Transcript PCR - University of Hawaii

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!