Discovery of PCR
During a late-night drive home in 1983, American biochemist Kary Mullis had a moment of inspiration and documented the idea that eventually earned him the Nobel Prize in Chemistry in 1993 on the back of a receipt. The simple concept aimed to replicate the DNA replication process in a laboratory test tube, generating new complementary DNA chains to the existing ones. Building on Sanger's DNA sequencing, Mullis realized that repetitive use of DNA polymerase could trigger a chain reaction, leading to the amplification of specific DNA segments. Before the discovery of the heat-stable DNA polymerase Taq, scientists struggled to optimize cyclic DNA amplification as polymerases couldn't withstand high-temperature denaturation. Taq's discovery allowed the amplification process to proceed without adding fresh polymerase in each cycle, making it more efficient and time-saving. In 1985, the journal Science reported the first use of Taq polymerase in the PCR. Since then, PCR has undergone robust and systematic improvements, becoming a pivotal tool in fields ranging from forensic evidence analysis and diagnostics to disease monitoring and genetic engineering, hailed as one of the most significant scientific advancements of the 20th century. In 1993, the first FDA-approved PCR kit was introduced to the market.
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Basic Steps of PCR
PCR is a technique used to amplify specific DNA segments from complex mixtures of template DNA. The experiment is straightforward and typically completed within a few hours. PCR reaction requires the template DNA to be amplified, DNA polymerase, primers, deoxyribonucleotide triphosphates (dNTPs), and PCR buffer. Primer design is crucial, requiring avoidance of binding to similar sequences, and online tools are available to assist in design.
Fig. 1 Basic Steps of PCR.
The basic steps of PCR include denaturation, annealing, and extension. In the denaturation step, template DNA is heated to 95°C, causing the two strands to separate. Next, in the annealing step, the temperature drops to 50-65°C, allowing primers to bind to the target DNA sequence. Finally, in the extension step, the temperature rises to 72°C, and Taq polymerase synthesizes a new DNA strand in the presence of primers. These three steps constitute a PCR cycle, which can be repeated 30-40 times, leading to a significant amplification of the target DNA.
Different Types of PCR
Quantitative Real-time PCR (qPCR)
qPCR stands as a cornerstone in the realm of PCR applications. This quantitative technique offers real-time insights into the amplification process and allows for on-the-fly detection of PCR products. Notably, it obviates the necessity for gel electrophoresis through the incorporation of non-specific fluorescent intercalating dyes like SYBR Green and sequence-specific fluorescent probes such as hydrolysis (TaqMan) probes.
Reverse Transcription-PCR (RT-PCR)
In the domain of gene transcription analysis, RT-PCR and its quantitative counterpart (RT-qPCR) emerge as stalwarts. RT-PCR involves the synthesis of a DNA/RNA hybrid, followed by the degradation of the RNA component. Successful execution hinges on the quality of the starting RNA. The methodology can be executed in either one-step or two-step configurations, each with distinct advantages.
Digital PCR (dPCR) and Droplet Digital PCR (ddPCR)
Digital PCR modifies the traditional approach by partitioning PCR reactions into numerous nanoliter-sized reactions or droplets. The subsequent analysis, leveraging Poisson statistics, discerns the percentage of positive droplets, signifying the target's presence. Droplet digital PCR (ddPCR) takes this a step further, utilizing water-oil emulsion for partitioning, simplifying outcome analysis.
Microfluidic PCR
In the pursuit of enhanced efficiency, Microfluidic PCR adopts microfluidic processing systems, harnessing microchannels and microchambers for PCR amplification. This technology marries speed, sensitivity, and minimal reagent consumption, rendering it particularly apt for real-time PCR applications, notably in diagnostics.
Applications of PCR
Gene Transcription
PCR proves invaluable in scrutinizing variations in gene transcription across cell types, tissues, and organisms. The process involves isolating RNA, reverse transcribing it into cDNA, and subsequently quantifying the original RNA levels.
Gene Typing
For detecting sequence variations in alleles of specific genes, PCR's application extends to gene typing. A notable instance is the gene typing of transgenic organisms, where primers amplify the transgenic or mutated portions.
Cloning and Mutagenesis
PCR's utility extends to cloning, with amplified DNA fragments inserted into vectors. This facilitates genetic material manipulation in bacterial strains. Site-directed mutagenesis, achieved through recombinant PCR, introduces point mutations.
Sequencing
In the realm of sequencing, PCR enriches template DNA. High-fidelity PCR ensures sequence accuracy. It plays a pivotal role in both Sanger sequencing and next-generation sequencing (NGS) library preparation.
In conclusion, the myriad applications and nuanced methodologies within the PCR landscape epitomize its pivotal role across scientific disciplines.
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