PCR Polymerase Chain Reaction Mimics the natural duplication
- Slides: 7
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.
PCR Polymerase Chain Reaction - 3 steps Denature ds. DNA Anneal primers to now ss. DNA primers designed and commercially synthesized annealing 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 & over After 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 amplification Your 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 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
PCR animations http: //www. people. virginia. edu/~rjh 9 u/pcranim. html http: //www. bio. davidson. edu/courses/Immunology/Fl ash/RT_PCR. html (uses reverse transcription as well to convert RNA into DNA before PCR)
