PCR: The Gold Standard Method in Molecular Diagnostics


Over the last few decades, the polymerase chain reaction (PCR) has revolutionized how we diagnose diseases and investigate their underlying causes at the genetic level. Reverse transcriptase-polymerase chain reaction (RT-PCR) is a highly sensitive, specific technique for studying differential gene expression, and real-time or quantitative reverse transcriptase PCR (real-time RT-PCR) provides valuable quantitative data that allows the user to see how much a gene has been activated or repressed. By profiling genes involved in specific pathways, diseases, or processes, one can put together a clear picture of the changes occurring in the body, potentially leading to the discovery of diagnostic and prognostic markers to enhance both curative and preventive medicine. PCR is used to make millions of copies of a target piece of DNA. It is a vital tool in modern molecular biology and has transformed scientific research and diagnostic medicine.

PCR was developed in the 1980s by Kary Mullis, who received the Nobel Prize in 1994. The discovery of PCR brought greater benefits and scientific developments such as genome sequencing, gene expressions in recombinant systems, the study of molecular genetic analyses, including the rapid determination of both paternity and the diagnosis of infectious diseases. PCR helps identify the DNA of interest rather than the whole genome. From a small genetic sample, the genotypes can now be determined, and as a result, many genetic disorders can be detected, diagnosed and monitored. PCR is an excellent technique for the rapid detection of pathogens. Real-time PCR is playing an ever-increasing role in clinical diagnostics and research laboratories. It is considered a rapid and accurate method due to its capacity to generate both qualitative and quantitative results.

Recent developments in molecular biology have revolutionized the detection and characterization of microorganisms in a broad range of medical diagnostic fields. Since its introduction, this PCR technology has revolutionized the field of biological research and established its numerous benefits in genetics and medical diagnostics. PCR enables the synthesis of specific DNA fragments using a DNA-polymerase enzyme, which takes part in the replication of the cellular genetic material. This enzyme synthesizes a complementary sequence of DNA, as a small fragment (primer) is connected to one of the DNA strands in the specific site. Primers limit the sequence to be replicated and the result is the amplification of a particular DNA sequence with billions of copies.

Components of PCR

i. DNA Template: The DNA template is the target sequence that we want to be copied.
ii. DNA Polymerase: It is a type of enzyme that synthesizes new strands of DNA complementary to the target sequence. Taq DNA polymerase is mostly used in PCR. It is derived from Thermus aquaticus. It does not usually denature in hot temperatures required in PCR.
iii. Primers: Primers are short oligonucleotides of DNA that are complementary to the target sequence. If we want to amplify a certain section of the gene, primers of the specific sequence must be used.
iv. Nucleotides (dNTPs or deoxynucleotide triphosphates): Adenine (A), Thymine (T), Guanine (G) and Cytosine (C) are building blocks for DNA replication.

How Does PCR Work?

The technique utilizes thermal cycling to accomplish three key steps: denaturation, annealing, and extension. A DNA template is mixed with a thermostable DNA polymerase such as Taq DNA polymerase, free deoxynucleotide triphosphate molecules (dNTPs), and short oligonucleotides called primers, which are complementary for the beginning and end of the sequence to be amplified. In the denaturation step, double-stranded dsDNA separates into single strands, and in the annealing step, primers bind to their complementary sequences. DNA polymerase then produces complementary DNA strands in the extension step. The size and the purity of the final DNA product can be analysed using gel electrophoresis.


Real-Time PCR

Real-time PCR has enabled the quantification process of DNA and RNA fragments. Moreover, qualitative detection is also possible. Real-time PCR allows the precise quantification of these nucleic acids with greater reproducibility. It is based on the concept of monitoring DNA amplification in real-time through the monitoring of fluorescence. It requires a thermocycler with an optical system to capture fluorescence and a computer with software capable of capturing the data and performing the final analysis of the reaction. The emission of fluorescence generates a signal that is directly proportional to the amount of amplified products. The fluorescent probes used are SYBR Green and TaqMan.

PCR As a Molecular Diagnostic Tool

PCR technology is considered a gold-standard method in molecular diagnostics.

I. In Virology

Recent advances in molecular biology have facilitated the detection and characterization of viral nucleic acids. PCR enables the amplification of the gene of interest. This provides a complete viral characterization, determining the subtype, genotype, variation, mutation and standards of genotypic resistance of these viruses. During this coronavirus pandemic, real-time RT-PCR serves as one of the most accurate laboratory methods for detecting, tracking and studying the COVID-19 coronavirus.

II. In Microbiology

Conventional PCR has been used for over a decade in clinical microbiology laboratory research to identify microbial pathogens. However, this technique has been restricted to the detection of microorganisms due to few reasons. Most tests based on conventional PCR involve multiple steps and, therefore, require careful expertise. Real-time PCR gives a rapid analysis with greater sensitivity and precision. To date, real-time PCR is the preferred method in detecting microorganisms.

III. In Dentistry

PCR has recently become a standard diagnostic and research tool in dentistry. It offers rapid detection of periodontal pathogens in subgingival samples. It is an excellent tool in identifying species associated with dental caries and their location in the ecological niches, thereby helping to clarify the progression of the carious process. PCR has a better detection pattern than traditional microbiological identification methods and exhibits greater specificity under optimized conditions.

The discovery of PCR enables the detection of microorganisms with increased sensitivity, precision and accuracy. RT-PCR and real-time RT-PCR are powerful tools to study changes in gene expression. They have already proven their utility for clinical applications in research laboratories. The pathway-focused analysis will continue to clarify many biological processes, and PCR technology will continue to advance. Ultimately, PCR technology will be one of the keys to achieving the goals of personalized medicine and the “bench-to-bedside” application of research discoveries through its ease of use, cost-effectiveness, and molecular power. In the future, medical advances will likely make use of PCR-based approaches for gene expression to yield early clues for diagnosis and prognosis and to monitor the effectiveness of treatments.