Kary Mullis, a man of intelligence once used to be a baker with the dream of earning his own money but not only was he good at cooking, he was also an extreme science enthusiast. This is why even though he had less experience in molecular biology or genetics, he joined the molecular biology department of Cetus Corporation in California with the position of UCSF.
Then an incident took place that created revolutionary inventions in the field of Genetics, Medicine, and Biology. In the year 1985, Kary Mullis invented the technique, Polymerase Chain Reaction, also known as PCR which helps in studying the core of every life on this earth called DeoxyriboNucleic Acid (DNA) in detail.
After the invention of DNA and its structure, multiple scientists studied in detail the human genome, but the major problem faced by all of them was the availability of such DNA samples. It could only be obtained in minute amounts which were not sufficient for detailed studies.
However, the introduction of PCR solved this issue. PCR amplifies the available sample of DNA into millions or billions through cycles. Due to this factor, it has been widely used since then in various medicinal, diagnostic, molecular, forensic, and research fields.
Principle and Working of Polymerase Chain Reaction
Most laboratories use PCR to amplify the DNA sequences to obtain a larger quantity of samples for research purposes. PCR is also known as Molecular Photocopying, as it photocopies the existing DNA strands. Kary B Mullis won the Nobel prize in 1993 for the invention of PCR.
PCR works on the principle of thermal cycling, which is a set of rapid cycles of heating and cooling of samples in a thermal cycle machine to permit temperature-dependent reactions.
Requirements for PCR
Thermal Cycler- The machine: It is the PCR machine inside which the reactions take place with samples. It has a thermal block with holes for loading the sample tubes containing reaction mixtures. It has the space to load three hundred plus vials usually distinctively set in three thermal blocks, along with preloaded coding features and screen to control the operation of the machine such as time set and program set.
DNA Polymerase: Polymerases are used in stabilizing the reaction so that the DNA would be successfully made at the high temperature required for the reaction. Hence the most important feature of a DNA polymerase is that it should be heat stable. A few of them are PFU polymerase, obtained from Pyrococcus furiosus, and TTH polymerase obtained from Thermus thermophilus. However, the aptest and common polymerase used is Taq DNA polymerase. It is obtained from Thermus Aquaticus, which possesses the capability of tolerating very high temperatures and is found in hot springs across the world. It was first invented in 1969 at the hot spring of Yellowstone National Park. It is also believed to be the most ancient form of bacteria. They are capable of making new proteins such as DNA in high temperatures without getting degenerated, due to which Taq Polymerase is made from this group of bacteria for PCR.
DNA Primers: Primers are short sequences of nucleotides that provide a starting point for DNA synthesis. They are attached at the end of DNA strands that are being multiplied which also helps in elongation. They are usually around 20 nucleotides in length. For a nucleotide to be used as a primer it should contain certain properties:
- Processivity: The rate at which polymerase makes DNA strands with the target DNA sample.
- Fidelity: Accuracy of new DNA strands that are being made.
- Persistence: Stability of polymerase at high temperature along with the primer.
- Primer uniqueness: Sequence must be definite and unique which is why it is difficult to make for an unknown sample.
- Melting temperature: It is selected on the number of base pairs(bp). DNA primers are always Oligonucleotides i.e., a polynucleotide whose molecule contains a relatively smaller number of nucleotides.
Deoxyribonucleic Triphosphates(dNTPs): Nucleosides bonded with three phosphate groups containing deoxyribose as the backbone are dNTPs. They are organic compounds used as the building blocks of DNA. They help in enhancing the production of new DNA strands.
Tris Buffer: Also known as Trisaminomethane buffers, they are used in TAE and TBE variations of buffers in solutions of nucleic acids. It helps in maintaining the pH of the reaction and has a melting point of 175 degrees celsius.
Magnesium Chloride: The salt form of magnesium enhances the activity of Taq DNA polymerase and acts as a co-factor of the reaction by increasing the rate of amplification of DNA.
Potassium Chloride: It neutralizes the charge present on the backbone of DNA and reduces repulsion between negatively charged DNA strands, i,e the primer and the template, and stabilizes the primer-template binding.
Bovine Serum Albumin: BSA helps in yielding results from the most impure form of sample DNA. It removes the contamination and enhances production. It also helps in the stabilization of enzymes during the storage of the samples. Sometimes Gelatin is used as an alternative to BSA or along with BSA.
Process of Polymerase Chain Reaction
PCR takes place in four different stages:
- Initialization: It is done only in the presence of DNA polymerase that requires manual heat activation by Hot Start PCR.
- Denaturation: The target DNA, the sample to be amplified is heated at a temperature range of 94-98 degrees celsius for one minute, leading to the separation of the double-stranded DNA into two single-stranded DNA due to the breakage of hydrogen bonds between the base pairs in the given high temperature.
- Annealing: The new strands are slowly built from the existing two template strands by attaching the primers in complementary directions(3’-5’: 5’-3’) using Taq polymerase. This is done by lowering the temperature to 50-54 degrees celsius.
- Elongation: The multiple strands are now forming with one old and one new strand of DNA and the cycle is repeated thirty to forty times for obtaining around billions of copies.
Types of Polymerase Chain Reaction (PCR)
PCR can be of various types with slight alterations in their making, but the principle remains the same for almost all of them. The major types include
- Real-time PCR: This is different from the typical PCR due to the presence of fluorescence reporter molecules in each reaction that yield increased fluorescence with increasing amounts of product DNA. The fluorescent agents used in this kind can be DNA binding dyes and certain fluorescent primers and proteins. RPCR helps to determine the initial number of copies of DNA template with accessory and high sensitivity over a wide dynamic range. It is applied hugely in Gene expression analysis and Cancer phenotyping.
- Quantitative Real-Time PCR (Q-RT PCR): Similar to RPCR, Q-RT PCR has the significance of reliable detection and measurement of product genes during each cycle of the PCR process.
- Reverse Transcription PCR (RT-PCR): It combines the reverse transcription of RNA to DNA and amplifies it using PCR. It is mainly used to measure the amount of specific RNA like in cases of highly infectious diseases caused by viruses such as Coronavirus.
Other types of PCRs include Multiplex PCR, Nested PCR, Long-range PCR, Single-cell PCR, Fast-cycling PCR, Methylation-specific PCR (MSP), Hot start PCR, High-fidelity PCR, etc.
Applications and Forensic Significance of PCR
Polymerase Chain Reaction had proved to be the best technique found till now in molecular inventions. It is mainly used in genetic fingerprinting. It is also applied in the medical field and has proved to cure lots of diseases such as cancer and those caused by dangerous viruses. It has also helped in the agricultural field in creating a wide variety of cross-bred species of crops.
In forensics, PCR brought in a huge change. Samples from crime scenes can be very limited. Hence concluding DNA evidence could have been highly difficult. But with the introduction of PCR, forensic experts could test and extract information by multiplying the little traces of DNA received from the crime scene.
Amplification of VNTRs and STR in it had added on to the improved assessment of evidence further on helping to eliminate the innocent people who were convicted and concluded to be the criminals.
It also helps in DNA paternity testing and to amplify DNA samples from bone or skeletal evidence. PCR also helps forensic research in the field of genetic concordance.