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PlanA. Link the technique of PCR to the basic knowledge about DNA replication
a. Review DNA replication in vivob. DNA replication in vitroc. Compare and contrast the in vivo and in vitro DNA replication
B. PCR as a technique for selective amplification of specific segments of DNA from a complex genomea. DNA replication cycles exponential amplification with doubling b. Steps of each cycle of replication
C. Important considerations for preparation of Genomic DNA from cheek cells for usea. Components of cell lysis solution and consideration of Mg+2 (Chelex)b. PCR reaction components
PCRBackground Information
• Review DNA replication in vivo • DNA replication in vitro• Compare in vivo & in vitro DNA
synthesis• Genomic DNA and cell lysis• Components of lysis mixture• PCR
• Steps • Reaction mixture components
• Human genomic DNA to be amplified (next lab)
Basic Lab Skills
• Genomic DNA preparation• Setting-up a PCR reaction• Analysis using DNA Gel
Electrophoresis (next lab)
Compare and contrast DNA Replication in vivo
PCR-DNA Amplification in vitro
DNA Replication • Initiation
Strand Separation by helicasePrimer synthesis by primase
• ElongationExtension and polymerization of nucleotides by DNA polymerase using dNTPs: dATP, dTTP, dCTP, and dGTP
• TerminationLeading strand- End of template strandLagging strand- Replace RNA primer with DNA strand & fill-in gaps with DNA ligase
Comparison of In vivo and In vitro reactions
In vivo In vitro
Strand separation –
Priming –
Elongation –
Preparation of genomic DNA
• Lyse or break cells by disrupting cell membranes using a detergent
• Digest with a protease to release naked DNA
• Separate DNA from digested proteins
PCR: polymerase Chain ReactionDNA amplification in a test tube
http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
http://www.dnalc.org/ddnalc/resources/animations.html
PCR animation Dolan learning Center• http://www.dnalc.org/
• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Polymerase Chain Reaction (PCR)http://www.biology.arizona.edu/
Target region of PCR amplificationAn Alu repeat Element inserted in intron 8 of the Tissue PlasminogenActivator (TPA gene) in some individuals
Position of PCR primers
Position of PCR primers
Individual genotypes
• cDNAhttp://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
• Plasmid cloning and transformationhttp://www.sumanasinc.com/webcontent/anisamples/molecularbiology/plasmidcloning_fla.html
• Steps of gene cloning http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Plasmid Cloning