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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 PCRQuantitate gene expression, collect data during growth/viral infection/etc.
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PCR Polymerase Chain Reaction - 3 steps
Denature dsDNAAnneal primers to now ssDNA
primers designed and commercially synthesizedannealing temp ~ Tm (melting temperature)usually 15-20 nt long
Polymerization (DNA synthesis - get 2 copies from 1)at 72 ˚C - optimal activity of Taq
One cycle can be repeated over & overAfter cycle 30, > 1 billion identical molecules (230 = 1.07 x 109)
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PCR
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DNA-based technology
Polymerase chain reaction (PCR)
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PCR Polymerase Chain Reaction
Amplify hemoglobin gene from rabbit - specifically the globin gene
Exons/Introns - Figure 3
Use PCR for amplificationYour DNA template to start is plasmid DNA (4700 bp circular DNA)DNA primers designed to amplify just globin gene
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PCR Polymerase Chain Reaction
Thursday before 10 amOne member of each group come to Rigge 232 to start PCR reaction; PCR reaction runs ~2 hrs
Thursday labSeparate PCR reaction on agarose gel to determine if correct size piece was amplifiedVisualize DNA on agarose gel - gel contains ethidium bromideEthidium Bromide fluoresces under UV lightEB intercalates into DNAPut gel on UV light source after electrophoresis
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PCR animations
http://www.people.virginia.edu/~rjh9u/pcranim.html
http://www.bio.davidson.edu/courses/Immunology/Flash/RT_PCR.html (uses reverse transcription as well to convert RNA into DNA before PCR)