Updated: 1/25/2018

Polymerase Chain Reaction (PCR)

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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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