LMP301H1 Lecture 22: Molecular Diagnostics

LMP301 Introduction to the Biochemistry of Human Disease Lecture 22 – Molecular Diagnostics  1949: discovery of restriction endonucleases, Southern, DNA sequencing, synthetic oligonucleotides  1953: Watson and Crick, DNA double helix  1958: isolation of DNA polymerase  1960s: hybridization  1970s: some kind of blotting based on the picture  1980s: PCR and RFLP  Sickle cell anemia o Detection by RFLP and Southern o Southern is very labor-intensive and takes over a week to finish  PCR to amplify DNA; more starting material reduces time to 1 day  PCR disadvantages: based on known sequence, contamination risk, may miss heterogeneous large insertion/deletion  Molecular diagnostics o Used in diagnosis, monitoring, therapeutic, predictive… reduce morbidity and mortality o A rapidly growing field  Sanger sequencing o Using DNA polymerase to synthesize complementary strand to ssDNA o Termination by 2’,3’-dideoxynucleotides o Analyze fragment using gel or capillary electrophoresis o Newer DNA sequencing now available  Hepatitis B o Monitoring antiviral therapy by measuring viral load o rtPCR: fluorescent probe with nearby counterdye probe – completed polymerization  separate of probes and emit fluorescence. Measure fluorescence as a quantitative value of products. Diseases Name Causes