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Transcript
PCR! This Book Belongs To: Robyn Inouye ABE Summer 2007 What is PCR? • Pandas Can Run? • Pandas Cook Rice? • Pandas Counting Rainbows? Polymerase Chain Reaction! Some History: • Invented in 1983 by Kary Mullis (while driving with his girlfriend) • Isolating and exponentially amplifying a fragment of DNA, via enzymatic replication • The sequencing of genes and the diagnosis of hereditary diseases, the identification of genetic fingerprints (used in forensics and paternity testing), the detection and diagnosis of infectious diseases, and the creation of transgenic organisms How It Works: Why PCR? • RT • T-DNA • RNA to DNA has only coding regions (no introns) • Make cDNA from mRNAlibraries • To check for insert of foreign DNA • Tom make copies of a gene of interest for insertion RT-PCR: • AMV reverse transcriptase from the avian myeloblastosis virus • Reverse Transcriptase (RT): A DNA polymerase enzyme that uses an RNA template to synthesize a complementary molecule of double stranded DNA • RNA template RT cDNA Amplify using PCR T-DNA Mutagenesis: • T-DNA=Transfer DNA • Mutagenesis=Creating mutations! • mutations are changes to the base pair sequence of genetic material (either DNA or RNA). Mutations can be caused by copying errors in the genetic material during cell division and by exposure to ultraviolet or ionizing radiation, chemical mutagens, or viruses Ti Plasmid Results: RT-PCR Group 2 • Restriction digest was a success! • Now we have our plasmids for cloning Group 3 Results: T-DNA Group 2 • Transfer of DNA a success! • “Mutant” genes found Thanks for Watching!
