Learning Objectives

• Describe the clinical utility of PCR in diagnosing infections.
• Master the three temperature-dependent steps: Denaturation, Annealing, and Elongation.
• Identify the essential reagents required for DNA amplification.

1. Overview & Clinical Utility

PCR is a molecular biology lab procedure used to amplify a desired fragment of DNA. It is highly sensitive, making it an ideal diagnostic tool for conditions where pathogen load may be low or antibody testing is unreliable.

• Neonatal HIV: Used because maternal IgG antibodies cross the placenta, which would cause a false positive on an ELISA.
• Herpes Encephalitis: PCR of the CSF is the gold standard for detecting HSV-1 or HSV-2.

2. The Three Steps of the Cycle

The reaction occurs in a thermal cycler that repeats these three steps to achieve exponential growth ().

• Step 1: Denaturation (~95°C)
  The double-stranded DNA (dsDNA) template is heated to break hydrogen bonds, separating it into two single strands.
• Step 2: Annealing (~55°C)
  The sample is cooled. DNA primers anneal to the specific sequence boundaries on the template.
• Step 3: Elongation (~72°C)
  The temperature is slightly increased. A heat-stable DNA polymerase (like Taq) adds dNTPs to the strand to replicate the sequence starting from the primers.

Activity

3. Reagents Summary

• DNA Template: Target sequence to be copied.
• DNA Primers: Short DNA fragments that provide a starting point for the polymerase.
• Heat-stable Polymerase: Often Taq polymerase; remains active during high-temperature denaturation.
• dNTPs: The “building blocks” (dATP, dTTP, dCTP, dGTP).

Activity