Genetics - Laboratory Techniques: PCR

Polymerase Chain Reaction (PCR)

  • PCR is a crucial laboratory technique used to amplify small quantities of DNA.
  • It gained prominence during the COVID-19 pandemic as a diagnostic tool.
  • The goal is to increase the amount of DNA in a sample, which is useful for downstream applications like gel electrophoresis.
  • PCR is a valuable diagnostic tool for viruses like HIV, herpes encephalitis, and COVID-19.

Steps of PCR

The PCR process involves three key steps:

1. Denaturation
  • The initial step is denaturation, where the DNA sample is heated to 9595 degrees Celsius.
  • This high temperature breaks the bonds between the two DNA strands, separating them.
2. Annealing
  • In the annealing step, the sample is cooled to 5555 degrees Celsius.
  • This is when the necessary ingredients for DNA replication are added:
    • DNA primers: These bind to the DNA strands.
    • Heat-stable polymerase (Taq polymerase): This enzyme is active at high temperatures and is typically sourced from thermal vents.
    • Deoxynucleotide triphosphates (dNTPs): These are the building blocks (nucleotide bases) that will be incorporated into a new DNA strand.
  • DNA primers anneal to the desired portions of the DNA strand, flanking the region to be amplified.
  • Usually, two primers are used to flank the desired region, amplifying the sequence in between.
3. Elongation
  • During elongation, the temperature is increased to 7272 degrees Celsius.
  • This allows the Taq polymerase to add dNTPs to the DNA primer, extending the DNA strand and amplifying the signal.
  • dNTPs are added in the three prime direction, synthesizing a new DNA strand.
  • After the first round of PCR, one strand of DNA has been replicated into two complete strands.

Amplification Process

  • The PCR process can be repeated multiple times to exponentially amplify the DNA.
  • Each cycle doubles the amount of DNA.

Reverse Transcriptase PCR

  • A special type of PCR, reverse transcriptase PCR, is used for amplifying mRNA instead of DNA.
  • Since PCR relies on DNA, mRNA must first be converted into complementary DNA (cDNA) before the standard PCR process can be applied.
  • This conversion is achieved through the addition of a reverse transcriptase enzyme.
  • The reverse transcriptase enzyme binds to the mRNA and creates a complementary strand of DNA (cDNA).
  • The resulting cDNA can then be used in the standard PCR process.

Review of PCR Steps

  1. Denaturation:
    • Heat the sample to a high temperature to break the bonds and separate DNA strands.
  2. Annealing:
    • Add DNA primers, deoxynucleotide triphosphates (dNTPs), and Taq polymerase.
    • Have primers anneal to DNA strands.
  3. Elongation:
    • Taq polymerase adds nucleotides in the three prime direction, creating a new strand of DNA.
    • This results in two new strands of DNA from the original.
  4. Repeat the process to obtain more DNA for analysis.