Polymerase chain reaction (PCR)

Purpose: PCR amplifies small segments of DNA, making it easier to detect and analyze genetic material. It’s highly sensitive and specific.

1. Basic Steps:
   • Denaturation (melting phase): Heating the DNA to separate strands (95°C).
   • Annealing: Cooling to allow primers to bind to the DNA template (50-65°C).
   • Extension (synthesis): DNA polymerase (usually Taq polymerase) adds nucleotides to the primers to synthesize new DNA (72°C).
   • This cycle is repeated multiple times (usually 20-40 cycles) to exponentially amplify the DNA.
2. Components:
   • Template DNA: The DNA to be amplified.
   • Primers: Short sequences that start the DNA synthesis.
   • DNA Polymerase: Enzyme that adds nucleotides to form new DNA.
   • Nucleotides (dNTPs): Building blocks of DNA.
   • Magnesium 1.5 - 4.5 mmol/L and buffer
3. Types of PCR:
   • Conventional PCR: Basic method to amplify DNA.
   • Real-Time PCR (qPCR): Quantifies DNA amplification in real-time, often using fluorescent dyes.
   • Reverse Transcriptase PCR (RT-PCR): Converts RNA into DNA (cDNA) before amplification, useful for detecting RNA viruses.
   • Multiplex PCR: Amplifies multiple targets in one reaction.

Clinical Applications of PCR:

1. Infectious Disease Diagnosis:
   • Detects pathogens like HIV, Hepatitis B/C, Mycobacterium tuberculosis, and SARS-CoV-2.
   • PCR is highly sensitive for viral load monitoring, essential for managing HIV or hepatitis.
2. Genetic Testing:
   • Used to identify genetic mutations in conditions like cystic fibrosis and BRCA1/BRCA2 mutations (hereditary breast cancer).
   • Detection of chromosomal translocations in leukemia (e.g., BCR-ABL in chronic myeloid leukemia).
3. Cancer:
   • Identifies oncogene mutations (e.g., KRAS, EGFR) and guides targeted therapies.
   • Minimal residual disease monitoring in hematologic cancers.
4. Forensic Science:
   • DNA fingerprinting for criminal investigations or paternity testing.
5. Transplant Medicine:
   • HLA typing for tissue matching in organ transplants.

Genetic markers such as microsatellites and single nucleotide polymorphisms (SNPs) are detected using PCR

RT-PCR is the standard for RNA virus detection, including COVID-19.

qPCR allows not only detection but quantification (e.g., viral load, cancer gene expression).

Clinical Correlate

In chronic myelogenous leukemia (CML), the presence of the Philadelphia chromosome translocation (t[9;22]) produces a BCR-ABL, an abnormal fusion protein with tyrosine kinase activity. It has been shown that monitoring the level of BCR-ABL mRNA with an RT-PCR in CML patients during therapy with Imatinib (a tyrosine kinase inhibitor) is helpful for both prognosis and management of therapy.