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