Polymerase chain reaction (PCR) | Khan Academy

Overview of PCR

  • Definition: Technique to amplify a specific DNA region in vitro.

  • Uses: Research, forensics, medical diagnostics.

Key Features

  • Taq Polymerase: Thermostable DNA polymerase derived from Thermus aquaticus.

  • Primers: Specific DNA sequences that flank the target region.

  • Temperature Cycles: Repeated cycles of temperature changes to amplify DNA.

Components of PCR

Taq Polymerase

  • Enzyme from heat-resistant bacterium (Thermus aquaticus).

  • Optimal performance at ~70°C for DNA synthesis.

Primers

  • Short, single-stranded DNA sequences (~20 nucleotides).

  • Complementary to sequences flanking the target DNA region.

Other Components

  • Template DNA: The DNA to be amplified.

  • Nucleotides (dNTPs): Building blocks for new DNA strands.

  • Cofactors: Assist enzyme functionality.

Steps of PCR

A. Denaturation (96°C)

  • Heat separates double-stranded DNA into single strands.

B. Annealing (55–65°C)

  • Primers bind to complementary sequences on single-stranded DNA.

C. Extension (72°C)

  • Taq polymerase synthesizes new DNA strands from primers.

D. Repetition

  • The cycle is repeated 25–35 times, doubling DNA in each cycle.

  • Exponential amplification results in millions/billions of copies in hours.

Visualization of PCR Products

  • Gel Electrophoresis: Separates DNA fragments by size using electric current.

  • DNA Ladder: Molecular weight marker for size comparison.

  • Staining: Stained DNA appears as bands under UV light.

Applications of PCR

  • DNA Cloning: Amplifying DNA for genetic engineering.

  • Medical Diagnostics: Identifying pathogens or genetic mutations.

  • Forensic Science: DNA profiling in crime scene investigations.

  • Genetic Testing: Detecting genetic disorders or paternity testing.

  • Ecology & Evolutionary Biology: Studying genetic diversity in populations.

PCR in Forensic Science

A. Short Tandem Repeats (STRs)

  • Highly polymorphic DNA regions for genetic fingerprinting.

  • STR analysis utilizes multiple markers for identification.

B. Accuracy in DNA Profiling

  • Standard forensic analysis uses 13 STR markers.

  • False positive probability: < 1 in 10 billion.

C. Legal & Humanitarian Impact

  • Exonerates wrongly accused individuals.

  • Identifies human remains from disasters.