Polymerase Chain Reaction - An Overview

Introduction

The polymerase chain reaction (PCR) is a common technique for quickly producing millions to billions of copies of a particular DNA sample, which enables researchers to amplify a very tiny amount of DNA (or a portion of it) to the point where it can be thoroughly examined. American scientist Kary Mullis of Cetus Corporation devised PCR in 1983. Thermal cycling is a technique used by PCR to multiply DNA through cycles of temperature variations. The essential elements for separating DNA strands, binding primers, and replicating DNA are primers and DNA polymerase. Taq polymerase is a commonly used heat-stable enzyme. This technique transformed the area of genetic research and is now essential to many scientific domains.

What Is the Principle of Polymerase Chain Reaction?

The DNA target is the portion of a DNA strand that is amplified during PCR. Most PCR techniques amplify DNA fragments that range in length from one-tenth to 10 kilobase pairs (kbp), while others even allow for the amplification of fragments as long as 40 kbp. Once the reaction reaches a limiting point, the amount of amplified product is governed by the substrates available.

Several components and reagents are needed for a basic PCR setup:

• DNA Template: DNA template that includes the DNA target area to amplify a DNA polymerase

• DNA Polymerase (Taq): An enzyme that polymerizes new DNA strands. Taq polymerase is used since it is heat resistant and is more likely to hold together during the process of high-temperature DNA denaturation.

• DNA Primers: Two DNA primers complementary to the three prime ends of the sense and antisense strands of the target DNA (DNA polymerase can only bind to and elongate from a double-stranded region of DNA; in the absence of primers, the polymerase cannot bind to a double-stranded initiation site).

• Deoxynucleoside Triphosphates (dNTPs): The building blocks that DNA polymerase utilizes to create a new DNA strand.

• Buffer Solution: The buffer solution produces the ideal conditions for the stability and activity of DNA polymerase.

• Cations: Monovalent cations, usually potassium (K) ions, and bivalent cations, usually magnesium (Mg) or manganese (Mn) ions; Mg is the most frequent, although Mn can be utilized for PCR-mediated DNA mutagenesis since a greater Mn concentration increases the error rate during DNA synthesis.

The PCR reaction typically occurs in tiny tubes with a volume of 10–200 μL within a heat cycler. For every chemical step, the tubes are heated and cooled by the cycler. A Peltier device, which enables simple electric current reversal for both heating and cooling, is included in many contemporary cycles. Rapid temperature variations are ensured using thin-walled tubes.

What Is the Procedure of Polymerase Chain Reaction?

Thermal cycles, or cycles of changing temperatures, are commonly used in PCR. Usually, there are two or three different temperature stages in each cycle. The procedure typically starts with a single step at a temperature of over 90 °C (194 °F), is followed by cycles, and concludes with a hold for the extension of the final product or temporary storage. Several variables, including the number of ions and dNTPs, primer melting temperature (Tm), and the DNA synthesis enzyme, affect the precise temperatures and lengths of time in each cycle.

Common stages in most PCR techniques consist of:

• Initialization: For heat-activated DNA polymerases, heat the chamber to 94–96 °C (201–205 °F) for one to 10 minutes.

• Denaturation: The first cycle involves heating to 94–98 °C (201–208 °F) for 20–30 seconds, causing DNA melting.

• Annealing: Lower temperature to 50–65 °C (122–149 °F) for 20–40 seconds. Primers bind to single-stranded DNA.

• Two different primers are used, each matching one of the single-stranded complements with the target region.

• Critical to find an optimal temperature for specificity and efficiency.

• Extension/Elongation: Temperature depends on DNA polymerase; Taq polymerase commonly uses 72 °C (162 °F). Polymerase adds dNTPs, synthesizing a new DNA strand complementary to the template.

• The time for elongation depends on the polymerase and target region length.

• At optimal conditions, DNA polymerases can polymerize a thousand bases per minute.

• Cycle: Denaturation, annealing, and elongation form one cycle. Multiple cycles amplify the DNA target exponentially.

• Final Elongation: Optional step at 70–74 °C (158–165 °F) for five to 15 minutes after the last cycle to ensure complete elongation.

• Final Hold: Cool to four to 15 °C (39–59 °F) for short-term storage of PCR products.

What Are the Applications of Polymerase Chain Reaction?

• Selective DNA Isolation: By amplifying specific areas of genomic DNA selectively, PCR allows the isolation of certain DNA fragments. This is important for several uses, including DNA cloning, forensic analysis, and the generation of hybridization probes. PCR yields a large amount of pure DNA, enabling examination with little initial material.

• PCR in DNA Sequencing: By identifying unknown sequences, PCR helps in DNA sequencing. Sanger sequencing may take advantage of amplified areas. This facilitates the insertion of DNA into vectors more quickly, which helps recombinant DNA technology. It is possible to quickly screen bacterial colonies for the appropriate DNA vector constructions. Forensic identification by genetic fingerprinting also makes use of PCR.

• DNA Amplification and Quantification: PCR is a technique that amplifies certain areas of DNA, making it possible to analyze tiny samples. In forensics and the study of ancient DNA, this is essential. Quantitative PCR, or qPCR, is used in gene expression research to measure DNA quantities. Accurate quantification is ensured throughout synthesis by real-time monitoring. Sensitive gene regulation assessment and RNA quantification are made possible by the combination of qPCR and reverse transcription (RT-qPCR).

• Applications in Medicine and Diagnostics: Prenatal testing, preimplantation genetic diagnosis, and genetic carrier screening all employ PCR. In tissue typing for organ transplantation, it is essential. Individualized therapy based on oncogene mutations is made possible by PCR-based assays, which help in cancer detection. Because PCR tests may identify malignant cells unique to translocations, they aid in the early diagnosis of malignant illnesses such as leukemia.

• Applications in Infectious Diseases: PCR offers a quick and accurate diagnosis for infections brought on by viruses or bacteria. It facilitates the discovery of antibiotic resistance and helps identify bacteria that are not cultivable. PCR testing tracks the transmission of illness throughout populations, identifies newly emerging virulent subtypes, and helps in the early diagnosis of viral diseases.

• Applications in Forensics: PCR-based genetic fingerprinting is commonly used in forensics to identify specific individuals. It supports the study of crime scenes, confirming or dismissing suspects. Common applications include DNA paternity testing and tracing adopted children's biological parents.

• Research Applications: PCR is a versatile tool for molecular genetics research. It contributes to conventional DNA cloning procedures, helps with DNA sequencing, and makes it easier to produce small DNA segments. PCR and sequence-tagged sites are useful for mapping genomes and coordinating findings from several labs.

Conclusion

Polymerase Chain Reaction (PCR) has revolutionized molecular biology, enabling rapid and accurate DNA amplification. This adaptable method has greatly increased the awareness of genetics and has uses in research, diagnostics, and forensics. The effectiveness, sensitivity, and wide-ranging effects of PCR highlight its critical position in the contemporary biological sciences.