
The polymerase chain reaction (PCR) is a standard method used to amplify the DNA fragments of interest in an automated process.
Some of the main applications of PCR include amplifying DNA for forensic tests. For example, Covid-19 tests, research, paternity tests, gene editing and many more.
The PCR process requires specific substrates and conditions:
- The DNA fragment of interest, which will be acting as the template DNA
- Taq DNA polymerase: This enzyme replicates DNA fragments. Taq polymerase is obtained from a particular type of bacteria in hot springs. As a result, Taq polymerase is tolerant of high temperature instead of human DNA polymerase, which denatures at high temperatures.
- Nucleotides: Free DNA nucleotides are the building blocks for the synthesis of new DNA strands.
- Primers: Primers are fragments composed of 18-30 nucleotides that complement the beginnings of each of the two DNA strands at either end. Their presence is essential for this process since Taq DNA polymerase can only bind to and replicate a double-stranded region of DNA. Primers bind to the beginnings of the two DNA strands and allow the recruitment of Taq polymerase.
- A thermocycler (or thermal cycler): An instrument that automatically increases and lowers the temperature of samples in a closed container according to a program.
The DNA fragments, primers, nucleotides, and Taq DNA polymerase are added to a tube then placed into the thermocycler.
The PCR steps
The PCR process involves three steps that are repeated in many cycles. These steps are:
- Denaturation: The thermocycler increases the temperature to 95°C. At this temperature, the hydrogen bonds between complete base pairs, holding the two DNA strands together, break. This results in the two strands of the DNA fragments separating.
- Annealing: The thermocycler cools down the content temperature inside the vessels down to 55°C. The primers then can bind to their complementary base sequence at the beginning of the DNA strands via hydrogen bonds. The primers allow for the recruitment of the Taq DNA polymerase to the DNA strands and hence provide the starting site for DNA synthesis. The primers prevent the two strands from simply re-joining each other by binding to the DNA fragment strands.
- Extension: The temperature is increased to 72°C. This is the optimum temperature for Taq DNA polymerase, which uses the free DNA nucleotides to replicate the strands. It adds complementary DNA nucleotides to the primer and carries on until it reaches the end of the fragment.
Fig. 1 - The three steps involved in polymerase chain reaction (PCR): denaturation, annealing, and elongation
At the end of each cycle, the number of DNA fragments doubles in size. For example, at the end of the first cycle, there would be two copies of the original fragment, and at the end of the second cycle, the number of copies would be four.
The process is repeated in many cycles to amplify the number of DNA fragment copies to the desired amount. This amount may depend on the type of application for which the DNA fragments will be used.
Below is the formula used for calculating the number of DNA molecules after multiple cycles of PCR.
n is the number of PCR cycles.
Advantages and disadvantages of PCR cloning
There are advantages and disadvantages to using the PCR cloning method.
Table 1. Advantages and disadvantages of PCR cloning.
| Advantages | Disadvantages |
Very rapid and has high throughput – can be used to produce millions of DNA copies in a few hours. | It cannot produce mRNA or proteins instead of in vivo systems. Disadvantages |
PCR only replicates the DNA of interest (the target gene), so there is no need to isolate the desired DNA fragment from the host DNA or other contaminants. |
It does not require any living systems. This lowers the costs, time, and technical skills needed for using PCR relative to the in vivo process. | It works best with only small DNA fragments. |
It can be used to create double-stranded DNA molecules from single DNA strands. This is particularly useful when creating DNA fragments using the reverse transcriptase enzyme. |
Benefits and risks of genetic engineering, including PCR
Genetic engineering has many applications and benefits. It also comes with some risks and ethical implications, which we need to consider. Here are some of the benefits and risks of recombinant DNA technology:
Table 2. Benefits and risks of genetic engineering.
| Benefits | Risks |
Bacteria may be genetically engineered to generate a variety of compounds such as antibiotics, hormones, and enzymes used to treat illnesses and disorders. | Antibiotic resistance marker genes are often introduced to genetically engineered bacteria to identify successfully transformed recombinant cells. These bacteria can potentially transfer their antibiotic resistance genes to dangerous pathogenic microbes. |
Genetically modified crops can also be made tolerant against pesticides and herbicides. This increases the cultivation yield and hence reduces the price of food. |
Certain genetic illnesses, such as cystic fibrosis (CF) and severe combined immunodeficiency (SCID), may be cured by replacing faulty genes. This treatment is called gene therapy. | Uncertainty of what long-term consequences gene editing and introducing new genetic material into nature can have on the ecosystem, both on a micro and macro level. |
Microorganisms can be utilized to reduce pollution. They can be modified to break down and digest oil slicks to remove toxic gases emitted by companies. | The ability to manipulate and exchange faulty genes brings on ethical questions on where the line should be drawn. Treating cystic fibrosis by gene editing is acceptable, but what about changing one’s features, such as eye colour, muscle tone or intelligence? |
Transgenic crops can resist environmental extremes, such as drought, cold, heat, salt, or contaminated soils. This allows crops to be cultivated economically in areas where they do not naturally grow. |
Polymerase Chain Reaction - Key takeaways
- In vitro: In vitro is Latin for “in the glass.” It involves using the polymerase chain reaction (PCR) in the lab.
- The PCR process requires certain substrates and conditions:
- The DNA fragment of interest
- Taq DNA polymerase
- Nucleotides
- Primers
- A thermocycler
- Three steps of PCR
- Denaturation
- Annealing
- Extension
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