1. Polymerase Chain Reaction
PCR
Polymerase Chain Reaction
Mimics the natural duplication of DNA during replication - protein enzyme is called DNA polymerase
Allows amplification of genetic information to ease in study of DNA, RNA, protein (genetic sequence of a protein expressed at very low levels in the cell can be amplified for studies of structure-function OR single gene sequence can be isolated)
Use of PCR in DNA fingerprinting, ID of mutations in genome, detect pathogenic organisms, etc.
PCR carried out at elevated temperatures - Use of thermostable DNA polymerase from thermophilic bacterium Thermus aquaticus (“Taq”)
Need to know something about sequence of DNA in order to design primers
Variation of traditional PCR:
REAL TIME PCR
Quantitate gene expression, collect data during growth/viral infection/etc.

2. Polymerase Chain Reaction - 3 steps
PCR
Polymerase Chain Reaction - 3 steps
Denature dsDNA
Anneal primers to now ssDNA
primers designed and commercially synthesized
annealing temp ~ Tm (melting temperature)
usually nt long
Polymerization (DNA synthesis - get 2 copies from 1)
at 72 ˚C - optimal activity of Taq
One cycle can be repeated over & over
After cycle 30, > 1 billion identical molecules (230 = 1.07 x 109)

3. PCR

4. DNA-based technology
Polymerase chain reaction (PCR)

5. Polymerase Chain Reaction
PCR
Polymerase Chain Reaction
Amplify hemoglobin gene from rabbit - specifically the b globin gene
Exons/Introns - Figure 3
Use PCR for amplification
Your DNA template to start is plasmid DNA (4700 bp circular DNA)
DNA primers designed to amplify just  globin gene

6. Polymerase Chain Reaction
PCR
Polymerase Chain Reaction
Thursday before 10 am
One member of each group come to Rigge 232 to start PCR reaction; PCR reaction runs ~2 hrs
Thursday lab
Separate PCR reaction on agarose gel to determine if correct size piece was amplified
Visualize DNA on agarose gel - gel contains ethidium bromide
Ethidium Bromide fluoresces under UV light
EB intercalates into DNA
Put gel on UV light source after electrophoresis

7. PCR animations
(uses reverse transcription as well to convert RNA into DNA before PCR)