PCR

Introduction

It is possible to take a very small sample of DNA and amplify it (or a part of it) to a large enough amount to study it in detail using the polymerase chain reaction (PCR), which is a method widely used to rapidly make millions to billions of copies (complete copies or partial copies) of a specific DNA sample. The American biochemist Kary Mullis invented the polymerase chain reaction (PCR), which was recognised with the Nobel Prize in Chemistry in 1993. Mullis and biochemist Michael Smith, who had developed other essential methods of manipulating DNA, were jointly awarded the prize in 1993.

PCR

The polymerase chain reaction (PCR) is essential to many procedures used in genetic testing and research, including the analysis of ancient DNA samples and the identification of infectious agents. PCR is a technique in which copies of very small amounts of DNA sequences are exponentially amplified through a series of temperature-changing cycles. PCR is now a common and often indispensable technique in medical laboratory research, with applications ranging from biomedical research to criminal forensics. It is used in a wide variety of applications, including biomedical research and criminal forensics.

Thermal Cycling

The majority of PCR methods rely on thermal cycling. When reactants are subjected to repeated cycles of heating and cooling, they undergo a variety of temperature-dependent reactions, such as DNA melting and enzyme-driven DNA replication, which are both beneficial to the organism. DNA polymerase is one of the two main components of the PCR reaction. The other component is primers (which are short single-strand DNA fragments known as oligonucleotides that have a complementary sequence to the target DNA region). 

Steps involved in PCR

1. Denaturation 

In a thermal cycler, the solution contained within the tube is heated to a temperature of at least 94°C (201.2°F). As a result of the heat, the hydrogen bonds in the original DNA sample are broken, and the DNA is separated into single strands (this is termed denaturation of double-stranded DNA).

2. Annealing 

Once the sample mixture has been cooled to between 50 and 60 degrees Celsius (122 to 140 degrees Fahrenheit), the DNA primers and DNA polymerase enzyme can bind to the individual strands of DNA that have been separated by the heat treatment (see Figure 1). (this is termed annealing of the primers). As a result of the heating process, the nucleotides (A, T, C, and G) in the added mixture solution will pair with the individual separated strands of DNA that were formed as a result of the process.

3. Extension 

Once they’ve been joined together, they form a new complementary strand of genetic material (termed extension of the DNA). As a result, from each of the single strands of the original sample molecule, a new duplicate double-stranded DNA molecule has been formed from the original single-stranded DNA molecule. The temperature fluctuates between 95°C and 50°C and 60°C. With the help of the thermal cycler, which automatically repeats the heating and cooling cycles of the process, the cycle is repeated approximately 35 to 40 times. With each heating/cooling cycle performed by the cycler, the length of the resulting DNA sequence increases twofold. In this way, what began as a single short segment of DNA from one sample can be amplified to form millions of copies after 35 doubling cycles, regardless of how small the segment of DNA was, to begin with.

Examples of the use of PCR

• DNA cloning for sequencing

• Gene cloning and manipulation

• Gene mutagenesis; construction of DNA-based phylogenies or functional analysis of genes Diagnosis and monitoring of genetic disorders 

• Amplification of ancient DNA 

• Analysis of genetic fingerprints for DNA profiling (for example, in forensic science and parentage testing)

• Detection of pathogens in nucleic acid tests for the diagnosis of infectious diseases

Principle

PCR is a technique for amplifying a specific region of a DNA strand (the DNA target). Although some techniques allow for amplification of DNA fragments up to 40 kbp in length, the majority of PCR methods amplify DNA fragments ranging in length from 0.1 to 10-kilo base pairs (kbp). The amount of amplified product produced is determined by the number of available substrates in the reaction, which becomes increasingly limited as the reaction progresses, as previously mentioned.

Procedure

• PCR is typically composed of a series of 20–40 repeated temperature changes, referred to as thermal cycles, with each cycle consisting of two or three discrete temperature steps, as is common in the industry.

• One or more temperature steps at extremely high temperatures are frequently performed before the cycling, which is followed by one hold at the end for final product extension or brief storage. 

• Several parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and deoxynucleotides in the reaction, and the melting temperature of the primers, influence the temperatures used and the length of time they are held at each temperature in a single cycling procedure.

Advantages

There are numerous advantages to using PCR. It is relatively simple to comprehend and use, and it produces results in a short period. In addition to being highly sensitive, this technique has the capability of producing millions to billions of copies of a specific product for sequencing, cloning, and other analyses. The advantages of qRT-PCR are the same as those of PCR, with the additional advantage of being able to quantify the synthesised product. Because of this, it has applications in the analysis of alterations in gene expression levels in tumours, microbes, and other disease conditions.

The polymerase chain reaction (PCR) is a very powerful and practical research tool. The polymerase chain reaction (PCR) is being used to determine the sequence of unknown etiologies of many diseases. The technique can aid in the identification of previously unknown viruses that are related to those that are already known, allowing us to gain a more complete understanding of the disease itself. If the procedure can be made even simpler, and sensitive non-radiometric detection systems can be developed, the polymerase chain reaction (PCR) will continue to hold a prominent position in clinical laboratories for many years to come.

Conclusion

It is possible to amplify DNA sequences using the polymerase chain reaction (PCR), which is a laboratory technique. The method entails the use of short DNA sequences known as primers to select the portion of the genome that will be amplified during the process. It is necessary to raise and lower the temperature of the sample repeatedly to aid a DNA replication enzyme in copying the target DNA sequence. The technique is capable of producing a billion copies of the target sequence in a matter of hours or even minutes.