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Updated: Jan 25 2018

Polymerase Chain Reaction (PCR)

  • PCR overview
    • Function
      • can amplify a selected region of DNA
    • Process
      • solution prepared containing
        • DNA primers specific for selected DNA region
        • DNA sample of interest
        • heat stable DNA polymerase
        • deoxyribonucleotides
      • denaturation of dsDNA by heating
      • annealing of DNA primer specific for region of interest and slowly cooling the solution
      • replication of DNA at the primer by heat stable DNA polymerase
      • repetition of the process several times
      • gel electrophoresis used to separate the various components of the solution
    • Clinical use
      • in all uses PCR functions to amplify the amount of DNA present in a sample
      • high specificity bacterial/viral infection testing
        • HIV
          • first step is ELISA (high sensitivity)
          • PCR is used to determine viral load
          • test examines the amount of viral DNA integrated into host cell DNA
          • advantages over ELISA
            • PCR becomes positive earlier in disease course
              • Positive ELISA result is dependent on antibody formation
            • PCR does not require that the patient have a competent immune system
              • ELISA requires the host to make antibodies
          • important specific cases when PCR should always be used
            • a newborn whose mother is HIV+
              • will have antibodies even if not infected, so ELISA does not work
            • when earliest possible detection is required
      • genetic identification
        • forensic/paternity testing
        • use of variable number tandem repeats (VNTRs) or short tandem repeats (STRs)
          • unique copies of non-coding regions of DNA between individuals
          • since they exist on both chromosomes, individuals have two copies at each locus
            • 1 paternal and 1 maternal
        • can only prove with certainty that the sample DOES NOT belong to the test subject
          • cannot prove with 100% certainty that DNA belongs to individual of interest because there is a small chance that someone shares the same VNTR or STR
      • direct mutation
        • if DNA region is known, PCR can amplify that region for sequencing
  • RT-PCR overview
    • Function
      • used to measure the amount of RNA present in a sample
    • Process
      • reverse transcriptase is added to solution containing RNA, dNTPs, primers for specific sequence of interest, and heat stable DNA polymerase
      • RNA is converted to DNA and DNA sequence of interest is amplified
      • When combined with quantitative (real-time PCR), the combined qRT-PCR (quantitative reverse transcription PCR) assesses the amount of RNA in the original sample by measuring the amount of amplified PCR product after a set number of PCR cycles, which is directly proportional to the concentration of RNA in the original sample
    • Clinical use
      • HIV viral load
        • measures transcriptional activity of the virus by detecting the amount of RNA present
        • gives a more detailed picture of the infection and treatment results
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