



The biotech sector involves a set of methods including the drugs, biologics, devices and other applications by integrating natural sciences and engineering principles. These biotech firms generally compete in established markets with novel products and technologies. Lab-based genome replication was tough to achieve since the techniques were inefficient and crude before the advent of PCR technology. However, the biotech industry started emerging in the late 1980s after the invention and approval of PCR technology. Since most of the products require replication or amplification of the genetic sequences, PCR helped to establish the biotech market. Hence, it has been a breakthrough in the world of life sciences. The simple initial design of the PCR technique had various improved versions later on with automated functioning. It not only improved the CAGR of the biotech industry but also helped the other fields to develop including the Human Genome Project, forensics, biodiversity, animal technology, and agriculture.





Image 1: Polymerase Chain Reaction



A biotech firm known as Cetus Corporation in the early 1970s was the first company to bring forward the concept of the PCR. A scientist working at Cetus Corporation known as Kary Mullis worked hard to achieve the DNA amplification using various molecular biology techniques. He first tried synthesizing oligonucleotide molecules manually and then tried evaluating them through automated sensitizer prototypes. Cloning was prevalent before PCR technology. However, PCR helped in improvising the technique. Cloning DNA was a time-consuming process. Thus, scientists tried to find a technique which would speed up the process generating more number of gene copies. PCR manifested this concept practically. The PCR technology was successful in the 1980s. Earlier the cloning techniques were “in vivo” cell-based. Now, most of the techniques involve in vitro polymerase chain reaction.





About Polymerase Chain Reaction (PCR):

In a process known as amplification, PCR produces millions of copies of short DNA segments through repeated cycles of denaturation, annealing, and extension. Thus, it generates a huge quantity of DNA to perform various tests. The PCR does not require the host organism. The amplified PCR products are known as amplified DNA or amplifiers. PCR is a complement to the cloning. However, it is not a complete replacement. PCR involves a principal of thermal cycling. The exposure of the reactants to repeated heating and cooling cycles is temperature dependent. It is a DNA melting and enzyme-based quick type of replication involving a selective amplification. Once a DNA gets amplified, it serves as a template in carrying out a series of reactions. It leads to a very high rate of DNA amplification. A PCR machine or a thermal cycler possesses a capacity to produce huge quantities of DNA fragments if the base pair sequence of the fragment is known. The PCR machine needs a very small quantity of the sample. It produces millions of copies of fragments.





Image 2: Steps involved in Polymerase chain reaction





Steps involved in PCR:

Three main steps of PCR include denaturation, annealing, and extension.

1. Denaturation: 0C. At this temperature, the hydrogen bonds break apart. The DNA double helix requires unwinding to undergo replication. Hence, the denaturation process primarily denatures the DNA double helix into a single-stranded structure. Denaturation process requires a temperature of 90-95C. At this temperature, the hydrogen bonds break apart.

2. Annealing: 0C. The primers are complementary DNA sequences. They involve annealing to the opposite strands of the template near the desired sequences. Thus, hybrid DNA molecules paired with strands get synthesized. Annealing leads to spontaneous alignment of the two complementary strands to form a double helix. The denatured solution is cooled to carry out the process of annealing. The primers anneal at 37-65C. The primers are complementary DNA sequences. They involve annealing to the opposite strands of the template near the desired sequences. Thus, hybrid DNA molecules paired with strands get synthesized. Annealing leads to spontaneous alignment of the two complementary strands to form a double helix.

3. Extension: 0C. We get the enzyme from thermophilic bacteria known as Thermus aquaticus. An enzyme known as Taq DNA polymerase extends the primers. The enzyme requires a temperature of 70-75C. We get the enzyme from thermophilic bacteria known as Thermus aquaticus.

Repetition of the heating and cooling cycles is a process involved in thermal cycling. Repeated thermal cycling results in an increase in the amount of unit-length DNA geometrically. Various thermal cyclers used in the industry include commercial applications for research purpose. However, there are limitations and strengths of this technology. Difficulty in the purification of gene fragments, cumbersome clone identification, and unattainable fragments with more than 100 Kb sizes are few limitations of the technology.





Applications of PCR:

The primary strength of PCR includes products easily obtained from a wide number of DNA samples. PCR is used to screen the DNA mutations, find SNPs and variations in the lengths of the microsatellites. PCR amplification requires a very small sample. A blood spot or a piece of hair is enough for the test. Hence, PCR has wide applications in the fields of forensics and archaeology. In clinical diagnostics, PCR involves detection of virus-induced diseases such as HIV, and virus-induced cancer. Thus it helps in treating the diseases. With the help of amplified samples, it becomes easy to run the gel and carry out the southern blot to reveal paternal and maternal relationships for conducting a successful DNA fingerprinting test.

With PCR technology, detection of pathogens, mutation screening, and genetic matching is easy. Human Genome Project achieved great heights due to PCR. It involves sequencing and bioinformatics apart from gene cloning. Site-directed mutagenesis and gene expression studies in genetic engineering require PCR. Plant genetic research essentially involves PCR.





PCR Market data:

The global industrial analysis and market opportunity studies reveal the expansion of the PCR market at a considerable CAGR. Hence there is a huge demand for PCR machines, reagents, kits, and other PCR consumables. Instruments including standard PCR systems, digital and RT-PCR systems have a great demand. The machine has high precision accuracy, reproducibility, and speed. Biotech companies in the Asia Pacific and North America expand in the PCR technology. More and more pathology labs have started using PCR for the detection of various diseases.





FDA approval for PCR:

Multiplex PCR, RT-PCR, and many other types of PCR gained approvals from the Food and Drug Administration, FDA every year. These assays further involve detection of HIV, HCV, HBV, Mycoplasma, and other infectious agents. FDA approved nucleic acid testing Cobas 6800 and 8800 PCR systems for studying the zika virus.

Digital PCR has opened up new avenues known as Bridged Nucleic Acid (BNA) technology. A bridged nucleic acid is a six-membered bridged structure. It consists of an N-O linkage. It is a nucleic acid analog with a higher binding affinity, single mismatch discrimination, and many other characteristics. BNA hybrids help to conduct sequence-specific hybridization and specific designing of probes for BNA.

RT-PCR or reverse transcriptase PCR involves a highly sensitive technique for detecting and quantifying RNA. Following steps are involved in RT-PCR:

A cDNA gets synthesized from an mRNA molecule using a primer and a reverse transcriptase enzyme. The cDNA is amplified using PCR. It is used for testing and quantifying RNA.

PCR also helps in plant genome analysis, editing and study of targeted mutagenesis.



References:

[1] Human Molecular Genetics 3, Volume 3, T. Strachan, Andrew P. Read

[2] Understanding PCR: A Practical Bench-Top Guide, Sarah Maddocks, Rowena Jenkins

[3] PCR Market data, Google news

[4] The Polymerase Chain Reaction, Kary B. Mullis, Francois Ferre, Richard A. Gibbs







