Post on 16-Jan-2016
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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.
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)
PCR
DNA-based technology
Polymerase chain reaction (PCR)
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
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
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)