Discuss the principle and the steps involved in real time PCR. Enlist any five applications of PCR in medicine.

Principle of Real-Time PCR

The core principle of PCR remains the same: enzymatic amplification of a specific DNA sequence using primers, a DNA polymerase, and nucleotides. However, real-time PCR incorporates fluorescent reporters that allow the detection of PCR product accumulation during each cycle. This is achieved through the use of fluorescent dyes or probes that bind to the amplified DNA. The intensity of the fluorescence signal is directly proportional to the amount of DNA produced. Unlike conventional PCR, which relies on end-point analysis (e.g., gel electrophoresis), real-time PCR provides continuous monitoring.

Steps Involved in Real-Time PCR

1. Master Mix Preparation: This includes the DNA template, primers (forward and reverse), a DNA polymerase (typically thermostable), dNTPs (deoxynucleotide triphosphates), a fluorescent reporter (dye or probe), and a buffer solution.
2. Initial Denaturation: The reaction mixture is heated to a high temperature (typically 95°C) for a specific duration (e.g., 5-10 minutes) to denature the double-stranded DNA template into single strands.
3. Cycling (Repeated 30-40 times): Each cycle consists of three steps:
   • Denaturation: Heating to 95°C to separate the DNA strands.
   • Annealing: Lowering the temperature (typically 55-65°C) to allow primers to bind to their complementary sequences on the single-stranded DNA.
   • Extension: Raising the temperature to the optimal temperature for the DNA polymerase (typically 72°C) to extend the primers and synthesize new DNA strands.
4. Fluorescence Detection: During the extension step, the fluorescent reporter binds to the newly synthesized DNA, and the fluorescence signal is measured by the real-time PCR instrument.
5. Data Analysis: The instrument generates amplification curves, which plot fluorescence intensity against cycle number. The cycle threshold (Ct) value, the cycle at which the fluorescence signal crosses a defined threshold, is used to quantify the initial amount of target DNA. Lower Ct values indicate higher initial DNA concentrations.

Fluorescent Reporting Methods

Two main methods are used for fluorescence detection:

• DNA-binding dyes (e.g., SYBR Green): These dyes bind to any double-stranded DNA, providing a simple and cost-effective method. However, they can also bind to non-specific PCR products, leading to inaccurate quantification.
• Fluorescent probes (e.g., TaqMan probes): These are sequence-specific oligonucleotides labeled with a fluorescent reporter and a quencher. The probe hybridizes to the target DNA, and during extension, the polymerase cleaves the probe, separating the reporter from the quencher and generating a fluorescent signal. This method is more specific than dye-based methods.

Applications of PCR in Medicine

1. Infectious Disease Diagnosis: qPCR is widely used to detect and quantify viral loads (e.g., HIV, Hepatitis B, COVID-19) and bacterial pathogens, enabling early and accurate diagnosis.
2. Cancer Detection and Monitoring: qPCR can detect cancer-specific mutations and gene expression changes, aiding in early cancer detection, prognosis, and monitoring treatment response.
3. Genetic Testing: qPCR is used for prenatal diagnosis, carrier screening, and pharmacogenomic testing to identify genetic predispositions to diseases and predict drug responses.
4. Organ Transplant Monitoring: qPCR can detect donor-specific DNA in the recipient's blood, allowing for early detection of organ rejection.
5. Gene Expression Analysis: qPCR is used to quantify mRNA levels, providing insights into gene regulation and cellular processes in various diseases.